Streptobacillus Moniliformis Clin

Clinical Microbiology Reviews

January 2007, Volume 20, Issue 1 , pp. 1-204

Instructions To Authors:

CLINICAL MICROBIOLOGY REVIEWS: 2007 INSTRUCTIONS TO AUTHORS Clin. Microbiol. Rev. 2007 20: 1-12. Reviews:

Sean P. Elliott Rat Bite Fever and Streptobacillus moniliformis Clin. Microbiol. Rev. 2007 20: 13-22. Anette Wohnsland, Wolf Peter Hofmann, and Christoph Sarrazin Viral Determinants of Resistance to Treatment in Patients with Hepatitis C Clin. Microbiol. Rev. 2007 20: 23-38. G. Balakrish Nair, Thandavarayan Ramamurthy, Sujit K. Bhattacharya, Basabjit Dutta, Yoshifumi Takeda, and David A. Sack Global Dissemination of Vibrio parahaemolyticus Serotype O3:K6 and Its Serovariants Clin. Microbiol. Rev. 2007 20: 39-48. Diane S. Leland and Christine C. Ginocchio Role of Cell Culture for Virus Detection in the Age of Technology Clin. Microbiol. Rev. 2007 20: 49-78. Florence Depardieu, Isabelle Podglajen, Roland Leclercq, Ekkehard Collatz, and Patrice Courvalin Modes and Modulations of Antibiotic Resistance Gene Expression Clin. Microbiol. Rev. 2007 20: 79-114. Carol A. Kauffman Histoplasmosis: a Clinical and Laboratory Update Clin. Microbiol. Rev. 2007 20: 115-132. M. A. Pfaller and D. J. Diekema Epidemiology of Invasive Candidiasis: a Persistent Public Health Problem Clin. Microbiol. Rev. 2007 20: 133-163. Vahab Ali and Tomoyoshi Nozaki Current Therapeutics, Their Problems, and Sulfur-Containing-Amino-Acid Metabolism as a Novel Target against Infections by "Amitochondriate" Protozoan Parasites Clin. Microbiol. Rev. 2007 20: 164-187. David J. Conway Molecular Epidemiology of Malaria Clin. Microbiol. Rev. 2007 20: 188-204.

CLINICAL MICROBIOLOGY REVIEWS, Jan. 2007, p. 1–12 Vol. 20, No. 1 0893-8512/07/$08.00ϩ0 doi:10.1128/CMR.00045–06 Copyright © 2007, American Society for Microbiology. All Rights Reserved. CLINICAL MICROBIOLOGY REVIEWS

2007 INSTRUCTIONS TO AUTHORS*

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Use of (O); the numeral one (1), the letter “el” (l), and the international units when dosing colistin will help letter “eye” (I); and a multiplication sign (ϫ) and the decrease confusion related to various formulations letter “ex” (x). Do not create symbols as graphics or use of the drug around the world. Antimicrob. Agents special fonts that are external to your word processing Chemother. 50:2274–2275. (Letter.) {“Letter” or program; use the “insert symbol” function. Set the page “Letter to the editor” is allowed but not required at the size to 81⁄2 by 11 inches (ca. 21.6 by 28 cm). Italicize or end of such an entry.} underline any words that should appear in italics, and 6. Fitzgerald, G., and D. Shaw. In A. E. Waters (ed.), indicate paragraph lead-ins in bold type. Clinical microbiology, in press. EFH Publishing Co., Authors who are unsure of proper English usage Boston, MA.* {Chapter title is optional.} should have their manuscripts checked by someone pro- 7. Forman, M. 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Arthritis Rheum. tions, published conference proceedings, meeting ab- 39(Suppl.):S185. {Meeting abstract published in jour- stracts from published abstract books or journal supple- nal supplement.} ments, letters (to the editor), and company publications, 9. Green, P. N., D. Hood, and C. S. Dow. 1984. Taxo- as well as in-press journal articles, book chapters, and nomic status of some methylotrophic bacteria, p. books (publication title must be given). Arrange the 251–254. In R. L. Crawford and R. S. Hanson (ed.), citations in alphabetical order (letter by letter, ignoring Microbial growth on C1 compounds. Proceedings of spaces and punctuation) by first author and number the 4th International Symposium. American Society consecutively. Provide the names of all the authors for for Microbiology, Washington, DC. each reference. All listed references must be cited par- 10. Odell, J. C. April 1970. Process for batch culturing. enthetically by number in the text. 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Distribution of fimbrial, phage and plasmid print references. associated virulence genes among poultry Salmo- nella enterica serovars, abstr. P-038, p. 445. Abstr. 1. Arendsen, A. F., M. Q. Solimar, and S. W. Ragsdale. 105th Gen. Meet. Am. Soc. Microbiol. American 1999. Nitrate-dependent regulation of acetate bio- Society for Microbiology, Washington, DC. {Ab- synthesis and nitrate respiration by Clostridium ther- stract title is optional.} moaceticum. J. Bacteriol. 181:1489–1495. 14. Stratagene. 2006. Yeast DNA isolation system: in- 2. Cox, C. S., B. R. Brown, and J. C. Smith. J. Gen. struction manual. Stratagene, La Jolla, CA. {Use the Genet., in press.* {Article title is optional; journal title company name as the author if none is provided for a is mandatory.} company publication.} 3. da Costa, M. S., M. F. Nobre, and F. A. Rainey. 2001. Genus I. Thermus Brock and Freeze 1969, 295,AL emend. Nobre, Tru¨per and da Costa 1996b, *A reference to an in-press ASM publication should state 605, p. 404–414. In D. R. Boone, R. W. Castenholz, the control number (e.g., CMR00577-07) if it is a journal and G. M. Garrity (ed.), Bergey’s manual of system- article or the name of the publication if it is a book. VOL. 20, 2007 2007 CMR INSTRUCTIONS TO AUTHORS 7

Online references must provide the same information NOT be included in the article. However, company that print references do, but some variation is allowed. URLs that permit access to scientific data related to the For online journal articles, posting or revision dates may study or to shareware used in the study are permitted. replace the year of publication, and a DOI or URL may be provided in addition to or in lieu of volume and page (iii) References related to supplemental material. numbers. Some examples follow. References that are related only to supplemental material hosted by ASM or posted on a personal/institutional 1. Charlier, D., and N. Glansdorff. September 2004, website should not be listed in the References section of posting date. Chapter 3.6.1.10, Biosynthesis of argi- an article; include them with the supplemental material nine and polyamines. In R. Curtiss III et al. (ed.), itself. EcoSal—Escherichia coli and Salmonella: cellular and molecular biology. ASM Press, Washington, DC. (iv) Referencing publish-ahead-of-print manuscripts. http://www.ecosal.org. {Note that each chapter has its Citations of ASM Accepts manuscripts should look like own posting date.} the following example. 2. Dionne, M. S., and D. S. Schneider. 2002. Screen- ing the fruitfly immune system. Genome Biol. 3: Wang, G. G., M. P. Pasillas, and M. P. Kamps. 15 REVIEWS1010. http://genomebiology.com/2002/3/4 May 2006. Persistent transactivation by Meis1 re- /reviews/1010. places Hox function in myeloid leukemogenesis mod- 3. Smith, F. X., H. J. Merianos, A. T. Brunger, and els: evidence for co-occupancy of Meis1-Pbx and Hox- D. M. Engelman. 2001. Polar residues drive associa- Pbx complexes on promoters of leukemia-associated tion of polyleucine transmembrane helices. Proc. genes. Mol. Cell. Biol. doi:10.1128/MCB.00586-06. Natl. Acad. Sci. USA 98:2250–2255. doi:10.1073/ pnas.041593698. If an author of an article cites an ASM Accepts manu- 4. Winnick, S., D. O. Lucas, A. L. Hartman, and D. Toll. script in his paper but wishes at the proof stage to 2005. How do you improve compliance? Pediatrics change the reference entry to that for the published 115:e718–e724. article, the following style should be used: NOTE: A posting or accession date is required for any Wang, G. G., M. P. Pasillas, and M. P. Kamps. online reference that is periodically updated or changed. 15 May 2006. Persistent transactivation by Meis1 re- places Hox function in myeloid leukemogenesis mod- (ii) References cited in the text. References to unpub- els: evidence for co-occupancy of Meis1-Pbx and Hox- lished data, manuscripts submitted for publication, un- Pbx complexes on promoters of leukemia-associated published conference presentations (e.g., a report or genes. Mol. Cell. Biol. doi:10.1128/MCB.00586-06. poster that has not appeared in published conference (Subsequently published, Mol. Cell. Biol. 26:3902– proceedings), personal communications, patent applica- 3916, 2006.) tions and patents pending, computer software, data- bases, and websites (home pages) should be made par- Other journals may use different styles for their pub- enthetically in the text as follows. lish-ahead-of-print manuscripts, but citation entries . . . similar results (R. B. Layton and C. C. Weathers, must include the following information: author name(s), unpublished data). posting date, title, journal title, and volume and page numbers and/or DOI. The following is an example: . . . system was used (J. L. McInerney, A. F. Holden, and P. N. Brighton, submitted for publication). Zhou, F. X., H. J. Merianos, A. T. Brunger, and D. M. Engelman. 13 February 2001, posting date. Polar . . . as described previously (M. G. Gordon and F. L. residues drive association of polyleucine transmem- Rattner, presented at the Fourth Symposium on Food brane helices. Proc. Natl. Acad. Sci. USA doi:10.1073 Microbiology, Overton, IL, 13 to 15 June 1989). {For /pnas.041593698. nonpublished abstracts, posters, etc.} . . . this new process (V. R. Smoll, 20 June 1999, Aus- tralian Patent Office). {For non-U.S. patent applications, Correspondent Footnote give the date of publication of the application.} . . . available in the GenBank database (http://www The complete mailing address, a single telephone .ncbi.nlm.nih.gov/Genbank/index.html). number, a single fax number, and a single e-mail address for the corresponding author should be included on the . . . using ABC software (version 2.2; Department of title page of the manuscript. This information will be Microbiology, State University [http://www.stu.micro]). published in the article as a footnote to facilitate communication, and the e-mail address will be used to notify URLs for companies that produce any of the products the corresponding author of the availability of proofs mentioned in your study or for products being sold may and, later, of the PDF file of the published article. 8 2007 CMR INSTRUCTIONS TO AUTHORS CLIN.MICROBIOL.REV.

Errata names or their symbols (folate, Ala, Leu, etc.) may also be used. The Erratum section provides a means of correcting Define each abbreviation and introduce it in paren- errors that occurred during the writing, typing, editing, theses the first time it is used; e.g., ‘‘cultures were or printing (e.g., a misspelling, a dropped word or line, grown in Eagle minimal essential medium (MEM).’’ or mislabeling in a figure) of a published article. Submit Generally, eliminate abbreviations that are not used Errata via Rapid Review (see “How To Submit Manu- at least three times in the text (including tables and scripts,” above). In the Abstract section of the submis- figure legends). sion form (a required field), put “Not Applicable.” Up- load the text of your Erratum as an MS Word file. Please see a recent issue for correct formatting. Not requiring introduction. In addition to abbreviations for Syste`me International d’Unite´s (SI) units of Authors’ Corrections measurement, other common units (e.g., bp, kb, and Da), and chemical symbols for the elements, the follow- The Author’s Correction section provides a means of ing should be used without definition in the title, sum- correcting errors of omission (e.g., author names or cita- mary, text, figure legends, and tables: DNA (deoxyribo- tions) and errors of a scientific nature that do not alter the nucleic acid); cDNA (complementary DNA); RNA overall basic results or conclusions of a published article (ribonucleic acid); cRNA (complementary RNA); (e.g., an incorrect unit of measurement or order of magni- RNase (ribonuclease); DNase (deoxyribonuclease); tude used throughout, contamination of one of numerous rRNA (ribosomal RNA); mRNA (messenger RNA); cultures, or misidentification of a mutant strain, causing tRNA (transfer RNA); AMP, ADP, ATP, dAMP, erroneous data for only a portion [noncritical] of the ddATP, GTP, etc. (for the respective 5Ј phosphates of study). Note that the addition of new data is not permitted. adenosine and other nucleosides) (add 2Ј-, 3Ј-, or 5Ј- For corrections of a scientific nature or issues involv- when needed for contrast); ATPase, dGTPase, etc. ing authorship, including contributions and use or own- (adenosine triphosphatase, deoxyguanosine triphos- ership of data and/or materials, all disputing parties phatase, etc.); NAD (nicotinamide adenine dinucleo- must agree, in writing, to publication of the Correction. ϩ tide); NAD (nicotinamide adenine dinucleotide, oxi- For omission of an author’s name, letters must be signed dized); NADH (nicotinamide adenine dinucleotide, by the authors of the article and the author whose name reduced); NADP (nicotinamide adenine dinucleotide was omitted. The editor who handled the article will be phosphate); NADPH (nicotinamide adenine dinucle- consulted if necessary. otide phosphate, reduced); NADPϩ (nicotinamide Submit an Author’s Correction via Rapid Review (see adenine dinucleotide phosphate, oxidized); poly(A), “How To Submit Manuscripts,” above). In the submis- poly(dT), etc. (polyadenylic acid, polydeoxythymidylic sion form, select Erratum as the manuscript type; there acid, etc.); oligo(dT), etc. (oligodeoxythymidylic acid, is no separate selection in Rapid Review for Authors’ etc.); UV (ultraviolet); PFU (plaque-forming units); Corrections, but your Correction will be published as CFU (colony-forming units); MIC (minimal inhibitory such if appropriate. In the Abstract section of the sub- concentration); Tris [tris(hydroxymethyl)aminometh- mission form (a required field), put “Not Applicable.” ane]; DEAE (diethylaminoethyl); EDTA (ethylene Upload the text of your Author’s Correction as an MS diaminetetraacetic acid); EGTA [ethylene glycol- Word file. Please see a recent issue for correct format- bis(␤-aminoethyl ether)-N,N,NЈ,NЈ-tetraacetic acid]; ting. Signed letters of agreement must be supplied as HEPES (N-2-hydroxyethylpiperazine-NЈ-2-ethanesul- supplemental material (scanned PDF files). fonic acid); PCR (polymerase chain reaction); and AIDS (acquired immunodeficiency syndrome). Ab- breviations for cell lines (e.g., HeLa) also need not be Abbreviations defined. The following abbreviations should be used without definition in tables: General. Abbreviations should be used as an aid to the reader, rather than as a convenience to the author, and therefore their use should be limited. Abbrevia- amt (amount) SE (standard error) tions other than those recommended by the Interna- approx (approximately) SEM (standard error of the tional Union of Pure and Applied Chemistry-Interna- avg (average) mean) tional Union of Biochemistry (IUPAC-IUB) (Biochemical concn (concentration) sp act (specific activity) Nomenclature and Related Documents, Portland Press, diam (diameter) sp gr (specific gravity) London, United Kingdom, 1992; available at http://www expt (experiment) temp (temperature) .chem.qmul.ac.uk/iupac/bibliog/white.html) should be exptl (experimental) tr (trace) ht (height) vol (volume) used only when a case can be made for necessity, such as mo (month) vs (versus) in tables and figures. mol wt (molecular weight) wk (week) It is often possible to use pronouns or to paraphrase no. (number) wt (weight) a long word after its first use (e.g., ‘‘the drug’’ or ‘‘the prepn (preparation) yr (year) substrate’’). Standard chemical symbols and trivial SD (standard deviation) VOL. 20, 2007 2007 CMR INSTRUCTIONS TO AUTHORS 9

Reporting Numerical Data nouns capitalized (e.g., Tobacco mosaic virus, Murray Valley encephalitis virus). When the behavior or manip- Standard metric units are used for reporting length, ulation of individual viruses is discussed, the vernacular weight, and volume. For these units and for molarity, use (e.g., tobacco mosaic virus, Murray Valley encephalitis the prefixes m, ␮, n, and p for 10Ϫ3,10Ϫ6,10Ϫ9, and Ϫ12 3 virus) should be used. If desired, synonyms may be 10 , respectively. Likewise, use the prefix k for 10 . added parenthetically when the name is first mentioned. Avoid compound prefixes such as m␮ or ␮␮. Use ␮g/ml ␮ Approved generic (or group) and family names may also or g/g in place of the ambiguous ppm. Units of tem- be used. perature are presented as follows: 37°C or 324 K. For enzymes, use the recommended (trivial) name When fractions are used to express units such as en- assigned by the Nomenclature Committee of the IUB as zymatic activities, it is preferable to use whole units, such described in Enzyme Nomenclature (Academic Press, as ‘‘g’’ or ‘‘min,’’ in the denominator instead of fractional Inc., New York, NY, 1992) and at http://www.chem or multiple units, such as ␮g or 10 min. For example, ‘‘pmol/min’’ is preferable to ‘‘nmol/10 min,’’ and ‘‘␮mol/ .qmul.ac.uk/iubmb/enzyme/. g’’ is preferable to ‘‘nmol/␮g.’’ It is also preferable that For nomenclature of restriction enzymes, DNA meth- an unambiguous form such as exponential notation be yltransferases, homing endonucleases, and their genes, used; for example, ‘‘␮mol gϪ1 minϪ1’’ is preferable to refer to the article by Roberts et al. (Nucleic Acids Res. ‘‘␮mol/g/min.’’ Always report numerical data in the ap- 31:1805–1812, 2003). plicable SI units. Genetic nomenclature should essentially follow the rec- For a review of some common errors associated with ommendations of Demerec et al. (Genetics 54:61–76, statistical analyses and reports, plus guidelines on how to 1966) and those given in the instructions to authors of the avoid them, see the article by Olsen (Infect. Immun. Journal of Bacteriology and Molecular and Cellular Biology 71:6689–6692, 2003). (January issues) and Eukaryotic Cell (February issue). To For a review of basic statistical considerations for vi- facilitate accurate communication, it is important that rology experiments, see the article by Richardson and standard genetic nomenclature be used whenever possible Overbaugh (J. Virol. 79:669–676, 2005). and that deviations or proposals for new naming systems be endorsed by an appropriate authoritative body. Review and/or publication of submitted manuscripts that contain Nomenclature new or nonstandard nomenclature may be delayed by the editor or the Journals Department so that they may be The spelling of bacterial names should follow the Ap- reviewed by the Genetics and Genomics Committee of the proved Lists of Bacterial Names (Amended) & Index of the ASM Publications Board. Before submission of manu- Bacterial and Yeast Nomenclatural Changes (V. B. D. Sker- scripts, authors may direct questions on genetic nomen- man et al., ed., ASM Press, Washington, DC, 1989) and the clature to the committee’s chairman: Maria Costanzo (e- validation lists and notification lists published in the Inter- mail: [email protected]). Such a consultation national Journal of Systematic and Evolutionary Microbiol- should be mentioned in the manuscript submission letter. ogy (formerly the International Journal of Systematic Bacte- riology) since January 1989. In addition, two sites on the World Wide Web list current approved bacterial names: Bacterial Nomenclature Up-to-Date (http://www.dsmz.de ILLUSTRATIONS AND TABLES /microorganisms/main.php?contentleft_idϭ14) and List of Prokaryotic Names with Standing in Nomenclature (http: Digital files that are acceptable for production (see //www.bacterio.cict.fr). If there is reason to use a name that below) must be provided for all illustrations on return does not have standing in nomenclature, the name should of the modified manuscript. (On initial submission, the be enclosed in quotation marks in the title and at its first entire paper may be submitted in PDF format.) use in the abstract and the text and an appropriate state- We strongly recommend that before returning their ment concerning the nomenclatural status of the name modified manuscripts, authors check the acceptability should be made in the text. “Candidatus” species should of their digital images for production by running their always be set in quotation marks. files through Rapid Inspector, a tool provided at the Names used for viruses should be those approved by following URL: http://rapidinspector.cadmus.com/mw/. the International Committee on Taxonomy of Viruses Rapid Inspector is an easy-to-use Web-based application (ICTV) and published in Virus Taxonomy: Eighth Report that identifies file characteristics that may render the of the International Committee on Taxonomy of Viruses image unusable for production. (C. M. Fauquet et al., ed., Elsevier Academic Press, San Illustrations may be continuous-tone images, line Diego, CA, 2005). In addition, the recommendations of drawings, or composites. Suggestions about how to en- the ICTV regarding the use of species names should sure accurate color reproduction are given below. generally be followed: when the entire species is dis- The preferred format for tables is MS Word; however, cussed as a taxonomic entity, the species name, like WordPerfect and Acrobat PDF are also acceptable (see other taxa, is italic and has the first letter and any proper the section on Tables below). 10 2007 CMR INSTRUCTIONS TO AUTHORS CLIN.MICROBIOL.REV.

Macintosh Illustrations File type Application File types and formats. As mentioned above, illustra- Black and white Color (CMYK)a tions may be supplied as PDF files for reviewing pur- Adobe Illustrator 6.0, EPS EPS poses only on initial submission; in fact, we recommend 7.0, 8.0, 9.0, 10.0, 11.0 this option to minimize file upload time. At the modifi- CS cation stage, production quality digital files must be Adobe InDesign 1.0 EPS EPS submitted: TIFF or EPS files from supported applica- Adobe PageMaker 6.5 EPS EPS Adobe Photoshop 4.0, TIFF TIFF tions or PowerPoint files (black and white only). Except 5.0, 5.5, 6.0, 7.0, 8.0 for figures produced in PowerPoint, all graphics submit- CS ted with modified manuscripts must be bitmap, gray- b Adobe Photoshop 5.0 TIFF N/A scale, or CMYK (not RGB). Halftone images (those LE ChemDraw Pro 5.0 EPS/TIFF EPS/TIFF with various densities or shades) must be grayscale, not Corel Photo-Paint 8.0 TIFF EPS bitmap. CorelDRAW 6.0, 8.0 EPS/TIFF EPS Color PowerPoint files are not accepted because the Deneba Canvas 6.0, 7.0, EPS/TIFF EPS application, designed for developing on-screen com- 8.0 puter presentations, uses the RGB color mode whereas Macromedia FreeHand EPS EPS 7.0, 8.0, 9.0 the printing process uses the CMYK color mode. Colors PowerPoint 98, 2001 PPTc N/Ab that are represented in a PowerPoint image may not be Prism 3 by GraphPad TIFF N/Ab reproducible on a printing press. Although black-and- b Synergy Kaleidagraph EPS N/A white Microsoft PowerPoint files are accepted, we do not 3.08, 3.51 recommend the use of PowerPoint. PowerPoint requires aColor graphics must be saved and printed in the CMYK mode, not RGB. users to pay close attention to the fonts used in their bASM accepts only black-and-white, not color, graphics created with Kaleida- graph, Adobe Photoshop 5.0 LE, Prism 3 by GraphPad, and PowerPoint. images (see the section on Fonts below). If instructions cFor instructions on saving PowerPoint files, refer to the Cadmus digital art for fonts are not followed exactly, images prepared for website at http://cjs.cadmus.com/da/index.jsp. publication are subject to missing characters, improperly converted characters, or shifting/obscuring of elements Windows or text in the figure. Use of PowerPoint is therefore not File type recommended for either color or black-and-white illustra- Application a tions. Black and white Color (CMYK) Acceptable file types and formats for production Adobe Illustrator 7.0, 8.0, EPS EPS are given in the charts below. More-detailed instruc- 9.0, 10.0, 11.0 CS tions for preparing illustrations are available at http: Adobe InDesign 1.0 EPS EPS //cjs.cadmus.com/da. Please review this information be- Adobe PageMaker 6.5 EPS EPS Adobe Photoshop 4.0, 5.0, TIFF TIFF fore preparing your files. If you require additional infor- 5.5, 6.0, 7.0, 8.0 CS mation, please send an e-mail inquiry to digitalart Adobe Photoshop 5.0 LE TIFF N/Ab @cadmus.com. ChemDraw Pro 5.0 EPS/TIFF EPS/TIFF Corel Photo-Paint 8.0, 9.0 TIFF EPS CorelDRAW 7.0, 8.0, 9.0 EPS/TIFF EPS Minimum resolution. It is extremely important that a Deneba Canvas 6.0, 7.0 EPS/TIFF EPS high enough resolution is used. Any imported images Macromedia FreeHand 7.0, EPS EPS must be at the correct resolution before they are placed. 8.0, 9.0 Note, however, that the higher the resolution, the larger PowerPoint 97, 2000, XP PPTc N/Ab Prism 3 by GraphPad TIFF N/Ab the file and the longer the upload time. Publication qual- SigmaPlot 8.01 EPS EPS ity will not be improved by using a resolution higher than the minimum. Minimum resolutions are as follows: aColor graphics must be saved and printed in the CMYK mode, not RGB. bASM accepts only black-and-white, not color, graphics created with Adobe Photoshop 5.0 LE, Prism 3 by GraphPad, and PowerPoint. 300 dpi for grayscale and color cFor instructions on saving PowerPoint files, refer to the Cadmus digital art 600 dpi for lettering website at http://cjs.cadmus.com/da/index.jsp. 1,200 dpi for line art 600 dpi for combination art (lettering and images) Image Manipulation Size. All graphics MUST be submitted at their in- Computer-generated images may be processed only tended publication size; that is, the image uploaded minimally. Processing (e.g., changing contrast, bright- should be 100% of its print dimensions so that no re- ness, or color balance) is acceptable only if applied to all duction or enlargement is necessary. Resolution must parts of the image, as well as to the controls, equally, and be at the required level at the submitted size. Include descriptions of all such adjustments and the tools used only the significant portion of an illustration. White (both hardware and software) must be provided in the space must be cropped from the image, and excess manuscript. Unprocessed data and files must be retained space between panel labels and the image must be by the authors and be provided to the editor on request. eliminated. VOL. 20, 2007 2007 CMR INSTRUCTIONS TO AUTHORS 11

Maximum width for a 1-column figure: 35⁄16 inches Point, Word, Excel) for color illustrations because (ca. 8.4 cm) they are restricted to the RGB color space. Maximum width for a 2-column figure: 67⁄8 inches (ca. 17.4 cm) Drawings Minimum width for a 2-column figure: 41⁄4 inches (10.8 cm) Submit graphs, charts, complicated chemical or math- Maximum height: 91⁄16 inches (23.0 cm) ematical formulas, diagrams, and other drawings as finished products not requiring additional artwork or type- Contrast. Illustrations must contain sufficient contrast setting. No part of the graph or drawing may be to withstand the inevitable loss of contrast and detail handwritten. All elements, including letters, numbers, inherent in the printing process. See also the section on and symbols, must be easily readable, and both axes of a color illustrations below. graph must be labeled. Keep in mind that the journal is published both in print and online and that the same Labeling and assembly. All final lettering, labeling, electronic files submitted by the authors are used to tooling, etc., MUST be incorporated into the figures. It produce both. cannot be added at a later date. If a figure number is When creating line art, please use the following guide- included, it must appear well outside the boundaries of lines: the image itself. (Numbering may need to be changed at 1. All art MUST be submitted at its intended publica- the copyediting stage.) Each figure must be uploaded as tion size. For acceptable dimensions, see the Size a separate file, and any multipanel figures must be as- section above. sembled into one file; i.e., rather than sending a separate file for each panel in a figure, assemble all panels in one 2. Avoid using screens (i.e., shading) in line art. It can piece and supply them as one file. be difficult and time-consuming to reproduce these images without moire´ patterns. Various pattern back- Fonts. To avoid font problems, set all type in one of grounds are preferable to screens as long as the pat- the following fonts: Helvetica, Times Roman, European terns are not imported from another application. If PI, Mathematical PI, or Symbol. All fonts other than you must use images containing screens, these five must be converted to paths (or outlines) in the • Generate the image at line screens of 85 lines application with which they were created. For proper per inch or lower. font use in PowerPoint images, refer to the Cadmus • When applying multiple shades of gray, differen- digital art website, http://cjs.cadmus.com/da/instructions tiate the gray levels by at least 20%. /ppt_disclaimer.jsp. • Never use levels of gray below 20% or above 70% as they will fade out or become totally black upon Compression. Images created with Macintosh appli- scanning and reduction. cations may be compressed with Stuffit. Images created 3. Use thick, solid lines that are no finer than 1 point in with Windows applications may be compressed with thickness. WINZIP or PKZIP. 4. No type should be smaller than 6 points at the final publication size. Color illustrations. Because of the requirements of print production, color illustrations must be in the 5. Avoid layering type directly over shaded or textured CMYK (cyan, magenta, yellow, black) color space. areas. The “normal” color mode for most computer software 6. Avoid the use of reversed type (white lettering on a is RGB (red, green, blue), which is also the color black background). space of your computer monitor. Since CMYK is a smaller color space (meaning it can define fewer col- 7. Avoid heavy letters, which tend to close up, and un- ors), colors often shift when an RGB file is converted usual symbols, which the printer may not be able to to CMYK. In particular, figures showing red or green reproduce in the legend. fluorescence and those with a significant range of col- 8. If colors are used, avoid using similar shades of the ors may be difficult or impossible to reproduce during same color and avoid very light colors. the printing process. Color illustrations must be supplied in the CMYK color mode, as either (i) CMYK TIFF images with a In figure ordinate and abscissa scales (as well as table resolution of at least 300 pixels per inch (raster files, column headings), avoid the ambiguous use of numbers consisting of pixels) or (ii) Illustrator-compatible EPS with exponents. Usually, it is preferable to use the Syste`me files with CMYK color elements (vector files, consist- International d’Unite´s (SI) symbols (␮ for 10Ϫ6, m for ing of lines, fonts, fills, and images). See the charts 10Ϫ3, k for 103, M for 106, etc.). A complete listing of SI above for a list of supported applications. symbols can be found in the International Union of Pure We cannot accept any Microsoft Office files (Power- and Applied Chemistry (IUPAC) publication Quantities, 12 2007 CMR INSTRUCTIONS TO AUTHORS CLIN.MICROBIOL.REV.

TABLE 1. Correlation between detection of V-Z viral antibody by Units and Symbols in Physical Chemistry (Blackwell Science, a Oxford, United Kingdom, 1993); an abbreviated list is neutralization and by EIA and IAHA available at http://www.iupac.org/reports/1993/homann No. of samples with V-Z Correlation Antibody virus-neutralizing antibody /index.html. Thus, a representation of 20,000 cpm on a (%) ﬁgure ordinate is to be made by the number 20 accompa- Positiveb Negative nied by the label kcpm. EIA When powers of 10 must be used, the journal requires Positive 50 4 94 that the exponent power be associated with the number Negative 3 64 shown. In representing 20,000 cells per ml, the numeral on the ordinate would be “2” and the label would be “104 cells IAHA Ϫ Positivec 37 0 ϫ 4 87 per ml” (not “cells per ml 10 ”). Likewise, an enzyme Negative 16 68 activity of 0.06 U/ml would be shown as 6 accompanied by the label 10Ϫ2 U/ml. The preferred designation would a Sera from individuals without evidence of a current V-Z virus infection. b Titer Ͼ 1:4. be 60 mU/ml (milliunits per milliliter). c Titer Ͼ 1:8.

Tables Presentation of Nucleic Acid Sequences Tables that contain artwork, chemical structures, or Nucleic acid sequences of limited length which are the shading must be submitted as illustrations in an ac- primary subject of a study may be presented freestyle in ceptable format at the modification stage. The pre- the most effective format. Longer nucleic acid sequences ferred format for regular tables is MS Word; however, must be presented as figures in the following format to WordPerfect and Acrobat PDF are also acceptable. conserve space. Print the sequence in lines of approxi- Note that a straight Excel file is not currently an ac- mately 100 to 120 nucleotides in a nonproportional ceptable format. Excel files must be either embedded (monospace) font that is easily legible when published in a Word or WordPerfect document or converted to with a line length of 6 inches (ca. 15.2 cm). If possible, PDF before being uploaded. If your modified manuscript contains PDF tables, select “for reviewing lines of nucleic acid sequence should be further subdi- purposes only” at the beginning of the file upload vided into blocks of 10 or 20 nucleotides by spaces within process. the sequence or by marks above it. Uppercase and low- Tables should be formatted as follows. Arrange the ercase letters may be used to designate the exon-intron data so that columns of like material read down, not structure, transcribed regions, etc., if the lowercase let- across. The headings should be sufficiently clear so ters remain legible at a 6-inch (ca. 15.2-cm) line length. that the meaning of the data is understandable with- Number the sequence line by line; place numerals, rep- out reference to the text. See the Abbreviations sec- resenting the first base of each line, to the left of the tion (p. 8) of these Instructions for those that should lines. Minimize spacing between lines of sequence, leav- be used in tables. Explanatory footnotes are accept- ing room only for annotation of the sequence. Annota- able, but more extensive table “legends” are not. tion may include boldface, underlining, brackets, boxes, Footnotes should not include detailed descriptions of etc. Encoded amino acid sequences may be presented, if the experiment. Tables must include enough informa- necessary, immediately above or below the first nucleo- tion to warrant table format; those with fewer than six tide of each codon, by using the single-letter amino acid pieces of data will be incorporated into the text by the symbols. Comparisons of multiple nucleic acid se- copy editor. Table 1 is an example of a well-con- quences should conform as nearly as possible to the structed table. same format. CLINICAL MICROBIOLOGY REVIEWS, Jan. 2007, p. 13–22 Vol. 20, No. 1 0893-8512/07/$08.00ϩ0 doi:10.1128/CMR.00016-06 Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Rat Bite Fever and Streptobacillus moniliformis Sean P. Elliott* Department of Pediatrics and Steele Children’s Research Center, University of Arizona Health Sciences Center, Tucson, Arizona

INTRODUCTION ...... 13 HISTORICAL ASPECTS...... 13 BIOLOGY ...... 14 Morphology...... 14 Growth Characteristics ...... 14 Pathogenesis ...... 14 EPIDEMIOLOGY...... 14 Geographic Distribution ...... 14 Animal Infectivity...... 15 Rats...... 15 Mice ...... 16 Other animals...... 16 Human Infectivity ...... 16 CLINICAL FEATURES ...... 16 Initial Symptoms...... 16 Disease Progression...... 16 Outcome ...... 17 Review of English Literature...... 18 Epidemiology ...... 18 Clinical ﬁndings...... 19 Complications...... 19 Differential Diagnoses ...... 19 Diseases...... 19 Sodoku...... 19 Haverhill Fever...... 19 U.S. experience ...... 19 United Kingdom experience ...... 20 DIAGNOSIS ...... 20 Culture...... 20 Fatty Acid Proﬁles ...... 20 Other Methods ...... 20 TREATMENT...... 20 CONCLUSIONS ...... 21 REFERENCES ...... 21

INTRODUCTION this has changed such that children now account for over 50% of the cases in the United States, followed by laboratory per- Disease following the bite of a rat has been known in India sonnel and pet shop employees. Over 200 cases of rat bite fever for over 2,300 years, but it has been described worldwide much have been documented in the United States, but this is likely a more recently as rat bite fever. This term describes two similar significant under-representation because rat bite fever is not a yet distinct disease syndromes caused by Streptobacillus monili- reportable disease. Further, rat bite fever has a nonspecific formis or Spirillum minus. Rat bite fever caused by S. monili- presentation with a broad differential diagnosis, and isolation formis is more common in North America, while S. minus and identification of its causative organism, S. moniliformis,is infection, also known as sodoku, is more common in Asia. not straightforward. Thus, the challenges of diagnosis and Streptobacillary rat bite fever, the subject of this review, is a broadened demographic exposure demand close attention to systemic illness classically characterized by relapsing fever, this disease and its causative organism by clinicians. rash, migratory polyarthralgias, and a mortality rate of 13% when untreated. Often associated with the bite of a wild or laboratory rat, rat bite fever historically has affected laboratory HISTORICAL ASPECTS technicians and the poor. As rats have become popular as pets, Rat bite fever was first reported in the United States in 1839 (89). An association with a specific pathogen was not reported * Corresponding author. Mailing address: Department of Pediatrics, University of Arizona Health Sciences Center, 1501 N. Campbell Ave., until 1914, when Schottmu¨ller described Streptothrix muris ratti, Tucson, AZ 85724-5073. Phone: (520) 626-6507. Fax: (520) 626-5652. isolated from a rat-bitten man (71). This association was con- E-mail: [email protected]. firmed in the United States in 1916 (9). In 1925, the organism

13 14 ELLIOTT CLIN.MICROBIOL.REV.

was renamed Streptobacillus moniliformis (48), a name that has onstrate growth because sodium polyanethol sulfonate typi- remained in general use since, although some reports refer to cally is not added (46, 68, 75). Once the organism has grown, Actinomyces or Actinobacillus muris (41, 87). A milk-associated confirmation of its identity occurs by conventional biochemical outbreak of disease occurred in Haverhill, MA, in 1926 and and carbohydrate fermentation analysis (Table 1). Serologic was described by Place and Sutton (60). The organism found at testing and gas-liquid chromatographic analysis of the fatty this time was named Haverhillia multiformis by Parker and acid profile have also been used and are discussed below. Hudson (55), although this most likely represents S. moniliformis disease. Any review of the literature regarding rat bite Pathogenesis fever is complicated by the near-simultaneous description of Spirillum minus, the primary cause of rat bite fever in Asia, Because of the relatively low incidence and low mortality which is known by many as sodoku. Unfortunately, some reports rate of rat bite fever when recognized and treated, little infor- discuss both organisms simultaneously, blurring the distinction mation describing the pathogenesis of S. moniliformis exists. between the two diseases and epidemiologic distributions that However, the organism appears to be capable of producing are, in fact, distinct. morphological findings not customarily associated with bacterial infections. Autopsy of rat bite fever victims demonstrates pronounced erythrophagocytosis, hepatosplenomegaly, inter- BIOLOGY stitial pneumonia, and lymph node sinus hyperplasia (72). En- Morphology docarditis and myocarditis have also been demonstrated, along with degenerative changes in the kidneys and liver (1). Radio- Streptobacillus moniliformis is a highly pleomorphic, filamen- logical data suggest that rat bite fever may be considered a tous, gram-negative, nonmotile, and non-acid-fast rod. It usu- cause of damage to physes and acrophyses, mimicking frostbite ally appears straight but may be fusiform and may develop damage (53), and clinical data suggest that S. moniliformis may characteristic lateral bulbar swellings. The organism is typically have a predilection for synovial and serosal surfaces (67). Bi- arranged in chains and loosely tangled clumps (Fig. 1). It varies opsy of skin lesions seen in rat bite fever has demonstrated ␮ ␮ in its dimensions, from 0.1 to 0.5 m by 2.0 to 5.0 m, up to 10 leukocytoclastic vasculitis (90). Experimental infection in mice ␮ ␮ to 15 m, with long, curved segments up to 100 to 150 m (65). (70) results in a progressive polyarthritis, beginning with S. moniliformis exists in two variant types, the normally occur- fibrinopurulent exudate within the joint space and adjacent peri- ring bacillary form and the inducible or spontaneously occur- ostea in the first 24 h of infection. This changes to a predomi- ring, cell wall-deficient L form, growing with a “fried-egg” nately macrophage presence on day 4, followed by periarticular colony morphology. The L form is considered nonpathogenic abscess and necrosis on day 7. Periostitis develops by 2 weeks and (28); spontaneous conversion between the two forms in vitro is followed by fibrous connective tissue proliferation after 3 has been reported and is felt by some to be responsible for weeks. The degree of polyarthritis depends on the size of the clinical relapses and resistance to therapy (72). inoculum. It is of concern that persistence of organisms within Spirillum minus, the other etiologic agent of rat bite fever, joint spaces at 3 months of infection may occur despite the clear- was discovered during the 19th century and initially named ance of organisms from blood, liver, and spleen (70). Spirocheta morsus muris or Sporozoa muris. It was renamed Spirillum minus in 1924. The organism is a short, thick, gram- negative, tightly coiled spiral rod which measures 0.2 to 0.5 ␮m EPIDEMIOLOGY and has two to six helical turns. Because Spirillum minus can- More than 2 million animal bites occur each year in the not be cultured on synthetic media, initial diagnosis relies on United States, and rats are responsible for approximately 1% direct visualization of characteristic spirochetes with Giemsa of these (30). Historically, the typical victim of rat bite fever stain, Wright stain, or dark-field microscopy (86). was a child under 5 years old living in poverty, and over 50% of reported cases in the United States were children (37, 65). Growth Characteristics Now that rats have become popular pets and study animals, the demographics of potential victims have broadened to include S. moniliformis is an extremely fastidious organism that children, pet store workers, and laboratory technicians. Over needs microaerophilic conditions to grow, making microbio- 200 cases of rat bite fever have been documented in this coun- logical diagnosis difficult. Optimal growth requires Trypticase try, but this represents a significant underestimate because soy agar or broth enriched with 20% blood, serum, or ascitic neither the disease nor its causative organism is reportable to fluid. The bacteria grow slowly (2 to 3 days) and may take as health departments. The youngest reported case of rat bite long as 7 days. Typical colonies have a “cotton ball” appear- fever was in a 2-month-old infant (72), and the oldest reported ance on media, while colonies on agar appear circular, convex, case occurred in an 87-year-old man (82). The risk of infection grayish, smooth, and glistening (69). After 5 days of growth, after a rat bite appears to be 10% (23, 35), and the mortality some colonies may demonstrate the “fried-egg” appearance rate of untreated rat bite fever is approximately 13% (65, 91). seen with the L form. Importantly, the 0.05% sodium polyanethol sulfonate (“Liquoid”) that is added to most commer- Geographic Distribution cial aerobic blood culture bottles as an anticoagulant inhibits the growth of S. moniliformis at a concentration as low as Most reports of S. moniliformis originate from the United 0.0125% (74). However, Trypticase soy agar or broth, resin States, although other Western Hemisphere reports have come bead culture systems, and anaerobic culture bottles may dem- from Brazil, Canada, Mexico, and Paraguay. Most European VOL. 20, 2007 RAT BITE FEVER AND STREPTOBACILLUS MONILIFORMIS 15

FIG. 1. Gram-stained smear of S. moniliformis on blood agar medium, demonstrating pleomorphic gram-negative bacilli in chains and clumps with irregular, lateral bulbar swellings (photo courtesy of L. Wilcox, Hamilton Regional Laboratory Medicine Program, and D. Yamamura, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada). reports come from the United Kingdom and France, but spo- States, most early reports originate from the eastern half of the radic reports from Norway, Finland, Germany, Spain, Italy, country. However, S. moniliformis now appears to have mi- Greece, Poland, Denmark, and The Netherlands also exist. grated to the West Coast (13, 32), and cases are documented Australia has also demonstrated some cases. Few reports from nationwide (34). Africa exist, other than one report of sodoku from Kenya (8) and two episodes of squirrel bite-associated disease in Nigeria Animal Infectivity (33), probably underestimating the presence of S. moniliformis. Most reports from Asia document cases of sodoku, caused by Rats. The rat appears to be the dominant natural reservoir Spirillum minus and not discussed here (91). Within the United of S. moniliformis, which likely is a member of the commensal 16 ELLIOTT CLIN.MICROBIOL.REV.

TABLE 1. Results of biochemical tests performed on the parent Human Infectivity strain and an L-phase variant of Streptobacillus moniliformisa The reported incidence of rat bite fever caused by S. mo- Result Test niliformis from laboratory rat bites is low. Of 65 cases of doc- Parent strain L-phase variant umented rat bite fever since 1938 that were reviewed for this Oxidase Negative Negative article, only 8 (12%) were attributed to a laboratory rat expo- Catalase Negative Negative sure. This likely does not represent the true incidence of dis- Indole Negative Negative ease in humans because of low clinical suspicion by clinicians Nitrate Negative Negative and the organism’s strict growth requirements. Similarly, the Hydrogen sulﬁde Negative Negative incidence of wild-rat-associated disease is seriously underesti- Arginine hydrolysis Positive Positive Methyl red Negative Negative mated, as not all cases of rat bite fever are associated with an Phenylalanine deaminase Negative Negative actual bite. S. moniliformis may also be acquired by handling of Citrate Negative Negative the animal or by exposure to its excreta or saliva. Nineteen of Urea hydrolysis Negative Negative the 65 reviewed cases (29%) documented no bite or known Esculin hydrolysis Weak reaction Weak reaction Glucose fermentation Positive Positive exposure, consistent with literature reports that 30% of pa- Galactose fermentation Weak reaction Positive tients report no known bite (15, 32). However, as stated above, Maltose fermentation Weak reaction Positive 10% to 100% of domestic rats and 50% to 100% of wild rats Mannose fermentation Weak reaction Weak reaction carry S. moniliformis, and a known bite causes infection ap- Other carbohydrates Negative Negative proximately 10% of the time. Thus, rat bite fever and rat TSI agar with serum (butt/slant) Acid/acid Acid/acid colonization with S. moniliformis represent a signiﬁcant public a Data are from references 17, 72, and 91. health threat that remains unrecognized.

CLINICAL FEATURES flora of the rat’s upper respiratory tract. Healthy rats may Rat bite fever is associated with three clinical syndromes in demonstrate the organism in cultures of the nasopharynx, lar- the literature. Rat bite fever caused by S. moniliformis infec- ynx, upper trachea, and middle ear (56). Healthy domesticated tion is the predominant form seen in the United States. Dis- or laboratory rats demonstrate S. moniliformis colonization ease caused by Spirillum minus is known as sodoku and occurs 10% to 100% of the time, while wild rats appear to be 50% to primarily in Asia. Ingestion of S. moniliformis via contaminated 100% colonized (16). Most rats are asymptomatically colo- food causes Haverhill fever, so named for the first description nized but occasionally may demonstrate signs and symptoms of of an outbreak in Haverhill, MA. disease. Mice. Because mice are a preferred animal model for research, a significant amount of effort has been expended to Initial Symptoms identify their risk of colonization and disease from S. monili- S. moniliformis-associated rat bite fever is a systemic illness formis, as summarized by Wullenweber (91). It is well docu- classically characterized by fever, rigors, and migratory poly- mented that laboratory mice may show symptoms of infection arthralgias. After exposure, the incubation period ranges from with S. moniliformis, ranging from septic lymphadenitis to poly- 3 days to over 3 weeks but typically is less than 7 days. Many arthritis and multiorgan microabscesses leading to septicemia, patients report symptoms suggestive of an upper respiratory cachexia, and death (30, 91). However, it appears that not all tract infection during this time. If a bite has occurred, it typi- strains of mice are equally susceptible to streptobacillosis and, cally heals quickly, with minimal residual inflammation and no in fact, many inbred strains demonstrate mild to no disease significant regional lymphadenopathy. Persistence of signifi- whatsoever. This is important from a laboratory personnel cant induration at the bite site should suggest an alternate health risk perspective, as some animals may be asymptomatic diagnosis, including sodoku. carriers with the potential to transmit disease via exposure to At disease onset, fevers begin abruptly and may range from saliva, as are rats. The persistence of S. moniliformis in mice is 38.0°C to 41°C. Rigors associated with fevers are prominent. debated in the literature and ranges from none (91) to 6 Fever may resolve in 3 to 5 days but can relapse. Other fre- months (70). Overall, there appears to be a low risk of rat bite quently reported symptoms in the initial phase of illness in- fever from the bite of a healthy, inbred laboratory mouse. clude headache, nausea, vomiting, sore throat, and severe my- However, this may be different if the bite is by an outbred algias. strain or wild mouse. Other animals. There are reports of infection or coloniza- Disease Progression tion in such potential pets as guinea pigs (44), gerbils (90), ferrets (31), cats (82, 91), and dogs (16, 57, 82). However, no As rat bite fever progresses, over 50% of patients develop confirmatory evidence exists to prove the risk of transmission migratory polyarthralgias. The severity of pain and the pres- from either cats or dogs. More likely, the latter two animals are ence of swelling and erythema indicate arthritis (38, 67, 80). colonized only transiently after attacking or eating a rodent Reports also document the presence of synovitis and nonsup- colonized with S. moniliformis (57). Rat bite fever in nonhu- purative arthritis suggestive of rheumatoid arthritis (40, 47, man primates (rhesus macaque and titi monkey) has been 67). The joints involved include both large and small joints of reported, and streptobacillary disease in turkeys and koalas has the extremities. Many patients experience arthritis of at least been demonstrated (83). the knee and ankle during their illness. Migratory polyarthral- VOL. 20, 2007 RAT BITE FEVER AND STREPTOBACILLUS MONILIFORMIS 17

FIG. 2. Petechial and purpuric lesions on the foot of a rat bite fever patient.

gia is the most persistent ﬁnding of rat bite fever, lasting bite fever. The rash may persist beyond the other, more acute, several years in some patients. symptoms. Approximately 20% of rashes desquamate, espe- Nearly 75% of patients develop a rash that may appear cially those with hemorrhagic vesicles (20). maculopapular, petechial, or purpuric (20) (Fig. 2). Hemor- rhagic vesicles may also develop on the peripheral extremities, Outcome especially the hands and feet, and are very tender to palpation (Fig. 3). Appearance of this rash, especially the hemorrhagic Untreated, rat bite fever has a mortality rate of approxi- vesicles, in the setting of an otherwise nonspeciﬁc set of disease mately 10%, ranging from 7% to 13% (15, 54, 65, 80, 91). signs and symptoms should strongly suggest the diagnosis of rat Reported causes of death include endocarditis, refractory peri- 18 ELLIOTT CLIN.MICROBIOL.REV.

FIG. 3. Hemorrhagic vesicles on the ﬁrst and third toes of a patient with advanced rat bite fever.

cardial effusion, bronchopneumonia, pneumonitis, periarteritis Review of English Literature nodosa, volvulus, and overwhelming septicemia, with organisms found in both the adrenal glands and bone marrow at Epidemiology. A review of the English language literature autopsy (14, 15, 62, 65, 72, 76). Although some patients appear reveals 65 discrete case reports that provide full descriptions of to show spontaneous recovery from serologically confirmed the clinical presentation (2, 3, 5–7, 13–16, 18, 21, 22, 24–27, 29, disease (5, 13), a lack of effective antibiotic treatment is highly 31, 33–36, 38–43, 45–47, 49–52, 54, 58, 61–65, 67–69, 77, 79, 80, associated with death. Initiation of an appropriate antibiotic 82, 84, 87, 90). Many additional cases are described within case regimen usually precipitates rapid resolution of acute symp- series in which signs and symptoms specific to each case are not toms. However, some patients experience prolonged migratory detailed (3, 54, 65, 69, 88). The 65 detailed cases were reported polyarthralgias, fatigue, and slowly resolving rash. from 1938 to 2005 and primarily come from the United States, VOL. 20, 2007 RAT BITE FEVER AND STREPTOBACILLUS MONILIFORMIS 19 although the United Kingdom, Europe, Canada, Australia, and rosis and secondary syphilis are also possible. It is of note that Nigeria are also represented. The patient ages range from 2 up to 50% of rat bite fever patients have a falsely positive months to 87 years. Fifty (77%) of the rat bite patients de- Venereal Disease Research Laboratory (VDRL) test; how- scribed were male. Twenty-six (40%) of the exposures oc- ever, a negative treponemal test can be used to rule out syph- curred from a wild rat, 8 (12%) were from a laboratory rat, and ilis. Many potential viral causes exist, although Epstein-Barr 3 (5%) were from a pet shop rat. Twenty-two (34%) of the virus, parvovirus B19, and coxsackieviruses are especially patients described a nonbite or nonrat exposure. The remain- prominent. Relapsing fevers may suggest Borrelia recurrentis, ing cases occurred in association with bites from a ferret (one), malaria, and typhoid fever. Noninfectious causes include col- mouse (one), squirrel (two), gerbil (one), and dog (one). lagen vascular diseases and drug reactions. Clinical findings. Symptoms described include fever (92%), Sodoku. Infection caused by rat bites in Asia is likely to be rash (61%), polyarthralgias (66%), myalgias (29%), nausea caused by Spirillum minus and is designated sodoku (so, rat; and vomiting (40%), headache (34%), and sore throat (17%). doku, poison). This entity differs from rat bite fever not only in The mean temperature achieved during the cases was 39.4°C. geographic distribution but also clinically. After an incubation Patients’ laboratory values reveal an average white blood cell period of approximately 14 to 18 days, the bite site becomes count of 12,200 per cubic millimeter, with a polymorphonu- indurated and may ulcerate, with associated regional lymph- clear cell and band form predominance. Only five patients adenopathy. Fevers have regular relapses separated by afebrile demonstrated white blood cell counts higher than 15,000 per periods lasting 3 to 7 days. Approximately 50% of patients cubic millimeter. More significantly, the average erythrocyte develop a violaceous red-brown macular rash which occasion- sedimentation rate was 69 mm per hour. Only four patients ally has plaques or urticarial lesions. Joint manifestations are had erythrocyte sedimentation rates below 15 mm per hour; rare (1, 27). these patients all had laboratory values obtained either very late in their clinical course or after recovery. Seven (10.8%) of Haverhill Fever the patients died, consistent with the published average mortality rate of 10%. Haverhill fever refers to an outbreak of epidemic disease resulting from S. moniliformis-contaminated milk. The first reports were from a 1926 outbreak in Haverhill, MA, and Complications described an illness termed erythema arthriticum epidemicum Published complications of rat bite fever include endocardi- caused by an organism named Haverhillia multiformis (55, 60). tis, myocarditis, pericarditis, systemic vasculitis, polyarteritis This organism was later shown to be identical to S. monilifor- nodosa, meningitis, hepatitis, nephritis, amnionitis, pneumo- mis (10). Patients with Haverhill fever develop signs and symp- nia, and focal abscesses (14, 24, 35, 54, 62, 78, 79, 83). Of these, toms identical to those of rat bite fever. However, the absence endocarditis is the best described and carries the highest mor- of rat exposure and the presence of a large number of patients tality rate (64). Seventeen patients with endocarditis associated with common temporal and geographic exposure should sug- with S. moniliformis infection have been described (14, 50, 58, gest Haverhill fever. 64, 68). A 1992 review of 16 of these patients (68) revealed that U.S. experience. Although Haverhill fever is named for the 8 of them had valvular disease prior to the onset of endocar- site of the first published description of epidemic S. moniliformis- ditis, most commonly rheumatic heart disease. Most cases were associated disease, an earlier outbreak likely occurred in 1925 defined by multiple positive blood cultures and had typical in Chester, PA (60). In this episode, approximately 400 cases symptoms of rat bite fever accompanied by murmur (100%), occurred with striking similarity of onset, symptoms, course, petechiae (13%), Osler’s nodes (13%), hepatosplenomegaly and epidemiologic relation to a milk supply. The following (33%), anemia (33%), and cardiac dysrhythmia (13%). Echo- year, 86 cases developed over a 4-week period in Haverhill, a cardiography was performed for four patients and demon- small manufacturing city. These cases were investigated by strated valvular vegetations in only two patients. The reported Place and Sutton (60), and the organism responsible was de- mortality rate associated with S. moniliformis endocarditis is scribed by Parker and Hudson (55). The ages of the patients 53% (68), and death may occur from 2 weeks to 3 years after ranged from 8 months to 54 years, and 41% were male. The symptom onset (58). However, a majority of these deaths oc- patients came from 39 families, representing 231 people and an curred in the absence of effective antimicrobial therapy (14). attack rate of 36%. The milk supply in every case came from one dairy, either directly or through four stores selling the dairy’s milk products. The milk from the dairy was not pas- Differential Diagnoses teurized. Although no cultures from the milk demonstrated S. Diseases. The differential diagnosis of symptoms typical of moniliformis, pasteurization of the milk products was associ- rat bite fever (fever, rash, polyarthralgias) is extensive (27, 54, ated with the end of the outbreak. 63, 78). Possible bacterial causes include sepsis from such bac- The onset of symptoms in the 1926 outbreak was acute and teria as Streptococcus pyogenes and Staphylococcus aureus, associated with sudden development of rigors, emesis, or se- disseminated gonorrhea, meningococcemia, Streptococcus pyo- vere headache. Initial symptoms resolved after 3 to 4 days and genes-associated diseases (scarlet fever, rheumatic fever, post- included fever (97%), vomiting (62%), headache (56%), chills streptococcal reactive arthritis), Lyme disease, ehrlichiosis, (55%), dizziness (16%), and irritability (8%). However, fever and brucellosis. Rickettsial infections, especially Rocky Moun- recurred 2 to 3 days later and was associated with the onset of tain spotted fever, must be considered in areas where such polyarthralgias and polyarthritis. Rash appeared from the first infections are endemic. Such spirochetal infections as leptospi- to the fifth day of disease, was most marked at the distal 20 ELLIOTT CLIN.MICROBIOL.REV.

extremities, and was mostly “rubelliform” in nature. The rash mals (4, 7, 11, 43, 85). A PCR assay specific for S. moniliformis progressed over 3 days to include hemorrhagic lesions and has been described by Boot et al. (11); it uses primers designed lasted an average of 6 days. Polyarthritis was the most persis- on the basis of 16S rRNA gene base sequence data of human tent symptom, lasting from 1 week to several months and and rodent strains of S. moniliformis (forward primer, 5Ј GCT severely limiting activity and weight bearing. Wrists and elbows TAA CAC ATG CAA ATC TAT 3Ј; reverse primer, 5Ј AGT were most frequently involved, followed, in order, by knees, AAG GGC CGT ATC TCA 3Ј). These primers showed 100% shoulders, fingers, and ankles. Associated laboratory findings complementarity to S. moniliformis ATCC 14674T and S. mo- demonstrated an average white blood cell count of 11,500 per niliformis ANL 370-1. The PCR assay generated a 296-bp prod- cubic millimeter, with 70% polymorphonuclear cells. Blood uct which, when discriminated by BfaI restriction enzyme cultures in 11 of 17 cases and joint fluid aspirate cultures in 2 treatment, generated three fragments (128, 92, and 76 bp) of 2 cases demonstrated an organism subsequently named Hav- specific to S. moniliformis. This assay has been used by others erhillia multiformis, with characteristics identical to S. monili- to examine both human- and animal-derived specimens and formis. Outcomes were uniformly excellent, with no deaths and has been found to distinguish S. moniliformis from other or- few permanent sequelae. Several patients, however, experi- ganisms with great accuracy (4, 11, 85). However, until such an enced chronic, recurring arthralgias. assay becomes more readily available, a diagnosis of S. monili- United Kingdom experience. A second reported outbreak of formis-associated rat bite fever requires a high clinical index of Haverhill fever occurred in 1983 in 208 children at a boarding suspicion coupled with the appropriate use of culture and school in Chelmsford, Essex, United Kingdom (59). In a de- attention to ruling out alternate diagnoses. scription of four cases from this outbreak (74), the clinical features were described as abrupt onset of fever with head- TREATMENT ache, peripheral erythematous rash, polyarthralgias, and subsequent sore throat. Initial diagnoses included viral illness (es- Penicillin is the treatment of choice for proven or highly sus- pecially coxsackievirus), meningococcal septicemia, and pected cases of rat bite fever. Tests of S. moniliformis antibiotic erythema multiforme. The point source of the outbreak ap- susceptibility by the disk diffusion method usually demonstrate peared to be raw milk, ingested by many students at the school. sensitivity to penicillins, cephalosporins, carbapenems, aztreo- Students developed symptoms at school and were sent home to nam, clindamycin, erythromycin, nitrofurantoin, bacitracin, tetra- recover from an apparent viral epidemic, thus explaining the cycline, teicoplanin, and vancomycin; intermediate susceptibility subsequent appearance of cases in London, Leeds, and Not- to aminoglycosides, fluoroquinolones, and chloramphenicol; and tingham. Blood cultures from the four described patients dem- resistance to trimethoprim-sulfamethoxazole, polymyxin B, and onstrated S. moniliformis, and the information was provided to nalidixic acid (69, 91). Antibiotic susceptibility tests performed by health care workers caring for other students to assist with broth macrodilution usually demonstrate the following MICs: diagnosis and management. penicillin, Ͻ0.03 ␮g/ml; cephalothin, Ͻ0.03 ␮g/ml; ceftriaxone, Ͻ0.03 ␮g/ml; vancomycin, 0.5 ␮g/ml; tetracycline, 0.25 ␮g/ml; ␮ ␮ DIAGNOSIS erythromycin, 2 g/ml; streptomycin, 8 g/ml; and gentamicin, 1 ␮g/ml (68). Only one penicillin-resistant S. moniliformis strain Culture has been demonstrated (81), and that was over 50 years ago. Growth characteristics are discussed separately (see “Biol- Adults with rat bite fever should receive 400,000 to 600,000 ogy,” above). IU/day (240 to 360 mg) of intravenous penicillin G for at least 7 days, but this dose should be increased to 1.2 million IU/day (720 mg) if no response is seen within 2 days (65). Children Fatty Acid Profiles should receive 20,000 to 50,000 IU/kg of body weight/day (12 Fatty acid profiles obtained by gas-liquid chromatography, to 30 mg/kg/day) of intravenous penicillin G for 5 to 7 days, together with characteristic growth, can be used for rapid iden- followed by 7 days of oral penicillin V, 25 to 50 mg/kg/day tification of S. moniliformis. The major cellular fatty acid (maximum, 3 g/day) divided four times per day (27, 73). For peaks are tetradecanoic acid (14:0), palmitic acid (16:0), penicillin-allergic patients, both streptomycin and tetracycline octadecanoic acid with linoleic acid (18:2) and oleic acid appear to be effective (61, 68), but erythromycin use has been (18:1), and stearic acid (18:0) (66, 69). High-resolution poly- associated with treatment failures (35). Cephalosporins have acrylamide gel electrophoresis in conjunction with computer also been used successfully (16, 20) and may be considered if analysis has also been used to distinguish and confirm cross-allergenicity with penicillin is felt to be unlikely. Other strains of S. moniliformis (19). antimicrobials may be considered, based on the in vitro susceptibility data presented above, but no published evaluations of their effectiveness exist. Other Methods Patients with S. moniliformis endocarditis require dual ther- Serologic assays and slide hemagglutination tests, although apy with high-dose penicillin G in combination with strepto- used historically (12, 55, 70) and in some animal research (10, mycin or gentamicin (50). The currently recommended dose 91), are currently not available for use with humans. Although for adults is 4.8 million IU/day (4.8 g) of intramuscular pro- these assays are sufficiently sensitive, the increasing demand caine penicillin G if the isolate is susceptible to 0.1 ␮g/ml. If for rapid, more-sensitive tests likely has detracted from their the isolate is more resistant, 20 million IU/day (12 g) of intra- utility. Molecular methods such as PCR show promise and venous penicillin G should be used (65, 68) for adults. Children have been used successfully with humans and laboratory ani- should receive 160,000 to 240,000 IU/kg/day (96 to 144 mg/kg/ VOL. 20, 2007 RAT BITE FEVER AND STREPTOBACILLUS MONILIFORMIS 21 day), up to the adult maximum of 20 million IU/day (12 g) (68, 1984. Brain abscess due to Streptobacillus moniliformis and Actinobacterium 73). Successful treatment of adults with a 4-week regimen has meyerii. Infection 12:262–264. 22. Downing, N. D., G. D. Dewnany, and P. J. Radford. 2001. A rare and serious been demonstrated (50). The appropriate treatment length for consequence of a rat bite. Ann. R. Coll. Surg. Engl. 83:279–280. children is not known, although 6-week regimens generally are 23. Etscorn, F., and D. D. Blodgett. 1987. Rat-bite fever in the animal laboratory: a precautionary note. Psychobiology 15:345–346. considered effective for other causes of bacterial endocarditis. 24. Faro, S., C. Walker, and R. L. Pierson. 1980. Amnionitis with intact amniotic The use of streptomycin appears to enhance activity against membranes involving Streptobacillus moniliformis. Obstet. Gynecol. 55:9S– the cell wall-deficient L forms of S. moniliformis (68); one 11S. 25. Fordham, J. N., E. McKay-Ferguson, A. Davies, and T. Blyth. 1992. Rat bite might anticipate that other aminoglycosides would provide the fever without the bite. Ann. Rheum. Dis. 51:411–412. same benefit. 26. Frank, W. P., and A. G. Bower. 1951. Rat bite fever—response to streptomycin therapy. Calif. Med. 74:42. 27. Freels, L. K., and S. P. Elliott. 2004. Rat bite fever: three case reports and CONCLUSIONS a literature review. Clin. Pediatr. 43:291–295. 28. Freundt, E. A. 1956. Experimental investigations into the pathogenicity of Rat bite fever, caused by S. moniliformis, is an under-recog- the L-phase variant of Streptobacillus moniliformis. Acta Pathol. Microbiol. Scand. 38:246–258. nized and under-reported disease characterized by abrupt on- 29. Gilbert, G. L., J. F. Cassidy, and N. M. Bennett. 1971. Rat-bite fever. Med. set of fever, rigors, and migratory polyarthralgias; it carries a J. Aust. 2:1131–1134. mortality rate of approximately 10%. Although S. moniliformis 30. Glaser, C., P. Lewis, and S. Wong. 2000. Pet-, animal-, and vector-borne infections. Pediatr. Rev. 21:219–232. is exquisitely susceptible to penicillin, most patients experience 31. Gordon, I. J., and E. S. Jones. 1999. I smell a rat. Hosp. Med. 60:682–683. treatment delays due to the nonspecific nature of the clinical 32. Graves, M. H., and M. M. 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Viral Determinants of Resistance to Treatment in Patients with Hepatitis C Anette Wohnsland, Wolf Peter Hofmann, and Christoph Sarrazin* Department of Internal Medicine II, Saarland University Hospital, Kirrbergerstrasse, 66421 Homburg/Saar, Germany

INTRODUCTION ...... 23 MECHANISMS OF RESISTANCE TO IFN-␣...... 24 IFN-␣-Based Antiviral Therapy...... 24 IFN-␣ Signal Transduction ...... 24 Approaches to In Vitro and In Vivo Studies of IFN-␣ Resistance...... 24 HCV Core and IFN-␣ Resistance...... 26 E2 and IFN-␣ Resistance ...... 26 CD81-binding sites/hypervariable region 2 ...... 26 Hypervariable region 1...... 26 PKR–eIF-2␣ phosphorylation homology domain ...... 27 NS3/4A and IFN-␣ Resistance ...... 27 Inhibition of the innate immune system ...... 27 NS3 protease sequence analysis...... 27 NS5A and IFN-␣ Resistance ...... 28 ISDR, complete NS5A region, and V3 region ...... 28 Interaction with interleukin-8 and the 2؅,5؅-oligoadenylate synthetase system ...... 28 Summary of NS5A and IFN-␣ resistance...... 28 MECHANISMS OF RESISTANCE TO RIBAVIRIN ...... 29 Ribavirin Sensitivity and Resistance In Vitro ...... 29 Ribavirin Sensitivity and Resistance In Vivo...... 29 Summary of Ribavirin and Treatment Resistance...... 31 MECHANISMS OF RESISTANCE TO AMANTADINE ...... 31 MECHANISMS OF RESISTANCE TO DIRECT ANTIVIRAL DRUGS...... 32 NS3/4A Protease Inhibitors...... 32 NS5B Polymerase Inhibitors ...... 33 CONCLUDING REMARKS...... 33 ACKNOWLEDGMENT...... 34 REFERENCES ...... 34

INTRODUCTION risk for the development of hepatocellular carcinoma (148). The current standard treatment for patients with chronic hep- Hepatitis C virus (HCV) is an enveloped, approximately atitis C consists of pegylated alpha interferon (IFN-␣) (PEG- 9.6-kb, positive-sense, single-stranded RNA virus and is clas- IFN) in combination with the nucleoside analogue ribavirin for sified in the family Flaviviridae (17). After translation from a 24 to 48 weeks and leads to a sustained virologic response in 54 large open reading frame, the polyprotein precursor is cleaved to 56% of cases (38, 83). Sustained virologic response is de- by viral and host peptidases, resulting in three structural pro- fined as undetectable HCV RNA by a sensitive assay (lower teins, termed core, envelope 1 (E1), and E2, and a protein Ͻ named p7, and six nonstructural (NS) proteins, termed NS2, detection limit of 50 IU/ml) at the end of a 24-week fol- NS3, NS4A, NS4B, NS5A, and NS5B. The F protein has been low-up period after the end of treatment. Patients who do not described as being a result of a ribosomal frameshift of the achieve a sustained virologic response may be found to be core-encoding genomic region (149). There is a noncoding region HCV RNA negative during therapy but may relapse thereafter of 324 to 341 nucleotides at the 5Ј end containing the internal or may be virologic nonresponders showing detectable HCV ribosome entry site (IRES) and a 3Ј noncoding region of variable RNA levels throughout the complete treatment period. length. Virologic response rates have been shown to depend on According to estimations of the WHO, over 170 million various host and viral factors such as age, weight, sex, race, individuals worldwide are infected with HCV (148). Chronic liver enzymes, stage of fibrosis, HCV genotype, and HCV HCV infection is responsible for inflammation of the liver, and RNA concentration at baseline (9, 38, 50, 62, 83, 94, 147a). To ϳ20% of patients progress to liver cirrhosis with an increased further improve sustained virologic response rates, different treatment approaches are currently under investigation. For example, individualized therapy durations on the basis of the * Corresponding author. Mailing address: Saarland University Hos- HCV RNA concentration at baseline and early during therapy pital, Department of Internal Medicine II, Kirrbergerstrasse, 66421 Homburg/Saar, Germany. Phone: 49 (0)6841-16-2 32 03. Fax: 49 are the subjects of clinical studies. In addition, triple therapy (0)6841-16-2 13 05. E-mail: [email protected]. with the addition of the antiviral drug amantadine to IFN-␣

23 24 WOHNSLAND ET AL. CLIN.MICROBIOL.REV. and ribavirin has been evaluated in multiple studies, leading to The best-understood IFN-mediated signaling and transcrip- contradictory results (7, 21, 82). Moreover, multiple new sub- tional activation pathway is called the Jak/signal transducer stances with different modes of action are being studied in and activators of transcription (STAT) pathway (Fig. 1). Thereby, ongoing clinical trials (e.g., albuferon, amantadine, Toll-like two cytoplasmic protein tyrosine kinases, Tyk2 and Jak1, be- receptor [TLR] agonists, and viramidine). So far, none of these longing to the Janus kinase family phosphorylate tyrosine res- drugs has been established as a standard treatment for hepa- idues of the IFN receptor that function as docking sites for titis C. STATs. The subsequent phosphorylation of the receptor-re- Most promising, results of phase 1/phase 2 clinical trials with cruited STATs by the Jak kinases promotes the formation of direct antiviral drugs such as inhibitors of HCV-speciﬁc NS3 heterodimers between STAT1 and STAT2, which further protease and HCV NS5B RNA-dependent RNA polymerase binds to a third protein, p48, to form the ISG factor 3 (RdRp) have recently been presented at international meet- (ISGF3) complex. This complex is translocated to the cell ings (60, 112a, 115, 120a, 125a, 155a). nucleus, where it binds to the IFN-stimulated response el- Resistance mechanisms that might explain how the virus ement in IFN-stimulated gene promoters, leading to the may circumvent the antiviral actions of IFN-␣, ribavirin, aman- expression of IFN effector proteins. HCV cell-based expres- tadine, and, most recently, new direct antiviral drugs have been sion models show inhibition of IFN-␣-induced signal trans- proposed in vitro and in vivo (20, 36, 41, 49, 70, 74, 139). This duction through the Jak/STAT pathway (22, 26, 55, 76). review discusses recent achievements of HCV treatment resis- Among all ISGs, the best characterized effector proteins are tance mechanisms based on in vitro experiments and clinical the double-stranded RNA-activated protein kinase (PKR) data. (41), the 2Ј,5Ј-oligoadenylate synthetase (92), and the Mx proteins (134). Besides its antiviral activities, IFN-␣ also affects processes that regulate cell growth and the modula- MECHANISMS OF RESISTANCE TO IFN-␣ tion of the immune response. IFN-␣-Based Antiviral Therapy

Considerable improvements in the treatment of patients in- Approaches to In Vitro and In Vivo Studies ␣ fected with chronic hepatitis C were achieved by the introduc- of IFN- Resistance ␣ tion of IFN- in its pegylated form and by the combination So far, several virus-encoded factors have been suggested with the nucleoside analogue ribavirin. Remarkably, the re- to be responsible for the inhibition of the antiviral effects of ␣ sponse rates of IFN- -based therapy are influenced mainly by IFN-␣ (39). The in vitro studies are based mainly on the the HCV genotype. Due to the lack of a proofreading activity recombinant expression of the whole HCV open reading of the HCV RNA-dependent RNA polymerase, different HCV frame or on the expression of individual HCV genes. The genomes have evolved and are classified into six genotypes expression of the whole HCV polyprotein has been shown to (130). The different genotypes can be further assigned to sub- inhibit the STAT1 function in the context of elevated levels types with homologies of approximately 80% (131). Within a of protein phosphatase 2A (PP2A). Overexpression of the given individual, a large number of closely related viral vari- catalytic subunit of PP2A resulted in the hypomethylation of ␣ ants, called HCV quasispecies, circulate. With all IFN- -based STAT1, leading to the increased binding of a protein inhib- treatment schedules, HCV genotype 1-infected patients itor of activated STAT, called PIAS. Increased binding of achieve much lower sustained rates of response (42 to 52%) to STAT1 to PIAS leads to reduced activation of ISGs (10, 26, antiviral therapy than those infected with HCV genotypes 2 55). For the HCV core, the envelope protein E2, the serine and 3 (78 to 86%) (38, 50, 83, 153). For patients infected with protease NS3/4A, and the NS5A protein, different mecha- HCV genotypes 4, 5, and 6, only limited data are available, but nisms were suggested to contribute to antiviral resistance sustained response rates seem to be slightly higher than those (11, 36, 41, 136, 139). for HCV genotype 1 (50 to 77%) (38, 83). No significantly Additionally, different HCV replicon systems have been uti- different virologic response rates between HCV subtypes were lized to study IFN-␣ resistance (77, 89). The HCV replicon reported (e.g., subtypes 1a and 1b). system is a well-defined and robust cell culture system for the The underlying mechanisms for the varying virologic re- replication of the nonstructural HCV genes. By definition, ␣ sponse rates to IFN- -based antiviral therapy between the replicons are molecules that are capable of self-amplification. different HCV genotypes are unknown. However, as response In the case of HCV, the first-generation replicons were derived rates are detected independently of any host or treatment from a cloned HCV genome by deleting the structural region factors, many studies focused on virus-encoded mechanisms of and inserting a selectable marker (neomycin phosphotransfer- ␣ resistance to IFN- -based therapy. ase, conferring G418 resistance). A second IRES element (en- cephalomyocarditis virus promoter) is introduced to allow the IFN-␣ Signal Transduction translation of the HCV nonstructural region. Transfection of synthetic RNAs derived from such a construct into the human IFNs are a family of cytokines belonging to the host’s natural hepatoma cell line HuH7 and G418 selection lead to cell lines immune response to various stimuli, particularly viral infec- carrying autonomously replicating HCV subgenomic RNA tion. After binding to their specific receptors on the target cell (77). By the development of HCV replicon systems, an addi- surface, an intracellular signaling cascade is activated, which tional tool to study interactions of the HCV proteins with the leads to the upregulation of IFN-stimulated genes (ISGs), re- host cellular machinery is available. sulting in the expression of multiple antiviral effector proteins. However, the translation of these in vitro experiments to the VOL. 20, 2007 RESISTANCE TO TREATMENT IN PATIENTS WITH HCV 25

FIG. 1. Potential strategies of the different HCV proteins to antagonize IFN therapy. (A) IFN resistance through inhibition of RIG-I and TLR3 by the HCV NS3/4A protein. Viral RNA binding to RIG-I and TLR3 leads to the activation of IRF3 and NF-␬B. Both transcription factors enter the nucleus and bind to target genes, resulting in the expression of antiviral defense proteins. The NS3/4A protein disrupts the activation of IRF3 and NF-␬B by the inactivation of Cardif, which is an adaptor protein in the RIG-I antiviral pathway, and through inhibition of Trif, an adaptor molecule in the TLR3 signaling pathway. (B) Disruption of the IFN-mediated Jak/STAT pathway. IFN binding to its receptor triggers the autophosphorylation of tyrosine residues through the Tyk2 and Jak1 kinases, which function as docking sites for STAT1 and STAT2. The subsequent phosphorylation of STAT1 and STAT2 promotes the formation of heterodimers between STAT1 and STAT2 and the assembly of the trimeric ISGF3 complex. After translocation of the ISGF3 complex to the cell nucleus, it binds to the IFN-stimulated response element (ISRE) in ISG promoters, leading to the expression of IFN effector proteins. The HCV core protein induces the expression of SOCs, which block Jak/STAT signaling through the IFN-␣/␤ receptor. The HCV NS5A protein induces the expression of IL-8, which blocks the assembly and function of the ISGF3 complex. The expression of the whole HCV polyprotein inhibits STAT1 function through elevated levels of PP2A, which is associated with an increased binding of STAT1 to PIAS and a reduced activation of ISGs. (C) IFN resistance through inhibition of PKR by the HCV E2 and HCV NS5A proteins. After IFN-␣ binding, autophosphorylation of PKR takes place, leading to the inhibition of the phosphorylation of eIF-2␣, which is responsible for the initiation of translation of cellular and viral proteins by facilitating Met-tRNA binding to the 40S ribosomal subunit. HCV E2 and HCV NS5A antagonize the effects of IFN-␣ by the inhibition of PKR.

situation in clinical practice has been difﬁcult. For example, the HCV isolates derived from patients with different sensitivities HCV isolates used in various HCV replicon systems are not to IFN-␣-based therapy. clinically characterized. So far, only HCV replicon systems As a consequence, the in vivo analysis of HCV treatment harboring genotype 1a/1b genomes and a recent HCV replicon resistance mechanisms are restricted to cloning and sequenc- system harboring the HCV genotype 2a genome of a patient ing approaches for HCV isolates derived from patient sera suffering from acute fulminant hepatitis C were developed. with known virologic responses to antiviral therapy. Today, the Moreover, selection of HCV replicon systems with resistance amino acid variabilities of HCV core protein and E2, NS3/4A, to IFN-␣ in vitro has not been successful so far. In this context, and NS5A proteins have been studied in correlation with the it would be worthwhile to construct HCV replicon systems with virologic treatment response. 26 WOHNSLAND ET AL. CLIN.MICROBIOL.REV.

TABLE 1. Clinical signiﬁcance of HCV mutations for sensitivity/resistance to antiviral therapy

Antiviral and Region analyzed Importance of amino acid mutations Reference(s) HCV protein IFN-␣ HCV core HCV complete open reading No correlation of mutations with virologic 29 frame treatment response in patients with HCV genotype 1 infection HCV E2 CD81-binding sites No correlation of mutations with virologic 61 treatment response in patients infected with HCV genotypes 1b and 3a PePHD No signiﬁcant correlation of mutations with 8, 42, 125, 147, 150 virologic treatment response in genotype 1- and genotype 3-infected patients For patients infected with HCV genotype 2, 116, 147 conﬂicting data about a correlation of viral PePHD variants and treatment response exist HVR1/E2 Correlation of quasispecies heterogeneity with 1, 15, 32, 123 response to antiviral therapy HCV NS3 Complete NS3 gene No correlation of mutations in patients infected 120 with genotype 1 with sustained virologic treatment response HCV NS5A ISDR High no. of mutations within the ISDR in patients 16, 29, 30, 69, 102, 104, infected with HCV genotypes 1a/1b 117, 121, 122 and 2 correlates with treatment response Complete NS5A gene High no. of mutations within the complete NS5A 124 sequence correlates with sustained virologic treatment response

Ribavirin HCV NS5B Complete NS5B gene F415Y mutation conferring resistance to ribavirin 151 in HCV replicon system; larger trials are needed to assess prevalence and relevance in vivo HCV NS5A Complete NS5A gene G404S and E442G mutations conferring resistance 107 to ribavirin in HCV replicon system; in vivo relevance is unknown

Amantadine, HCV p7 Complete p7 gene No correlation of amino acid variations with 87 virologic treatment response in patients with genotype 1 infection

HCV Core and IFN-␣ Resistance two conformational CD81-binding sites located in the carboxy- terminal part of the E2 protein that overlap with parts of The HCV core protein was shown to induce the expression hypervariable region 2 (HVR2) were described (108). It was of suppressor of cytokine signaling 3 (SOCS-3) and SOCS-1 in hypothesized that various capabilities for the binding of the E2 cultured cells, which in turn may antagonize IFN-␣ action by protein to CD81 may depend on amino acid substitutions blocking the Jak/STAT pathway (11) (Fig. 1). Sequence anal- within the CD81-binding sites, and HVR2 and may thus con- ysis of the HCV core gene in patients with chronic hepatitis C tribute to different treatment outcomes. However, sequencing and different sensitivities to IFN-␣-based therapy has not been of the putative CD81-binding sites within the HCV E2 gene published so far. However, in one study, the complete open reading frame of HCV was sequenced to determine the genetic showed no association between specific amino acid variations ␣ basis of resistance to IFN-␣ (29). A comparison of the pairs of and the HCV RNA concentration or response to IFN- -based IFN-␣-resistant and IFN-␣-sensitive HCV isolates from therapy (61). Thus, given the uncertainty for the predicted three patients infected with genotype 1b revealed only two CD81-binding sites and the negative results of that study, at amino acid differences within the core region in two of three present, no importance of amino acid variability within the E2 patients at different codons. In conclusion, genetic varia- protein for the CD81 receptor interaction in correlation with ␣ tions of the highly conserved HCV core protein seem not to the response to IFN- -based therapy could be shown. be relevant in determining IFN-␣ sensitivity in chronic hep- Hypervariable region 1. The HCV amino acid quasispecies atitis C (Table 1). heterogeneity within HVR1 of the E2 protein has been correlated with the virologic response to antiviral therapy in multiple studies (Table 1). While a lower HCV quasispecies hetero- E2 and IFN-␣ Resistance geneity was associated with spontaneous recovery in patients CD81-binding sites/hypervariable region 2. The human with acute hepatitis C (31), in patients with chronic hepatitis C, CD81 receptor has been proposed to be a candidate receptor conflicting data concerning the significance of HVR1 quasi- for HCV. The E2 glycoprotein is involved in virus entry, and species diversity and complexity in correlation with virologic VOL. 20, 2007 RESISTANCE TO TREATMENT IN PATIENTS WITH HCV 27 response were published. Many studies showed that a higher tial E2 PePHD interaction with PKR may explain general degree of variability in the HVR1 quasispecies correlates with differences in the IFN-␣ sensitivities of HCV isolates of dif- lower response rates to IFN-␣ treatment (46, 59, 63, 68, 90, 96, ferent HCV genotypes (genotype 1 versus genotypes 2 and 3) 100, 105, 128). However, no such correlation was observed in but is highly conserved in HCV isolates from patients without other studies (72, 78, 97, 119). The discrepancies of these an individual correlation to the outcome of IFN-␣-based ther- results may be related to (i) HCV genotype distribution in apy. Investigations with the recently established full-length different studies, (ii) methodologies to assess quasispecies het- HCV cell culture system with E2 genes of different genotypes erogeneity (i.e., band shift assays or sequencing), (iii) different may further resolve the relevance of the PePHD/PKR inter- assessments and quantifications of quasispecies heterogeneity, action (145). and/or (iv) different definitions of treatment outcome. How- ever, in more recent studies using extensive cloning and sequencing approaches, in patients with chronic hepatitis C, a NS3/4A and IFN-␣ Resistance lower HVR1 heterogeneity of HCV quasispecies before antiviral therapy and changes in HCV E2 quasispecies early during Inhibition of the innate immune system. Expression of the therapy also seem to be associated with higher rates of viro- HCV NS3/4A serine protease was associated with diminished logic response (1, 15, 32, 123). The lower heterogeneity of phosphorylation and nuclear translocation of IFN regulatory HCV quasispecies in patients with a subsequent sustained vi- factor 3 (IRF3), leading to the reduced expression of IFN- rologic response to IFN-␣-based therapy may indicate a more induced genes (36) (Fig. 1). IRF3 functions as a key activator effective control of the emergence of viral variants by the of the ISGs that limits virus replication at multiple points immune system during successful treatment. Interestingly, this within the replicative cycle. Among these genes, ISG56 has seems to be restricted to neutralizing antibodies directed to- been identified as being a suppressor of HCV translation wards HVR1, while within other regions (e.g., NS5A), a higher through the disruption of viral IRES function independently of number of amino acid variations in the HCV quasispecies is the PKR pathway (47, 135, 146). NS3/4A was shown to evade associated with the virologic response to IFN-␣-based therapy the early immune response by the disruption of the retinoic (see below). acid-inducible gene I (RIG-I) signaling pathway (35). Nor- PKR–eIF-2␣ phosphorylation homology domain. The HCV mally, this pathway is induced by virus-produced double- E2 glycoprotein was reported to be involved in mediating stranded RNA, resulting in the activation of type I IFN, by IFN-␣ resistance through the inhibition of PKR (140) (Fig. 1). phosphorylation and activation of preexisting transcription fac- PKR is a primary regulator of the IFN-␣-induced antiviral and tors such as NF-␬B and IRF3 (151). Recently, a new adapter antiproliferative effects of the host cell. Once activated by protein in the RIG-I pathway, called Cardif, was identified. ␣ IFN- , autophosphorylation of PKR takes place and inhibits Cardif was shown to be targeted and inactivated by the HCV ␣ the phosphorylation of the subunit of eukaryotic initiation NS3/4A protease (85). The other described strategy of NS3/ ␣ factor 2 (eIF-2 ), which is responsible for facilitating Met- 4A, to block the expression of defense genes, is based on tRNA binding to the 40S ribosomal subunit (118). This results binding to TLR3. TLR3 functions as a signaling receptor for in the decreased translation of cellular or viral proteins (54). A extracellular and intracellular double-stranded RNA (2) and sequence of 12 amino acids within the E2 gene that displays transmits signals to activate the type I IFN response through its high homology to several autophosphorylation sites within the adaptor molecule, called Trif (also known as TICAM-1). Trif regulatory domain of the PKR and to its natural substrate, functions as a linkage between TLR3 and the kinases respon- eIF-2␣, which is called the PKR–eIF-2␣ phosphorylation ho- sible for activating IRF3 and NF-␬B. NS3/4A mediates the mology domain (PePHD), was identified. Within the E2 pro- cleavage of this adaptor protein, resulting in the inhibition of tein of HCV subtype 1a/1b isolates, inhibition of PKR was the antiviral response (73) (Fig. 1). demonstrated through interactions with the PePHD by abol- NS3 protease sequence analysis. Sequence analysis of the ishing its kinase activity and blocking its inhibitory effect on NS3 gene derived from patients infected with HCV genotype 1 viral protein synthesis in vitro (139, 140). Interestingly, the revealed no significance of the NS3 protein variability in cor- PePHD of the most resistant HCV subtype, subtype 1a/1b, ␣ shares higher homology with the PKR autophosphorylation relation with the response to IFN- -based therapy (Table 1). sites than genotypes 2 and 3, suggesting that the interaction of Overall, the amino acid sequence within the NS3 protein as the PePHD with PKR specifically enhances resistance to anti- part of the NS3/4A serine protease complex was highly con- viral therapy of HCV genotype 1 isolates compared with ge- served (120). In the HCV replicon system, several mutations notypes 2 and 3. Several studies that aimed to investigate within the NS3 gene that lead to enhanced replication effi- whether the PePHD shows a correlation between mutation ciency were detected. Interestingly, in HCV isolates from pa- rates and treatment responses could not observe significant tients harboring identical amino acid substitutions in the NS3 mutations during IFN-␣ treatment for isolates of HCV geno- protein (R1283G, P1112R, and S1496M), a slower decrease of types 1a/b and 3a (8, 42, 125, 147, 150). For HCV genotype 1, HCV RNA during IFN-␣-based therapy was observed (120). the PePHD region has turned out to be highly conserved, and To confirm these findings and to address the overall impor- no clinical relationship between the amino acid sequence of tance of NS3 protein variability with regard to HCV replication the PePHD and the outcome of IFN-␣ therapy could be dem- efficiency and sensitivity to IFN-␣, a phenotypic evaluation onstrated. For genotypes 2a and 2b, conflicting data about a should be performed using the HCV replicon assay before any putative correlation of PePHD polymorphism and IFN-␣ re- conclusions regarding the importance for the treatment of sistance exist (116, 147) (Table 1). Taken together, the poten- patients with chronic hepatitis C can be drawn. 28 WOHNSLAND ET AL. CLIN.MICROBIOL.REV.

NS5A and IFN-␣ Resistance gion 3 (V3) within the carboxy-terminal part of the NS5A protein was found to correlate with the treatment response in ISDR, complete NS5A region, and V3 region. The first data HCV genotype 1a/1b isolates, which is currently under further to support a significant role for amino acid variations within investigation (14, 98, 124). Furthermore, it must be assumed the HCV NS5A protein and the response to IFN-␣-based that additional mutations outside the NS5A gene but within therapy came from Japan. In 1995 and 1996, a correlation of a ␣ other genes of the HCV open reading frame are important for high number of mutations in a so-called IFN- sensitivity- determinations of IFN-␣ sensitivity. For example, the isolated determining region (ISDR) comprising 40 amino acids within replacement of the NS5A gene in the HCV replicon system the carboxy-terminal part of the NS5A protein and sustained with sequences from clinical responder and nonresponder pa- ␣ virologic response to IFN- therapy in HCV genotype 1b- tients did not change the in vitro sensitivity to IFN-␣ (6). infected patients was described by Enomoto et al. (29, 30) For HCV genotype 2 isolates, a correlation between the (Table 1). In the first report, all patients with at least four number of mutations within the ISDR and treatment response mutations within the ISDR achieved a sustained virologic re- was also observed (67, 91), while for HCV genotype 3a isolates, sponse in comparison to a prototype reference sequence. In no such association was reported for relatively small patient addition, an inverse correlation between an increasing number cohorts reported in several studies (117, 125). -of mutations within the ISDR and the HCV RNA concentra- Interaction with interleukin-8 and the 2؅,5؅-oligoadenylate syn tion at baseline was observed (30). Subsequently, the correla- thetase system. Since a direct interaction of PKR with the tion of ISDR mutations with treatment response was investi- NS5A protein has not convincingly been demonstrated so far gated by different groups in Japan, Europe, and the United (44), other mechanisms by which the NS5A protein could in- States. Studies from Japan were generally able to confirm the hibit the action of IFN-␣ were investigated. Recently, the ex- strong correlation between ISDR mutations and treatment pression of the NS5A protein was associated with the induction response (16, 69). Groups from Europe and the United States of interleukin-8 (IL-8) and the subsequent inhibition of IFN-␣ were initially not able to verify the importance of ISDR mu- activity in vitro (64, 109, 110) (Fig. 1). Interestingly, in patients ␣ tations in IFN- sensitivity (18, 65, 84, 99, 133, 154), and in with HCV genotype 1 infection and with a sustained virologic subsequent studies, it turned out that HCV genotype 1b iso- response to IFN-␣-based therapy, levels of IL-8 in blood that lates with multiple mutations within the ISDR are rare in were significantly lower than those of virologic nonresponders Europe and the United States compared to those found in were also observed (88). Japan (104, 117, 121, 122). In further studies with larger pa- Aside from the above-described intracellular interactions of tient cohorts and in a meta-analysis, the positive correlation of the HCV NS5A protein, multiple studies on numerous mod- an increasing number of ISDR mutations with a sustained ulations of cell processes by the NS5A protein were published virologic response to IFN-␣-based therapy could be confirmed (4). Recently, the structure of an N-terminal zinc-binding do- (102, 104, 117, 121, 122). After reports of the existence of a main of the NS5A with protein, RNA, and membrane inter- PKR-binding domain within the NS5A protein comprising the action sites was described (141). Overall, the NS5A protein ISDR in the amino-terminal part (40, 41), the clinical impor- seems to be a promiscuous protein with the function of an tance of mutations within this PKR-binding domain was inves- important regulator of HCV replication and multiple intracel- tigated in additional studies. However, in several studies, no lular interactions. specific mutation within the carboxy-terminal part of the PKR- Furthermore, an interaction of the NS5A protein with the binding domain in correlation with IFN-␣ treatment response 2Ј,5Ј-oligoadenylate synthetase leading to the inhibition of was observed (43, 93, 124). Furthermore, in in vitro studies of the antiviral activity of IFN-␣ in vitro was described. However, the expression of the complete HCV open reading frame, no the HCV core protein seems rather to activate the 2Ј,5Ј-oligoad- colocalization of NS5A and PKR could be demonstrated (37). enylate synthetase system (95, 136). Activation of 2Ј,5Ј-oligo- To summarize the results of the different studies from West- adenylate synthetase leads to the cleavage of single-stranded ern countries, in the majority of patients with a sustained UA and UU dinucleotides by RNase L, and differences in the virologic response to IFN-␣-based therapy, no or only a small frequencies of UA and UU dinucleotides between full-length number of mutations within the ISDR were observed. Thus, prototype of HCV genotype 1 and those of genotypes 2 and 3, for these patients, sensitivity to IFN-␣ cannot be explained by which may explain general differences in the IFN-␣ sensitivities the ISDR hypothesis. However, in several in vitro studies of between these genotypes, were described (51, 52). 2Ј,5Ј-Oligo- the cellular expression of clinically characterized NS5A genes, adenylate synthetase serum levels were not associated with it was shown that the NS5A protein, independently of the virologic responses in patients with chronic hepatitis C (92), presence of ISDR mutations, was able to lower the protection whereas a specific polymorphism within the 2Ј,5Ј-oligoadenyl- of previously IFN-␣-induced cells against infection with a sur- ate synthetase gene was associated with the outcome of anti- rogate virus (111, 132). viral therapy (66). Nothing is known about the frequency of Therefore, mutational analysis by sequencing the entire UA and UU dinucleotides before and during IFN-␣-based NS5A gene was performed to assess the significance of NS5A therapy in correlation with HCV RNA clearance. mutations outside the ISDR/PKR binding domain (14, 28, 98, Summary of NS5A and IFN-␣ resistance. Taken together, 124). In a study that reported the analysis of 70 full-length the highest grade of clinical evidence suggests that mutations NS5A sequences, the overall number of mutations within the within the ISDR of the NS5A protein are involved in sensitiv- NS5A protein was highly correlated with the treatment re- ity/resistance to IFN-␣-based therapy in HCV genotype 1b- sponse (124) (Table 1). Furthermore, in three studies, a local infected patients. A meta-analysis of 28 studies from Japan, accumulation of mutations around the so-called variable re- Europe, and the United States clearly showed the correlation VOL. 20, 2007 RESISTANCE TO TREATMENT IN PATIENTS WITH HCV 29 between an increasing number of mutations within the ISDR E2 HVR1 and NS5A and invariant regions such as NS5B, and an increased probability of a sustained virologic response underlining the presence of ribavirin-induced mutagenesis to IFN-␣-based therapy on the basis of 1,230 isolates from (19). These results for the NS5B region (61a) and for the single patients. Since an inverse correlation between the num- NS5A ISDR region using a subgenomic HCV replicon system ber of ISDR mutations with HCV RNA serum concentrations (138) were confirmed by others. Lanford et al. reported error- was also shown in several of these studies, it is likely that prone replication of HCV using a colony formation assay. NS5A-mediated regulations of replication efficiency may ex- Thus, it was shown that the colony-forming efficiency of HuH7 plain the clinical correlation with treatment response. As for cells decreased after cells were transfected with total RNA the other HCV proteins, in vitro analysis of HCV viral variants from cultures harboring the HCV replicon system treated with with defined mutational patterns must be performed to con- increasing concentrations of ribavirin (71). Accordingly, Zhou firm this hypothesis. et al. reported a decrease in colony formation efficiency after ribavirin treatment (156). However, in that study, the muta- MECHANISMS OF RESISTANCE TO RIBAVIRIN tional frequency of the NS5A gene was found to be increased only when HCV replicon cells were treated by a combination Ribavirin (1-␤-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is of IMPDH inhibitors and ribavirin but not by ribavirin alone. a guanosine analogue that was synthesized more than 35 years Pfeiffer and Kirkegaard provided in vitro evidence that re- ago and that possesses broad-spectrum antiviral activity against sistance to ribavirin can be conferred by distinct mechanisms several RNA and DNA viruses in vitro (129). The first pilot by using both a poliovirus cell culture system and the HCV study addressing the utility of ribavirin for oral antiviral ther- replicon system (106, 107). By passaging HeLa cells transfected apy in patients with chronic hepatitis C was published in 1991 with poliovirus cDNA that were treated with ribavirin, a single (114), which was followed by several controlled trials showing amino acid replacement of glycine with serine at position 64 that ribavirin monotherapy led to a decline in alanine amino- (G64S) located in the finger domain of poliovirus RdRp was transferase levels in a significant proportion of patients (23, selected. Interestingly, ribavirin-resistant poliovirus RdRp dis- 27). However, in the majority of patients receiving ribavirin played an increased fidelity of RNA synthesis that may as a monotherapy, no significant reduction of HCV RNA was ob- result reduce the frequency of ribavirin misincorporation. Ac- served, suggesting that ribavirin monotherapy has no or only cordingly, by passaging HuH7 cells harboring the HCV repli- marginal effects on HCV replication in vivo in some patients con system that were treated with increasing concentrations of (103). In recent years, ribavirin was combined with PEG-IFN, ribavirin, those authors generated ribavirin-resistant HCV rep- leading to substantially improved sustained virologic response licon cells. Resistance was not conferred by mutations in the rates in patients with chronic hepatitis C. HCV RdRp, and surprisingly, two other mechanisms of riba- Data from pivotal randomized trials suggest that the benefit virin resistance were reported. On the one hand, the resistant of adding ribavirin to PEG-IFN treatment results predomi- cell lines showed reduced ribavirin import in the cells, most nantly from the prevention of virologic relapse. However, the likely by the downregulation of a nucleotide transporter. On distinct antiviral mechanisms of ribavirin are poorly under- the other hand, those authors could detect mutations within stood, and at present, four different mechanisms of action have the NS5A region, G404S, and E442G that were present only been proposed: (i) enhancement of the host adaptive antiviral in the ribavirin-resistant HCV replicon cell lines but not in the immune response (113, 137), (ii) inhibition of host IMP dehy- ribavirin-sensitive cell lines (107) (Table 1). The NS5A protein drogenase (IMPDH) (33), (iii) direct inhibition of HCV NS5B has been shown to interact with the HCV NS5B RdRp in vitro, RNA-dependent RNA polymerase (12, 80), and, more re- and those authors suggested that the NS5A mutations may cently, (iv) RNA virus mutagenesis and “error catastrophe” alter NS5B polymerase activity. (Fig. 2). For the latter hypothesis, it has been reported that ribavirin acts as an RNA virus mutagen, thereby leading to Ribavirin Sensitivity and Resistance In Vivo an increased mutational frequency that exceeds the mutational threshold of viral fitness and drives RNA viruses into To translate the in vitro evidence of ribavirin-induced mu- error catastrophe. By using a model of poliovirus polymer- tagenesis to clinical settings, accumulating data from patients ase, Crotty et al. showed that the misincorporation of riba- with chronic hepatitis C who received either ribavirin mono- virin nonspecifically templates the incorporation of cytidine therapy or IFN-␣–ribavirin combination therapy were recently and uridine with equal efficiency, thus exerting mutagenic published. Schinkel et al. were the first to evaluate nucleotide activity, which is strongly correlated with a decrease in po- substitutions of the NS5A region by a direct sequencing ap- liovirus infectivity (20). proach comparing mutation rates in serum samples from patients who had not responded to IFN-␣ monotherapy and who ␣ Ribavirin Sensitivity and Resistance In Vitro were subsequently treated with IFN- and ribavirin combination therapy (126). As the mutation rate was not increased and Several in vitro studies provided evidence that ribavirin may no significant phylogenetic sequence divergence was detect- also cause error catastrophe in HCV infection. Using a full- able in the NS5A region during combination treatment com- length binary HCV replicon system treated with increasing pared to IFN-␣ monotherapy, those authors concluded that concentrations of ribavirin, Contreras et al. showed that the ribavirin does not induce mutations, possibly rendering the error generation rate, defined as the number of nucleotide virions more sensitive to IFN-␣. In two other studies, the substitutions divided by the number of nucleotides sequenced, mutagenic effect of ribavirin monotherapy was studied by ei- increased significantly in both naturally variant regions such as ther direct sequencing of the NS5B region or quasispecies 30 WOHNSLAND ET AL. CLIN.MICROBIOL.REV.

FIG. 2. Different proposed antiviral mechanisms of ribavirin. (A) Enhancement of the host adaptive antiviral immune response. Ribavirin modulates the balance of T-helper 1 (TH1) and TH2 responses by enhancing TH1 and inhibiting TH2 cytokine production. (B) Inhibition of host IMPDH. Intracellular ribavirin is converted by cellular kinases to ribavirin monophosphate (RMP), ribavirin diphosphate (RDP), and ribavirin triphosphate (RTP). Ribavirin monophosphate is a competitive inhibitor of IMPDH, which leads to the depletion of the intracellular GTP pool necessary for viral RNA synthesis. (C) Direct inhibition of HCV NS5B RdRp. The phosphorylated form of ribavirin, ribavirin triphosphate, binds the nucleotide-binding site of polymerases, thereby competitively inhibiting viral replication. Moreover, ribavirin is utilized and incorporated by viral RNA polymerases including the HCV RdRp opposite cytosine or uridine, which can result in a signiﬁcant block to RNA elongation. (D) RNA virus mutagenesis and error catastrophe. Ribavirin acts as an RNA virus mutagen, thereby leading to an increased mutational frequency that exceeds the mutational threshold of viral ﬁtness and drives RNA viruses into lethal mutagenesis and error catastrophe. CTL, cytotoxic T lymphocyte. TNF-␣, tumor necrosis factor alpha.

analyses of the NS3 and NS5A regions at several time points. C-to-T transition mutations that may be specific for the mis- In both studies, no significant increase in the mutation rate was incorporation of ribavirin during RNA synthesis was observed. observed (79a, 105a). In concordance with those findings, Asahina et al. recently However, in a recent study, a moderate but significant in- reported significantly increased mutation rates by direct se- crease of the NS5B and NS3 quasispecies heterogeneity was quencing of the NS5A and NS5B regions in patients who reported during the first weeks of ribavirin monotherapy (61a). were treated with ribavirin monotherapy for 4 weeks fol- In that study, an increase in the proportions of G-to-A and lowed by IFN-␣–ribavirin combination treatment for 24 VOL. 20, 2007 RESISTANCE TO TREATMENT IN PATIENTS WITH HCV 31 weeks when samples collected prior to treatment, at base- viral therapy may help to gain more insights into ribavirin line, and at the end of ribavirin monotherapy were com- sensitivity and resistance (24, 57). pared (5). Furthermore, the highest mutation rates within the NS5A region and nonsynonymous NS5A mutations were detected in those patients who achieved a sustained re- MECHANISMS OF RESISTANCE TO AMANTADINE sponse to combination therapy. In another study that compared NS5B quasispecies of Amantadine, a tricyclic amine, has antiviral activity against a mainly HCV subtype 1a-infected patients before and after broad range of viruses. Used for patients with chronic hepatitis ribavirin monotherapy, a weak but statistically not significant C, amantadine had no antiviral effect when it was given as increase in the error generation rate was detected in samples monotherapy in different studies (3, 45, 127). Placebo-con- ␣ from patients treated with ribavirin compared to controls that trolled trials with the combination treatment of IFN- with or were treated with IFN-␣ or placebo (152). Interestingly, a without amantadine have shown no significantly higher sus- nucleotide substitution leading to an amino acid change tained virologic response rates in patients who received aman- ␣ from phenylalanine to tyrosine at NS5B position 415 tadine (56, 142, 155). Meta-analyses of all studies with IFN- (F415Y) emerged in all five HCV subtype 1a-infected pain combination with amantadine showed contradictory results tients treated with ribavirin. By constructing HCV sub- (22, 81). Triple therapy with PEG-IFN, ribavirin, and amanta- genomic replicons with both the NS5B 415F and the NS5B dine showed improved sustained virologic response rates in 415Y amino acid substitutions, those authors demonstrated patients who were previously nonresponders but showed no in vitro that the treatment of replicon cells with ribavirin significant benefit in naı¨ve patients or relapsers in comparison reduced the HCV RNA levels of the NS5B 415F replicon with standard combination therapy (7, 13, 22, 34, 82, 143). but not of the NS5B 415Y replicon (Table 1). In silico Additional studies with PEG-IFN, ribavirin, and higher doses analyses showed that the NS5B 415 amino acid position of amantadine to verify a potential antiviral effect of amanta- maps to the thumb subdomain of NS5B and that the re- dine in patients with chronic hepatitis C virus genotype 1 in- placement of the phenylalanine with tyrosine may narrow fection are under way. the putative RNA-binding pocket in the central tunnel of As a potential target for amantadine, Griffin et al. identified the HCV RdRp, thus affecting polymerase function. Those the HCV p7 protein (48). The small, highly hydrophobic HCV authors concluded that the selection of the NS5B F415Y p7 protein is located downstream of the HCV structural pro- substitution during ribavirin therapy represents a mutation teins and is inconsistently cleaved from the E2 protein (25). that confers resistance to ribavirin. In other cohorts, how- The HCV p7 protein was identified as being a calcium ion ever, this proposed NS5B resistance mutation was less prev- channel in artificial lipid bilayers and in membranes of mam- alent and was not associated with a lack of response to malian cells (48, 49). Amantadine abrogates the activity of this antiviral therapy (20a). Furthermore, it has to be noted that channel, similar to its ability to inhibit the M2 protein, an the tyrosine residue at NS5B position 415 is the consensus integral membrane protein of influenza A virus (53). Further- residue found in almost all HCV genotypes but not HCV more, a study of mutated p7 variants showed that p7 is involved subtype 1a. in the release of infectious virus particles. Mutated p7 proteins An earlier study examined the possibility of selecting riba- and p7 deletion mutants strongly impaired virus release in virin-responsive or -resistant variants in patients receiving riba- transfected HuH7 cells (134a). virin monotherapy for at least 12 months. Single-stranded con- In a recent study, the clinical importance of amino acid formation polymorphism analysis of the HVR1 and NS5A and variations within the HCV p7 protein for the response to ␣ NS5B regions followed by direct sequencing or cloning and IFN- -based antiviral therapy with and without amantadine in sequencing before treatment and at 6 and 12 months of ther- patients with HCV genotype 1 infection was investigated. apy was conducted and compared to data for untreated con- Overall, the HCV p7 protein was highly conserved, and no trols (112). Phylogenetic analyses did not show any clustering significant association of amino acid variations within HCV p7 of related variants in correlation with ribavirin treatment, sug- with the virologic treatment response in patients who received gesting that no selection of responsive or resistant variants amantadine was observed (87) (Table 1). Interestingly, in pa- occurred during ribavirin monotherapy. tients with HCV subtype 1b infection and combination therapy with amantadine, a trend for a more frequent presence of amino acid substitution L20F was detected in virologic nonre- Summary of Ribavirin and Treatment Resistance sponders compared with virologic responders. By in silico modeling, amino acid position 20 was located towards the p7 Taken together, conflicting results concerning mechanisms channel lumen, and the L20F substitution may impair the that confer resistance to ribavirin both in vitro and in vivo have amantadine interaction by changes in the size and shape of the been published so far. Additional experimental and clinical p7 ion channel pore. Taken together, there is no evidence for data are needed to elucidate the role of ribavirin as a mutagen a clear additional antiviral effect of amantadine for first-line in hepatitis C virus infection. Furthermore, despite the appar- therapy of patients with chronic hepatitis C, and results of ent existence of ribavirin resistance in cell culture, the a priori future studies, especially with higher amantadine doses, presence of ribavirin resistance and possible mechanisms that have to be awaited. Together with these studies as well as in confer ribavirin resistance have to be confirmed in patients in vitro systems (e.g., HCV replicon or patch-clamp assay), undergoing antiviral therapy. As recently shown, mathematical the potential importance of the L20F mutation should be modeling of the ribavirin effect on HCV kinetics during anti- addressed (87). 32 WOHNSLAND ET AL. CLIN.MICROBIOL.REV.

TABLE 2. Phase 1/2 HCV NS3/4A protease inhibitors and resistance mechanisms

Inhibitora Company and study Description Resistance mechanisms Reference(s) BILN2061 Bo¨hringer-Ingelheim, Peptidomimetic inhibitor, noncovalent In the replicon system, resistance is 74, 75, 79 phase 1 binding; clinical development was conferred by mutations at positions stopped because of cardiac toxicity R155Q, A156S/T, and D168A/V observed in animal studies VX-950 Vertex, phase 2 Peptidomimetic inhibitor, ␣-ketoamide, In the replicon system, resistance is 74, 75 (Telaprevir) monotherapy and combination conferred by mutation at position A156S therapy with IFN By sequence analysis, resistance conferred 125a by mutations at positions V36, T54, R155, and A156 SCH503034 Schering-Plough, Peptidomimetic inhibitor, ␣-ketoamide, In the replicon system, low to moderate 144 phase 2 monotherapy and combination resistance is conferred by mutations at therapy with IFN positions T54A, V170A, and A156S; longer exposure or selection led to the selection of a more resistant variant, A156T By sequence analysis, only selection of 120a HCV isolates with mutations at position T54 in single patients

a The inhibitor target is HCV NS3/4A protease.

MECHANISMS OF RESISTANCE TO DIRECT NS3/4A Protease Inhibitors ANTIVIRAL DRUGS During short-term monotherapy with the protease inhibitors Based on the availability of an HCV replicon system and BILN2061 (2 days), SCH503034, and VX-950 (14 days), a the detailed knowledge about the structure and function of decrease in HCV RNA concentrations of between 1.5 and 4

HCV proteins, in recent years, a subset of new compounds log10 HCV RNA IU/ml was observed in patients with chronic comprising direct inhibitors of HCV enzymes like protease, HCV genotype 1 infection. BILN2061 and VX-950 especially helicase, and polymerase were developed. The most ad- showed high antiviral efficacy, with HCV RNA levels below the vanced new antiviral agents are directed against the HCV detection limit of a highly sensitive assay (Ͻ10 IU/ml) in indi- NS3/4A serine protease and the HCV-specific NS5B RNA- vidual patients at the end of therapy. However, after the end of dependent RNA polymerase. Different antiviral agents for dosing, HCV RNA levels increased to concentrations at base- the specific inhibition of the HCV NS3/4A protease and the line in all patients (60, 70, 112b, 115, 155a). For the protease HCV NS5B polymerase are currently in phase 1/phase 2 inhibitor BILN2061, studies of patients infected with geno- trials for treatment of patients with chronic hepatitis C types 2 and 3 were published. Interestingly, a generally less (Tables 2 and 3), and multiple additional direct antiviral pronounced antiviral activity in patients infected with geno- drugs are in preclinical development. So far, results of phase types 2 and 3 was observed. This shows a high probability of 1 studies are available for three protease inhibitors (BILN2061, specificity of direct antiviral drugs for a special genotype VX-950, and SCH503034) and three polymerase inhibitors (va- caused by the substantial HCV sequence variability between lopicitabine, R1479, and HCV796). different genotypes (60, 115). In comparison with IFN-␣-based

TABLE 3. Phase 1/2 HCV NS5B polymerase inhibitors and resistance mechanisms

Inhibitora Company and study Description Resistance mechanism(s) Reference(s) NM283 Idenix, phase 2 Nucleoside polymerase inhibitor NM283 Sequence analysis of the HCV NS5B 86, 101 (valopicitabine) (prodrug of 2Ј-C-methylcytosine gene of several drug-resistant ͓NM107͔) replicons deﬁned a single substitution of the highly conserved S282T mutation that confers resistance to nucleosides containing 2Ј-methyl functionality R1626 Roche, phase 1 Nucleoside polymerase inhibitor Unknown 115a 4Ј-acidocytidine (R1479) HCV796 ViroPharma/Wyeth, phase 1 Nonnucleoside polymerase inhibitor One mutation, C316Y, was detected 144a by sequence analysis of the HCV NS5B gene of patients with resistance

a The inhibitor target is HCV NS5B polymerase. VOL. 20, 2007 RESISTANCE TO TREATMENT IN PATIENTS WITH HCV 33 therapy, the antiviral efficacy for blocking viral production is gated using NM107 and a bovine viral diarrhea virus in vitro significantly higher in patients treated with the protease inhib- infection assay. Bovine viral diarrhea virus is a pestivirus re- itor BILN2061 (Ͼ99% versus 55 to 95%) (58). Combination lated to HCV, and sequencing analysis of variants that are therapy with SCH503034 or VX-950 and PEG-IFN leads to a resistant to NM107 revealed that resistance was due to an higher decline in the HCV RNA viral load compared with that S405T substitution, which is the homologous mutation of of protease inhibitor monotherapy (120a). Restoration of the S282T in HCV NS5B (9a). Taken together, only limited data IFN signal transduction pathway, which was shown to be par- on HCV polymerase inhibitors are available today. So far, the tially blocked by the NS3/4A protein, may be responsible for antiviral activity of the drugs tested in patients with chronic the improved effect of IFN-␣ under specific inhibition of the hepatitis C seems to be generally weaker than that of several NS3/4A protease (36). Due to the error-prone nature of the HCV protease inhibitors, but this may be overcome by in- HCV RNA-dependent RNA polymerase together with highly creased specificity and higher doses of HCV polymerase inhib- efficient replication, the selection of isolates that are resistant itors. In the future, it will be highly interesting to investigate to compounds such as HCV NS3/4A protease and NS5B poly- the significance of mutations in different HCV genes of pa- merase inhibitors could be a major limitation for the efficiency tients treated with IFN-␣ in combination with direct antiviral of direct antiviral therapies in patients with chronic hepatitis C. drugs (potential IFN-␣ resistance regions together with targets In the subgenomic HCV replicon system, different mutations of protease and polymerase inhibitors). in the HCV NS3 serine protease domain (R155Q, A156S/T, D168A/V, T54A, and V170A) that confer different levels of CONCLUDING REMARKS resistance to BILN 2061, VX-950, and SCH503034 were identified (74, 75, 79, 144) (Table 2). In patients treated with the Several HCV proteins have been associated with mecha- protease inhibitor VX-950, selection of resistant variants with nisms of resistance to different antiviral drugs in acute and viral breakthrough during a 14-day treatment period was ob- chronic hepatitis C. In the vast majority of the multiple studies served. By highly sensitive sequence analysis of the HCV qua- addressing this issue, either in vitro mechanisms together with sispecies before treatment, at the end of treatment, and during HCV prototype isolates or amino acid sequence data for dif- follow-up, different amino acid positions (V36, T54, R155, and ferent HCV proteins obtained from patients undergoing anti- A156) with mutations that confer different levels of resistance viral therapies have been investigated. This restriction should to VX-950 were described (125a). For treatment with be overcome in the future. SCH503034, no analysis of the HCV quasispecies during ther- As a main viral strategy to circumvent the effects of IFN-␣, apy was performed, and by direct sequencing, only the selec- the inhibition of PKR by the E2 and NS5A proteins in in vitro tion of HCV isolates with mutations at position T54 in single studies has been proposed. However, for both proteins, no patients was reported (120a, 155a). Whether the combination concluding data in follow-up studies have been presented, and of different direct antiviral drugs as well as combination ther- the overall importance of PKR inhibition is uncertain. More apy with PEG-IFN could prevent the development of resis- recently, the inhibition of signaling molecules of the innate tance remains to be clarified in future studies. Overall, it is immune response (Cardif and Trif) by the NS3/4A protease clear that for HCV infection, a rapid development of resis- has been described. This mechanism may explain the high rate tance as in human immunodeficiency virus infection is possible of HCV persistence after acute hepatitis C virus infection. and that an efficient initial blocking of HCV replication with However, the potential importance of the virologic response to high plasma levels of the protease inhibitor is essential for the IFN-␣-based therapy in patients with chronic hepatitis C has to long-term success of this treatment option. be investigated in future studies. In the majority of clinical studies investigating amino acid NS5B Polymerase Inhibitors mutations of different HCV proteins, no correlation of specific mutations or the number of mutations with the response to Results of phase 1/phase 2 studies with the HCV NS5B IFN-␣-based antiviral therapy was detected. While, in all prob- RNA polymerase inhibitor valopicitabine (NM283) showed a ability, the amino acid variability of HCV proteins reflects ␣ decrease in the HCV RNA concentration of 1 to 2 log10 HCV different sensitivities to IFN- -based antiviral therapy, it be- RNA IU/ml in the treatment of naı¨ve patients or IFN-␣ non- came clear that simply counting mutations generally may not responders (44a). Interim analysis of ongoing studies also be sufficient for the detection of functional differences, which showed improved antiviral efficacy for combination therapy in turn may result in different treatment outcomes. However, with NM283 and PEG-IFN (98a). NM283 is the oral prodrug multiple studies and a meta-analysis have conclusively shown of 2Ј-C-methyl-cytidine (NM107) and is cleaved to the free that for the NS5A ISDR, indeed, just the number of mutations nucleoside that is converted to the active triphosphate by the is significantly associated with the response to IFN-␣-based cellular machinery. No in vivo resistance data for NM283 are therapy, and future studies should aim to elucidate the under- available yet, but it was shown that the 2Ј-C-methyl-nucleoside lying functional mechanisms of this phenomenon. NM107 is susceptible to resistance development, as shown in None of the proposed antiviral effects of ribavirin have been replicon systems and isolated polymerase assays. Sequence conclusively proofed so far. For the error catastrophe mecha- analysis of the HCV NS5B gene of several drug-resistant rep- nism, several supportive data exist, and also, analysis of HCV licons defined a single replacement of the highly conserved kinetics in patients with relatively low IFN-␣ efficacy argues in serine 282 with threonine (S282T) that confers resistance to favor of this hypothesis. However, the decline of alanine amino- nucleosides containing 2Ј-methyl functionality (86, 101). Re- transferase levels despite significant changes in HCV RNA cently, the development of resistance to NM283 was investi- concentrations during ribavirin monotherapy is not well ex- 34 WOHNSLAND ET AL. CLIN.MICROBIOL.REV. plained by a sole RNA virus mutagenic effect of ribavirin, and inhibits interferon alpha signaling in the liver of transgenic mice. Gastro- thus, additional mechanisms of action have to be assumed. enterology 124:1465–1475. 11. Bode, J. G., S. Ludwig, C. Ehrhardt, U. Albrecht, A. Erhardt, F. 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Global Dissemination of Vibrio parahaemolyticus Serotype O3:K6 and Its Serovariants G. Balakrish Nair,1* Thandavarayan Ramamurthy,2 Sujit K. Bhattacharya,2 Basabjit Dutta,2 Yoshifumi Takeda,3 and David A. Sack1 ICDDR,B: Centre for Health and Population Research, Mohakhali, Dhaka 1212, Bangladesh1; National Institute of Cholera and Enteric Diseases, P-33, CIT Road, Scheme XM, Beliaghata, Calcutta 700 010, India2; and Cine-Science Laboratory, Itabashi, Tokyo, Japan3

INTRODUCTION ...... 39 THE MYSTERIOUS ORIGIN OF THE O3:K6 SEROTYPE...... 40 SEROVARIANTS OF THE O3:K6 SEROTYPE...... 41 IS THE SPREAD OF O3:K6 AND ITS SEROVARIANTS A PANDEMIC? ...... 41 CLONALITY OF O3:K6 ISOLATES AND SEROVARIANTS ...... 42 GLOBAL SPREAD ...... 43 HOW ARE O3:K6 ISOLATES AND THEIR SEROVARIANTS DIFFERENT?...... 43 ENVIRONMENTAL ISOLATION...... 44 ECOLOGY AND EPIDEMIOLOGY...... 45 METHODS FOR DETECTION...... 45 CONCLUSION...... 46 ACKNOWLEDGMENTS ...... 46 REFERENCES ...... 46

INTRODUCTION urease production (an unusual phenotype for V. parahaemolyticus) and the trh gene exists (38). Enteroinvasiveness of the Vibrio parahaemolyticus, a gram-negative halophilic bacte- bacterium has been reported for a rabbit model, in which the rium, is recognized as a worldwide cause of food-borne gas- organism invaded, colonized, and produced inflammation in troenteritis, particularly in the Far East, where seafood con- the small intestine (13). In patients in the acute stage of V. sumption is high. The halophile was first identified as a cause parahaemolyticus infection, the inflammatory response in the of food-borne illness in Japan in 1950, when 272 individuals gut and in the circulation is less severe than that observed in became ill and 20 died after the consumption of semidried patients with shigellosis but more severe than that seen in juvenile sardines (27). V. parahaemolyticus causes three major patients with cholera (77). It has been shown that gastroenter- syndromes of clinical illness, i.e., gastroenteritis, wound infec- itis caused by V. parahaemolyticus results in strong systemic tions, and septicemia. The most common syndrome is gastro- and mucosal B-cell responses to TDH and lipopolysaccharide; enteritis; the symptoms include diarrhea with abdominal both antigens also induce an increase in the presence of im- cramps, nausea, vomiting, headache, and low-grade fever (34). munoglobulin M antibody-secreting cells, which suggests that Sometimes the diarrhea is bloody, with stools described as this is a primary response to the antigen (77). In a recent study, “meat washed” since the stool is reddish watery stool (77) but it was shown that irrespective of TDH production, V. para- unlike that seen in dysentery caused by Shigella species or in haemolyticus profoundly disturbs epithelial barrier function in amebiasis. The mean incubation period for V. parahaemolyti- Caco-2 cells due to the involvement of another virulence cus infection is 15 h (range, 4 to 96 h). The illness is self- factor(s) (53). The overall mechanism of pathogenesis by V. limiting and of moderate severity and lasts an average of 3 days parahaemolyticus, however, remains unclear. in immunocompetent patients. Most gram-negative pathogens disrupt the normal physiol- V. parahaemolyticus strains that are isolated from diarrheal patients produce either the thermostable direct hemolysin ogy of the intestinal mucosa by inducing cytoskeleton rear- (TDH), the TDH-related hemolysin (TRH), or both, while rangements, proinflammatory responses, and/or cell death. hardly any isolates from the environment have these properties Many of these cellular events are caused by bacterial effector (34, 83, 87). An isolate producing TDH is referred to as Kana- proteins, which are delivered into intestinal cells that directly gawa positive and can be identified by ␤ hemolysis on a special modulate the activities of host cell proteins and are secreted agar known as Wagatsuma blood agar (87, 93). TDH has been and translocated into host cells through the bacterial type III shown to have hemolytic, enterotoxic, cardiotoxic, and cyto- secretion systems (TTSSs). The TTSS macromolecular assem- toxic activities (34, 68, 83, 93). A strong correlation between bly is a needle-like complex composed of more than 20 proteins, and its components are highly conserved among bacteria. Genes encoding the TTSS apparatus are generally found on the chromosomal pathogenicity islands or plasmids (35, 99). In * Corresponding author. Mailing address: Enteric Microbiology Unit, Laboratory Sciences Division, ICDDR,B: Center for Health and several bacteria, the genes encoding TTSS-secreted proteins Population Research, Mohakhali, Dhaka 1212, Bangladesh. Phone: are located outside the gene clusters encoding the TTSS ap- 880-2-9886464. Fax: 880-2-88125289. E-mail: [email protected]. paratus (35). The specific properties of the effectors and their

39 40 NAIR ET AL. CLIN.MICROBIOL.REV. symptomatic effects on the host vary widely (35). Based on the and 71 different K types (37). The serotyping scheme was assembly and functions of different proteins, the TTSSs in developed using strains of clinical origin. Early investigations animal pathogens are classified into the following three major carried out by Baross et al. (6) with 20 bacteriophages against groups: (i) the Ysc-plus-Psc system, (ii) the 1-plus-Mxi/Spe V. parahaemolyticus showed no correlation between O and K system of the Salmonella pathogenicity island, and (iii) the serotypes in the lytic pattern, and hence they were assumed to enterohemorrhagic Escherichia coli system (22). have a wide host range. The first phage typing scheme for V. In V. parahaemolyticus, two sets of genes, for TTSS1 and parahaemolyticus was formulated in 1992 with 46 phages be- TTSS2, were identified on the large and small chromosomes, longing to morphological groups II, IV, and V (49). However, respectively (54, 76). V. parahaemolyticus TTSS1 is a Ysc-plus- for these phages the specificities for the serotypes have also not Psc system and is similar to the TTSSs of Yersinia spp. and been established. Libinzon et al. (51) tested 34 V. parahaemo- Pseudomonas aeruginosa in the number of genes, their order, lyticus phages isolated from the Black Sea. Except for one, and the identity of each protein (54, 61, 73). However, no which was specific for O5:K15, the majority of the phages were homologue of the effector protein genes has been found in the also found to lyse Vibrio alginolyticus strains. Phage typing TTSS1 region. Four new TTSS1-secreted proteins were iden- studies conducted during an O3:K6 outbreak in Vladivostok tified in V. parahaemolyticus, and one was located in the small showed types 1, 2, 7, and 10 with V. parahaemolyticus strains chromosome (73). TTSS2 is detected only in Kanagawa-posi- and phage types 2, 4, 5, and 7 with V. alginolyticus strains (85). tive V. parahaemolyticus strains and is not similar to any par- Due to the lack of specificity, the phage typing scheme for V. ticular TTSS of other bacteria (76). The GϩC contents of the parahaemolyticus is not customary in international practice. In DNA regions of TTSS1 and TTSS2 suggest that the former is recent years, a variety of molecular typing techniques, such as intrinsic in V. parahaemolyticus while the latter has a feature of ribotyping, pulsed-field gel electrophoresis (PFGE), and mul- laterally transferred DNA (54). Both TTSSs in V. parahaemo- tilocus sequence typing (MLST) techniques, as alluded to later lyticus may act as effectors during infections, as mutational in this review, have been used successfully to study the molec- studies and an adenylate cyclase fusion assay with TTSS1 ular epidemiology of the organism. showed it to be involved in the cytotoxicity in HeLa cells, while TTSS2 has a role in enterotoxicity in a rabbit model (73, 76). It THE MYSTERIOUS ORIGIN OF THE O3:K6 SEROTYPE was assumed that both TTSSs might act synergistically in the pathogenesis of V. parahaemolyticus (73). During active surveillance of diarrheal etiologies among hos- V. parahaemolyticus is widely disseminated in estuarine, ma- pitalized patients in Calcutta, a city in the northeastern part of rine, and coastal environments throughout the world (42) and India, an increase in hospital admissions of patients with V. has been detected as far north as Alaska (91). Water temper- parahaemolyticus gastroenteritis was observed beginning in ature, salinity, zooplankton blooms, tidal flushing, and dis- February 1996. Analysis of the strains revealed that a unique solved oxygen play an important role in dictating its spatial and serotype, O3:K6, which was not previously isolated during sur- temporal distribution (45). This pathogen is typically not re- veillance in Calcutta, accounted for 50 to 80% of infections in covered from estuarine waters during winter months in tem- the following months (69). The O3:K6 isolates had identical perate zones, when the water temperature is too low for its genotypes (tdh positive, trh and urease negative) and nearly existence. Water temperatures have been shown to influence identical arbitrarily primed PCR (AP-PCR) profiles and the growth of V. parahaemolyticus (44, 46, 47, 88), and the shared similar antibiotic sensitivity patterns (69). importance of water temperature in the epidemiology of in- From the literature, it seems that the first O3:K6 isolate was fections is reflected by the fact that most outbreaks occur isolated from a traveler returning from Indonesia to Japan in during the warmer months. In tropical countries, in contrast, 1995 (69). Curiously, the seventh pandemic strain of Vibrio chol- the seasonality of V. parahaemolyticus is less defined, with erae O1, which is of the El Tor biotype, also had its origin in the infection occurring throughout the year. Studies in Calcutta island of Sulawesi in Indonesia (43). However, based on hospital have shown that both marine and freshwater fishes provide admissions, the first localized cluster of cases of O3:K6 occurred ideal substrates for the survival and proliferation of V. para- in Calcutta starting in February 1996. Thus, the epidemiological haemolyticus. The isolation of V. parahaemolyticus from market setting in Calcutta at that time was conducive to infecting a larger samples of freshwater fishes was attributed to cross-contami- population. Food-borne outbreak statistics for Taiwan revealed nation due to mishandling at fishmongers’ stalls (81). Most V. that the O3:K6 serotype could have emerged as early as October parahaemolyticus outbreaks that occurred between 1973 and 1995, when it was responsible for a single outbreak with three 1998 in the United States occurred during the warmer months isolates (15). Four tdh-negative O3:K6 isolates obtained in Japan and were attributed to seafood, particularly oysters and other between 1983 and 1988 grouped with the unique O3:K6 cluster shellfish, and the median attack rate among persons who con- (Ͼ75% similarity) when recently examined by AP-PCR and sumed the implicated seafood was 56% (63). Many investiga- showed toxRS sequences identical to that of an O3:K6 clone, tions have shown that marine mollusks are associated with the leading Okura et al. (70) to speculate that the O3:K6 isolates spread of toxigenic V. parahaemolyticus (10, 15, 24, 32, 59). might have originated from these nonpathogenic strains after The primary basis of classification of strains of V. parahae- acquisition of the tdh gene. Therefore, at this point it appears that molyticus is a serotyping scheme, which depends on the anti- the progenitors of the O3:K6 isolate might have originated in the genic properties of the somatic (O) and capsular (K) antigens. environs of Japan. The serotyping scheme for V. parahaemolyticus is a combina- The regional dominance of a specific serotype of V. para- tion of O and K antigens, and serotyping is done using com- haemolyticus has occasionally been reported. Early investiga- mercially available antisera that include 11 different O antigens tions in Calcutta revealed the dominance of serotype O1:K56 VOL. 20, 2007 VIBRIO PARAHAEMOLYTICUS O3:K6 AND ITS SEROVARIANTS 41 among diarrheal cases (12) and in index cases and carriers (75, TABLE 1. Chronology of appearance of Vibrio parahaemolyticus 84). Along the western coasts of Mexico and the United States, O3:K6 and its serovariants in different countries V. parahaemolyticus O4:K12 was the dominant serotype caus- Serotype Country (yr of isolation) Reference(s) ing infections (1). The O3:K6 clustering of cases, however, O3:K6 India (1996) 17, 69 differs from the previously reported clustering of single V. Vietnam (1997) 16 parahaemolyticus serotypes in possessing additional attributes. Laos (1997) 57 One is the ability to rapidly increase hospitalizations in areas Indonesia (1997) 69 where it prevails and the other is to become the dominant United States (1997–1998) 23, 25 serotype, supplanting other serotypes of V. parahaemolyticus in Korea (1997–1998) 57 Chile (1998 and 2004) 21, 30 the given area. Both of these attributes were not observed Taiwan (1996–1999) 15, 101 before for V. parahaemolyticus. For example, in the Aichi Pre- Bangladesh (1998–2000) 7 fecture in Japan, the percentage of outbreaks by V. parahae- Japan (1998) 103 molyticus O3:K6 increased from 3% for the period 1988 to Thailand (1999) 17, 50 Russia (2001) 85 1995 to 75% for the period 1996 to 2001 (104). France (2004) 78 Mozambique (2004) 3 O4:K68 India (1998) 18 SEROVARIANTS OF THE O3:K6 SEROTYPE Thailand (1999) 17 Bangladesh (1998 and 2000) 7 The development of a specific method to facilitate the rapid Vietnam (1998) 16 Mozambique (2004) 3 identification of O3:K6 isolates (57) led to the serendipitous find- O1:K25 India (1998) 18, 57 ing of other serotypes, such as O4:K68, O1:K25, and O1:KUT Thailand (1999) 50 (untypeable), that had toxRS sequences, AP-PCR profiles, ri- Vietnam (1998–1999) 16 botypes, and PFGE profiles identical to those of the O3:K6 se- Bangladesh (1999–2000) 7 O1:KUT India (1998) 18 rotype (17, 18, 57). The discovery of the filamentous phage des- Bangladesh (1998 and 2000) 7, 57 ignated f237 in O3:K6 isolates (62) led to the development of O4:K12 Thailand (1998–1999) 50 another specific PCR method, which targeted ORF8 of f237, Vietnam (1998–1999) 16 claimed to be a specific genetic marker of O3:K6 isolates. Later, Chile (2004) 30 it was found that other serotypes that were not like the O3:K6 O1:K41 Thailand (1998–1999) 50 Vietnam (1998–1999) 16 isolates also carried ORF8 (39). In addition to O4:K68, O1:K25, O1:K56 Vietnam (1998–1999) 16 and O1:KUT, another serotype, O6:K18, which shared high mo- O3:K75 Vietnam (1998–1999) 16 lecular identity with an O3:K6 isolate, was detected in Taiwan O4:K8 Vietnam (1998–1999) 16 (102). Therefore, from a single O3:K6 serotype, other serotypes O4:KUT Vietnam (1998–1999) 16 O5:KUT Vietnam (1998–1999) 16 that had identical genotypes and molecular profiles to those of India (2004) Dutta and Ramamurthy O3:K6 isolates emerged, and these were collectively referred to as (unpublished data) “serovariants” of O3:K6 isolates (57). These serotypes appeared O5:K17 India (2002) Dutta and Ramamurthy to have diverged from the O3:K6 isolates by alteration of the O:K (unpublished data) antigens and were postulated to be clonal derivatives of the O5:K25 India (2002) Dutta and Ramamurthy (unpublished data) O3:K6 serotype. MLST data have further confirmed the finding O1:K33 India (2002) Dutta and Ramamurthy that multiple serotypes occur in a single genetic lineage (18, 19, (unpublished data) 57). The acquisition of additional serotypes of the pandemic O2:K3 India (2002) Dutta and Ramamurthy strain may be a selected response to host immunological pressure (unpublished data) OUT:KUT India (2003–2004) Dutta and Ramamurthy (19). Eleven O:K serotypes were detected among the strains iso- (unpublished data) lated during a survey of diarrhea patients in Khanh Hoa Province, O3:KUT India (2003–2004) Dutta and Ramamurthy Vietnam, and all were found to be closely related to O3:K6 (16). (unpublished data) To date, 21 serotypes that are similar to the O3:K6 serotype have O3:K5 India (2004) Dutta and Ramamurthy (unpublished data) been identified (Table 1) by a variety of molecular typing tech- O4:K4 India (2004) Dutta and Ramamurthy niques. (unpublished data) Table 2 shows the chronology of appearance of the various O4:K10 India (2004) Dutta and Ramamurthy serotypes in Calcutta that are similar to the O3:K6 isolates (unpublished data) recorded from February 1996. Clearly, the vigor with which the O6:K18 Taiwan (2005) 96 O3:K6 isolates appeared in the beginning of 1996 is on the wane. This matches the epidemiology of cholera, which generally settles into an endemic pattern of seasonal outbreaks IS THE SPREAD OF O3:K6 AND ITS separated by periods of quiescence after passage of a pandemic SEROVARIANTS A PANDEMIC? wave through a geographic region (62). The more recent serotypes, which have molecular traits similar to those of O3:K6, When the Calcutta O3:K6 isolates were compared with an do not seem to have the propensity for elevating hospital archived collection of O3:K6 strains isolated between 1982 and admissions due to V. parahaemolyticus gastroenteritis observed 1996 from travelers returning to Japan, it was found that the with O3:K6 and the earlier serovariants of O3:K6. Some kind Calcutta O3:K6 isolates were identical to the isolates obtained of decay in the epidemic process seems to be evident. from travelers from 1995 onwards but differed from O3:K6 strains 42 NAIR ET AL. CLIN.MICROBIOL.REV.

TABLE 2. Emergence of O3:K6 serotype of Vibrio parahaemolyticus and its serovariants in Calcutta from January 1994 to December 2004a

No. of isolates Total no. of Serotype 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 isolates O3:K6 70 33 12 20 4 1 2 9 1 152 O1:KUT 6 5 3 4 2 3 23 O4:K68 7 5 1 2 15 O1:K25 1 22 1 6 18 48 O5:K25 11 O1:K33 11 O2:K3 11 OUT:KUT 11 2 O3:KUT 27 9 O3:K5 11 O4:K4 11 O4:K10 11 O5:KUT 22 O5:K17 11

Total 70 39 24 26 29 7 15 12 36 258

a Data in this table are from references 14, 15, 52, and 64 and from Dutta and Ramamurthy (unpublished data). isolated prior to 1993. This comparison also fortuitously revealed each other and distinct from O3:K6 isolates obtained before that isolates of O3:K6 like those found in Calcutta were already 1995 and from non-O3:K6 serotypes. The O3:K6 isolates ap- prevalent in Indonesia, Thailand, and Singapore, since travelers pearing after 1995 carried the tdh but not the trh gene, did not returning from these countries were infected (69). Evidence sup- produce urease, and were defined by a positive group-specific porting the hypothesis that the O3:K6 serotype emerged only PCR (GS-PCR) based on the mismatched nucleotides at seven recently was furnished by Matsumoto et al. (57), who showed that base positions of the toxRS gene sequences and ORF8 from the O3:K6 isolates from clinical sources in Taiwan, Laos, Japan, Thai- f237 phage. Ribotyping revealed a certain degree of instability land, Korea, and the United States in 1997 and 1998 were iden- in the early Calcutta O3:K6 isolates, reflected as genomic re- tical to the Calcutta O3:K6 strains. Based on this evidence, they assortment during their initial period of existence (5). Accord- concluded that the unique O3:K6 isolate and its serovariants were ing to biochemical fingerprinting by a PhenePlate system (PhP- causing a pandemic. Such a widespread occurrence of one clonal 48; PhenePlate Microplate Techniques, Stockholm, Sweden) type of V. parahaemolyticus was unprecedented and spurred great based on the kinetic measurement of the fermentation of se- interest. lected reagents, the pandemic strains belonged to the same In retrospect and following the trend of the spread of O3:K6 biochemical phenotype, whereas the nonpandemic strains were isolates over the past decade, it seems that use of the word heterogeneous (79). Thirty-five isolates of V. parahaemolyticus “pandemic” is somewhat misleading and might be a bit of a strong from different countries and belonging to different serotypes term in a true epidemiological sense. The Webster’s dictionary (O3:K6, O4:K68, and O1:KUT) showed identical ribotypes definition of pandemic describes it as “occurring over wide geo- and PFGE patterns, with some exceptions, including a Japa- graphic areas and afflicting an exceptionally high proportion of nese tdh-negative O3:K6 strain and a U.S. clinical O3:K6 isolate the population.” Although the O3:K6 serotype of V. parahaemo- (18). At least two different ribotype patterns were observed lyticus has been isolated over a wide geographic area, supplanting among O3:K6 isolates from the United States and Asia, and most the other serotypes of V. parahaemolyticus in the process, it has of the strains from the 1998 Galveston Bay outbreak were differ- not affected an exceptionally high proportion of the population, ent from those isolated in New York and parts of Asia (28). The and it apparently has caused little, if any, mortality. In fact, com- 11 serotypes of V. parahaemolyticus obtained from surveillance in pared to the seventh pandemic El Tor biotype V. cholerae O1 (80) Vietnam were shown to be closely related, regardless of the or compared to V. cholerae O139 (65), V. parahaemolyticus is a ORF8 genotype, using AP-PCR and PFGE (16). pretty rare disease. In this review, we use the term “pandemic” MLST provided strong molecular evidence for the clonal only to allude to the description investigators have used in their origin of V. parahaemolyticus O3:K6 and revealed that isolates publications, but as explained above, we are of the opinion that within such a clonal group may acquire previously identified the spread of O3:K6 and its serovariants is not a pandemic in the serotypes of V. parahaemolyticus. The MLST study also con- real sense. firmed genetic diversity among the V. parahaemolyticus strains that prevailed before O3:K6 and genetic uniformity between CLONALITY OF O3:K6 ISOLATES O3:K6 and its serovariants in spite of their serotype diversity AND SEROVARIANTS (16). All of the O3:K6 isolates collected from 1983 to 1993 from diverse countries around the Indian Ocean were related. Starting with AP-PCR (17, 18, 57, 69), a variety of molecular However, the sequence types (STs) of all 11 other serotypes techniques, including ribotyping and pulsed-field gel electro- were distinct. In contrast, the four distinct serotypes O3:K6, phoresis (18, 25, 101, 105), revealed that O3:K6 isolates from O4:K68, O1:KUT, and O1:K25 were clonally related. Fifty-one widely different geographic areas were genetically similar to of 54 isolates had the ST 1,1,1,1 (19). VOL. 20, 2007 VIBRIO PARAHAEMOLYTICUS O3:K6 AND ITS SEROVARIANTS 43

FIG. 1. Global dissemination of the unique O3:K6 isolate of Vibrio parahaemolyticus and its serovariants.

GLOBAL SPREAD northern city of Antofagasta, Chile, from November 1997 to March 1998, and other outbreaks occurred during the summer At about the same time as the Calcutta occurrence, infor- months of 2004 and 2005, mainly in Puerto Montt, a region mation on steadily increasing numbers of V. parahaemolyticus with usually cold waters (21, 26, 30). Interestingly, a recent infection in Japan was documented. The number of food- retrospective analysis of V. parahaemolyticus strains isolated borne infections caused by this pathogen in 1998 in Japan from several places in Peru showed the prevalence of the doubled compared to that in 1997 and exceeded the number of O3:K6 serotype and other pandemic serovariants of V. para- infections by Salmonella, which previously was the dominant haemolyticus, with the earliest O3:K6 isolate being recovered cause of food-borne infections in Japan (103). An increase in as early as 1996, the same year that the pandemic O3:K6 food-borne disease outbreaks was recorded in Taiwan in 1996, serotype was identified in Calcutta, India (29). The majority of and this increase correlated with the high rate of isolation of V. the V. parahaemolyticus strains isolated during an outbreak of parahaemolyticus O3:K6 (15, 100). During 1995 in Taiwan, the acute enteric disease in Vladivostok, Russia, in 1997 belonged O3:K6 serotype accounted for only 0.6% of V. parahaemolyti- to serotype O3:K6 (85). More recently, the O3:K6 serotype cus infections, and this level abruptly increased to 50.1% in was isolated from hospitalized diarrhea patients in Mozam- 1996 and reached a peak of 83.8% in 1997 (15). From 1996 bique, ushering its spread into the African continent (3). V. onwards, the O3:K6 serotype was isolated from diarrhea pa- parahaemolyticus O3:K6 strains similar to the pandemic clone tients admitted to the ICDDR,B hospital in Dhaka, Bang- have been isolated from the coasts of Spain and France (55, ladesh (7, 15, 57). Between May and June 1998, 416 persons in 78). A diarrheal outbreak caused by serotype O3:K6 was re- 13 states reported having gastroenteritis after eating oysters ported from Calcutta, India (82). The incidence of tdh-positive harvested from Galveston Bay, Tex. All 28 available stool sam- strains of V. parahaemolyticus in patients in Hangzhou, China ples yielded V. parahaemolyticus O3:K6 isolates which closely (107), has been reported, but the molecular traits of the iso- resembled the Asian O3:K6 isolates by PFGE (23). During lates were not examined. Clearly, global dissemination of a July to September 1998, an outbreak of V. parahaemolyticus specific clone of V. parahaemolyticus is apparent, and at the O3:K6 infections associated with the consumption of oysters time of writing of this review, this clone has spread into Asia, and clams harvested from Long Island Sound occurred among America, Africa, and Europe (Fig. 1). residents of Connecticut, New Jersey, and New York (10, 11). Significantly, before this series of outbreaks in the United States, V. parahaemolyticus serotype O3:K6 was not reported in HOW ARE O3:K6 ISOLATES AND THEIR this country (23). Subsequently, O3:K6 isolates obtained in SEROVARIANTS DIFFERENT? 1997 and 1998 from clinical sources in Taiwan, Laos, Japan, Thailand, Korea, and the United States were found to share Since the discovery of the unique O3:K6 serotype of V. nearly identical AP-PCR profiles (57). parahaemolyticus, efforts have been made to determine the Outbreaks caused by the O3:K6 serotype occurred in the factor that endows these isolates with the ability to rapidly 44 NAIR ET AL. CLIN.MICROBIOL.REV. increase hospitalizations and to become the dominant sero- encoding a putative phage protein and has the insertion gene type. The initial efforts were centered on whether the O3:K6 sequence TTCTTCAG at its 5Ј and 3Ј ends (54, 72). Bioinfor- isolates produced more TDH than the other existing serotypes matic study of the whole genome sequence of an O3:K6 isolate of V. parahaemolyticus. Such a difference in production of (RIMD2210633) revealed the exclusive presence of four TDH was not evident (69, 101), but later studies revealed one genomic islands, termed V. parahaemolyticus islands (VPaIs), amino acid polymorphism in the tdh open reading frame in the pandemic group, which may represent DNAs acquired (ORF) that appeared to differentiate the O3:K6 isolates by the pandemic group that increased its fitness either in the

(Gly109) from non-O3:K6 strains (Asp109) (105). Interestingly, aquatic environment or in the ability to infect humans (36). sequence polymorphisms were also observed in the putative The direct repeats present in VPaIs 1, 4, and 5 and those in tdh promoter region of these isolates (99), but the significance VPaI 6 are located in the large and small chromosomes, re- of these differences was not clear given that there was no spectively. Moreover, the phage-like integrases show that these discernible difference in the amounts of TDH produced by VPaIs were acquired by horizontal gene transfer (36). Clearly, O3:K6 and non-O3:K6 strains of V. parahaemolyticus (69). O3:K6 isolates seem to have acquired some attributes that are These findings match well with an early report from Calcutta not seen in non-O3:K6 isolates, but so far the determining indicating that the clinical symptoms of patients infected by factor(s) remains elusive. A methyltransferase gene carried by O3:K6 isolates did not differ significantly from those of pa- a 23-kb novel pathogenicity island-like element was identified tients infected by non-O3:K6 serotypes. O3:K6 isolates com- among pandemic V. parahaemolyticus strains (95). However, its pared to non-O3:K6 strains that were tdh positive were shown specific advantages in virulence traits among pandemic strains to demonstrate an enhanced ability to swarm over agar surface have not yet been established. plates, and the presence of magnesium appeared to further stimulate swarming (105). There were, however, no significant ENVIRONMENTAL ISOLATION differences between survival rates under the same environmental stresses, such as extreme temperatures, low pH, and high The detection of virulent V. parahaemolyticus organisms salinity, for O3:K6 and non-O3:K6 strains of V. parahaemolyti- against a background of numerically greater numbers of avir- cus (69, 101). ulent V. parahaemolyticus organisms has remained a daunting The V. parahaemolyticus phage f237 is similar to the CTX problem. Undifferentiated total V. parahaemolyticus counts are filamentous phage of V. cholerae O1 (94), but instead of the therefore used as an indicator for the control of food contam- ctxAB genes, f237 has ORF8, and therefore it was thought that ination and the prevention of infection. Clearly, this is inade- ORF8 may play a significant role (like the profound role that quate, as reflected in the outbreaks caused in the United cholera toxin has in the disease cholera) in increasing the States, where despite bacteriologic monitoring at harvest sites virulence of O3:K6 isolates. Based on the similarity of the and despite the number of V. parahaemolyticus organisms be- motifs of the predicted amino acid sequence of ORF8 to those ing lower than the permissible most probable number of of the plx gene of Drosophila, which encodes a novel adhesion 10,000, the outbreaks could not be prevented (23). Attempts to molecule (106), Nasu et al. (67) speculated that ORF8 encodes isolate O3:K6 from the environment and from seafood have an adherence protein and that strains possessing this gene not met with much success. could be more adhesive to host intestinal cells or to the sur- An immunomagnetic separation technique targeting differ- faces of marine plankton. In vitro adherence and cytotoxicity ent K antigens was established for food poisoning investiga- studies with human epithelial cells showed that O3:K6 isolates tions of V. parahaemolyticus (89). Application of this tech- exhibited statistically higher levels of adherence and cytotox- nique, targeting the K6 antigen for the identification of O3:K6 icity to host cells than did non-O3:K6 isolates (105). Studies in isolates, was successfully demonstrated using clinical and en- Bangladesh, however, showed that ORF8 could not be de- vironmental samples (31, 92). An extensive environmental tected in several O3:K6 isolates from hospitalized diarrhea study in Japan combining a tdh-specific PCR method, chromo- patients (7). genic agar medium, and the most-probable-number method Recently, Okura et al. (71) identified, cloned, and sequenced showed that isolates similar to the Calcutta O3:K6 isolates a 930-bp AP-PCR fragment that was unique to the O3:K6 were widely distributed throughout the Japanese coastal envi- isolates and their serovariants; the sequence of this fragment ronment. Of the 19 strains examined, 14 were tdh and GS-PCR was found to be 80% homologous to the Mn2ϩ and Fe2ϩ positive and showed the same AP-PCR profile as the reference transporter of the NRAMP family of Vibrio vulnificus. Again, a O3:K6 isolates (32). Studies conducted at Kii Channel, To- specific function was not attributed to this fragment. In O3:K6 kushima, Japan, further confirmed this finding (33). However, isolates of V. parahaemolyticus, a histone-like DNA-binding neither the O3:K6 serotype nor any of the ribogroups associ- protein, HU-␣, with a C-terminal amino acid sequence differ- ated with the O3:K6 serotype was found among any of the ent from those in other strains of V. parahaemolyticus, has been environmental or food isolates examined, suggesting that the identified (72, 98). Further study has revealed that the gene O3:K6 serotype has not become established in the United encoding this protein has a 16-kb insert at the 3Ј terminus of States (25). It was felt that the O3:K6 serotype did not have an the ORF, but the effect of this insertion sequence on the environmental reservoir in the United States and that the or- activity of the HU-␣ protein in relation to a change in patho- igin and spread of this organism may have occurred via ship genicity is unknown at this time (72, 98). The functional role of ballast water. Cargo ships entering the Gulf of Mexico were a histone-like protein in streptococci was shown to be associ- thought to be responsible for the introduction of the Latin ated with tissue inflammation (86). The 16-kb insertion se- American epidemic strain of V. cholerae O1 into Gulf Coast quence might have evolved from a phage, as it includes a gene waters in 1991 (58). O3:K6 isolates have also been isolated VOL. 20, 2007 VIBRIO PARAHAEMOLYTICUS O3:K6 AND ITS SEROVARIANTS 45 from the aquatic environs of Bangladesh (40) and from the kitchen by V. parahaemolyticus-contaminated fish brought east coast of India (24). O3:K6 and other serovariants seem to from markets are thought to be the most likely routes of have established an ecological niche in Asia (4). It appears transmission in this setting (75). Early ecological studies have that, in some regions, aquatic birds act as reservoirs of V. shown the occurrence of V. parahaemolyticus in freshwater parahaemolyticus during the winter (60). However, more infor- plankton and in freshwater fishes (81). The survival of V. para- mation should be generated in identifying the pandemic strains haemolyticus in freshwater ecosystems has been shown to be from such nonhuman reservoirs. Environmental surveys have transient and dependent on a biological host (81). shown that the application of sensitive techniques is essential for isolating specific pathogenic strains, such as O3:K6 isolates, METHODS FOR DETECTION from environmental and seafood samples. The emergence of the O3:K6 serotype and its serovariants ECOLOGY AND EPIDEMIOLOGY and its widespread distribution have necessitated the development of specific methods to detect such strains. Serotyping The origin and subsequent spread of the O3:K6 isolates of V. enabled the detection of the O3:K6 isolates in Calcutta. Al- parahaemolyticus must be the consequence of coincidental though serotyping is a relatively easy technique for identifying events occurring at the right time and at the right place. Is the clusters of cases caused by a specific serotype and also for emergence of O3:K6 isolates and their serovariants the conse- tracking their spread, the cost of antisera is prohibitive and quence of the effects of global warming? For several of the therefore limits their availability and applicability. In the Cal- reported outbreaks, especially during the period 1996 to 1998, cutta episode, the identification of the clustering of the O3:K6 elevated environmental temperatures have been ascribed as a serotype did not occur in real time because a V. parahaemo- cause (23, 59). The year 1998 was part of the El Ninõ years, lyticus antiserum kit was not available at that time at the Na- when elevated seawater temperatures were shown to influence tional Institute of Cholera and Enteric Diseases in Calcutta, the incidence of V. cholerae (20) and other diarrheal diseases and the strains were confirmed and serotyped later at the (14). In 2005, serotype O6:K18 caused a diarrheal outbreak in Osaka Prefecture Institute of Public Health, Osaka, Japan. Alaska (59); however, its genetic relatedness with the O3:K6 The API-20 E identification system is used in many labora- isolate was not established. The Alaskan outbreak was associ- tories for the identification of enteric bacteria. For the identi- ated with warming of ocean waters (59). Among the food- fication of V. parahaemolyticus, it appears that the use of spe- borne disease outbreaks in 13 provinces of China during 2003, cific concentrations of NaCl makes a considerable difference in about 40% of the patients were infected with V. parahaemo- the identification of clinical (0.85%) and environmental (2.0%) lyticus (52). Are environmental conditions becoming conducive strains (56). Over the past few years, PCR-based detection for the proliferation of pathogens like V. parahaemolyticus?It techniques, including GS-PCR (52) and orf8 PCR (57, 64, 67), would at least seem so because V. parahaemolyticus has been have been developed to specifically detect the O3:K6 serotype recognized since the 1950s, and raw oysters and seafood have and its serovariants. The development of GS-PCR was a mile- been consumed from time immemorial. Yet 1996 to 1998 stone in simplifying the identification of the O3:K6 isolate and seemed to be particularly unpleasant years for humans, espe- also for detecting other serotypes that share identical molec- cially for those who had an inclination for oysters, which is the ular traits (57). Inconsistencies between the results of the most important source of infection of V. parahaemolyticus in toxRS and ORF8 PCRs related to serotypes that were not like the United States and the Far East. We still do not understand the O3:K6 isolates or their serovariants and that were toxRS the epidemiology of V. parahaemolyticus infections. In Viet- negative but positive by ORF8 PCR were reported (74). Ad- nam, the incidence of V. parahaemolyticus stopped abruptly ditionally, some of the O3:K6 isolates from Bangladesh iso- without meteorological changes or changes in water supply and lated between 1998 and 2000 (7) and from Vietnam (16) were sanitation, and the reasons for this abrupt interruption in negative by orf8 PCR, indicating that neither the toxRS nor the transmission are not clear (90). In recent times, there have ORF8 sequence is a reliable gene marker for the definite been occurrences of other extraordinary events in relation to identification of the pandemic group. A real-time PCR assay pathogenic Vibrio species. In the summer of 1996, a major targeting the orf8 gene was shown to be specific and sensitive outbreak of systemic V. vulnificus infections started among for the detection of pandemic O3:K6 strains (97). However, Israeli fish market workers and fish consumers (8). Molecular this assay may not be useful for the detection of pandemic analysis showed that this strain evolved by hybridization of the strains that are devoid of orf8. Pyrolysis metastable atom bom- genomes of two existing nonpathogenic forms of V. vulnificus, bardment mass spectrometry was shown to identify various which apparently led to the emergence of an epidemic caused phenotypic characteristics of V. parahaemolyticus (96). By tar- by the newly evolved pathogenic variant (9). Similarly, the geting specific phenotypic markers, this technique may differ- appearance of new hybrids between the classical and El Tor entiate pandemic and nonpandemic V. parahaemolyticus biotypes of V. cholerae O1 has been reported from Matlab, strains. Bangladesh (66), and from Mozambique (2). Okura et al. (70) developed a novel multiplex PCR assay The transmission and epidemiology of V. parahaemolyticus specific for the O3:K6 isolates and their serovariants that suc- infections in places such as Calcutta, India, and Bangladesh are cessfully distinguished these stains from other V. parahaemo- entirely different because seafood is never eaten raw and fresh- lyticus strains by yielding two distinct PCR products, for tdh water fish is preferred over seawater fish by the local popula- (263 bp) and the toxRS/new sequence (651 bp). A PCR-based tion. Contamination of freshwater fish by seawater fish at the assay was developed, employing an oligonucleotide primer pair fish market and secondary contamination of other foods in the derived from the group-specific sequence of an arbitrarily 46 NAIR ET AL. CLIN.MICROBIOL.REV. primed PCR fragment which was located in the genome en- 5. Bag, P. K., S. Nandi, R. K. Bhadra, T. Ramamurthy, S. K. Bhattacharya, M. coding a hypothetical protein. The assay distinguished the Nishibuchi, T. Hamabata, S. Yamasaki, Y. Takeda, and G. B. Nair. 1999. Clonal diversity among recently emerged strains of Vibrio parahaemolyticus O3:K6 isolates and their serovariants from other V. parahae- O3:K6 associated with pandemic spread. J. Clin. Microbiol. 37:2354–2357. molyticus strains by yielding a 235-bp specific amplicon (70). 6. Baross, J. A., J. Liston, and R. Y. Morita. 1978. Ecological relationship between Vibrio parahaemolyticus and agar-digesting vibrios as evidenced by Khan et al. (48) reported that all of the O3:K6 strains isolated bacteriophage susceptibility patterns. Appl. Environ. Microbiol. 36:500– from the Texas and New York outbreaks yielded an 850-bp 505. DNA fragment along with other amplicons in enterobacterial 7. Bhuiyan, N. A., M. Ansaruzzaman, M. Kamruzzaman, K. Alam, N. R. Chowdhury, M. Nishibuchi, S. M. Faruque, D. A. Sack, Y. Takeda, and repetitive intergenic consensus PCR. The primers designed G. B. Nair. 2002. Prevalence of the pandemic genotype of Vibrio parahae- from this 850-bp gene sequence were specifically identified in molyticus in Dhaka, Bangladesh, and significance of its distribution across O3:K6 isolates with a 327-bp amplicon, whose function and different serotypes. J. Clin. Microbiol. 40:284–286. 8. Bisharat, N., and R. Raz. 1996. Vibrio infection in Israel due to changes in homology remain unknown. However, this PCR method was fish marketing. Lancet 348:1585–1586. not validated. Using the detection of an insertion mutation in 9. Bisharat, N., D. I. Cohen, R. M. Harding, D. Falush, D. W. Crook, T. Peto, ␣ and M. C. Maiden. 2005. Hybrid Vibrio vulnificus. Emerg. Infec. Dis. 11: the HU- ORF, a PCR technique was reported for the iden- 30–35. tification of not only the O3:K6 serotype but also other sero- 10. Centers for Disease Control and Prevention. 1998. Outbreak of Vibrio variants, such as O1:K25, O1:KUT, and O4:K68 (72, 98), which parahaemolyticus infection associated with eating raw oysters—Pacific Northwest, 1997. Morb. Mortal. Wkly. Rep. 47:457–462. had molecular traits identical to those of the O3:K6 isolate. 11. Centers for Disease Control and Prevention. 1999. Outbreak of Vibrio Genes located on VPaIs 4, 5, and 6 (36) can be targeted for the parahaemolyticus infection associated with eating raw oysters and clams specific detection of pandemic strains by PCR. However, the harvested from Long Island Sound—Connecticut, New Jersey, and New York, 1998. Morb. Mortal. Wkly. Rep. 48:48–51. specificity and sensitivity are not yet established for routine use 12. Chatterjee, B. D., and T. Sen. 1974. Vibrio parahaemolyticus serotypes in in the laboratory. Use of microarray technology with specific Calcutta, India. Bull. W. H. O. 50:559–561. 13. Chatterjee, B. D., A. Mukherjee, and S. N. Sanyal. 1984. Enteroinvasive amplification and oligonucleotides was shown to be useful for model of Vibrio parahaemolyticus. Indian J. Med. Res. 79:151–158. the detection and identification of pathogenic bacteria, includ- 14. Checkley, W., L. D. Epstein, R. H. Gilman, D. Figueroa, R. I. Cama, J. A. ing V. parahaemolyticus (41). Targeting specific genes in this Patz, and R. E. Black. 2000. Effect of El Nino and ambient temperature on hospital admissions for diarrhoeal diseases in Peruvian children. Lancet microarray technique will differentiate pandemic and nonpan- 355:442–450. demic strains. 15. Chiou, C. S., S. Y. Hsu, S. I. Chiu, T. K. Wang, and C. S. Chao. 2000. Vibrio parahaemolyticus serovar O3:K6 as cause of unusually high incidence of food-borne disease outbreaks in Taiwan from 1996 to 1999. J. Clin. Micro- CONCLUSION biol. 38:4621–4625. 16. Chowdhury, A., M. Ishibashi, V. D. Thiem, D. T. N. Tuyet, T. V. Tung, B. T. The emergence and spread of the O3:K6 isolate and its Chien, L. V. Seidlein, D. G. Canh, J. Clements, D. D. Trach, and M. Nishibuchi. 2004. Emergence and serovar transition of Vibrio parahaemo- serovariants offer an invaluable opportunity to examine factors lyticus pandemic strains isolated during a diarrhea outbreak in Vietnam that abet and perpetuate events like this. Many aspects of between 1997 and 1999. Microbiol. Immunol. 48:319–327. O3:K6 and its serovariants are still unknown. For example, we 17. Chowdhury, N. R., S. Chakraborty, B. Eampokalap, W. Chaicumpa, M. Chongsa-Nguan, P. Moolasart, R. Mitra, T. Ramamurthy, S. K. Bhatta- do not know the factors that triggered the genesis of the O3:K6 charya, M. Nishibuchi, Y. Takeda, and G. B. Nair. 2000. Clonal dissemi- serotype and the bacterial factors that are involved. We also do nation of Vibrio parahaemolyticus displaying similar DNA fingerprint but not know the mechanism of formation of serovariants and belonging to two different serovars (O3:K6 and O4:K68) in Thailand and India. Epidemiol. Infect. 125:17–25. those extraneous factors that drive this event. 18. Chowdhury, N. R., S. Chakraborty, T. Ramamurthy, M. Nishibuchi, S. Yamasaki, Y. Takeda, and G. B. Nair. 2000. Molecular evidence of clonal Vibrio parahaemolyticus pandemic strains. Emerg. Infect. Dis. 6:631–636. ACKNOWLEDGMENTS 19. Chowdhury, N. R., O. C. Stine, J. G. Morris, and G. B. Nair. 2004. Assess- Vibrio parahaemolyticus The core donors to the International Centre for Diarrheal Disease ment of evolution of pandemic by multilocus sequencing typing. J. Clin. Microbiol. 42:1280–1282. Research, Bangladesh (ICDDR,B) supported this work. Current do- 20. Colwell, R. R. 1996. Global climate and infectious diseases: the cholera nors providing unrestricted support include the aid agencies of the paradigm. Science 274:2025–2031. Governments of Australia, Bangladesh, Belgium, Canada, Japan, 21. Cordova, J. L., J. Astorga, W. Silva, and C. Riquelme. 2002. Characteriza- Kingdom of Saudi Arabia, The Netherlands, Sweden, Sri Lanka, Swit- tion by PCR of Vibrio parahaemolyticus isolates collected during the 1997– zerland, and the United States. The funding sources had no involve- 1998 Chilean outbreak. Biol. Res. 35:433–440. ment in the study design, interpretation, or decision to submit the 22. Cornelis, G. R., and F. Van Gijsegem. 2000. Assembly and function of type manuscript. III secretory systems. Annu. Rev. Microbiol. 54:735–774. 23. Daniels, N. A., L. MacKinnon, R. Bishop, S. Altekruse, B. Ray, R. M. Hammond, S. Thompson, S. Wilson, N. H. Bean, P. M. Griffin, and L. REFERENCES Slutsker. 2000. Vibrio parahaemolyticus infections in the United States, 1. Abbott, S. L., C. Powers, C. A. Kaysner, Y. Takeda, M. Ishibashi, S. W. 1973–1998. J. Infect. Dis. 181:1661–1666. Joseph, and J. M. Janda. 1998. Emergence of a restricted bioserovar of 24. Deepanjali, H., S. Kumar, I. Karuasagar, and I. Karunasagar. 2005. 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Role of Cell Culture for Virus Detection in the Age of Technology Diane S. Leland1* and Christine C. Ginocchio2,3 Indiana University School of Medicine, Department of Pathology and Laboratory Medicine, Indianapolis, Indiana1; North Shore LIJ Health System Labs, Lake Success, New York2; and North Shore University Hospital, Department of Laboratory Medicine, Manhasset, New York3

INTRODUCTION ...... 49 VIRUS ISOLATION IN TRADITIONAL CELL CULTURES...... 50 NEWER CELL CULTURE FORMATS...... 55 CRYOPRESERVED CELL CULTURES ...... 55 CENTRIFUGATION-ENHANCED INOCULATION AND PRE-CPE DETECTION OF VIRUSES IN CELL CULTURES ...... 56 VIRUS ISOLATION IN COCULTURED CELLS...... 57 VIRUS ISOLATION IN TRANSGENIC CELL LINES...... 60 NONCULTURE METHODS COMPARED TO VIRUS ISOLATION IN CELL CULTURES...... 64 IF Methods ...... 64 Non-IF Methods...... 66 Molecular Methods...... 68 APPLICATIONS OF VIRAL DIAGNOSTIC TECHNIQUES ...... 70 Testing for Respiratory Viruses...... 71 Exceptions in Respiratory Virus Testing...... 71 Samples from persons with immunosuppression...... 71 Samples from persons considered clinically as having possible cases of avian inﬂuenza or SARS or exposure to other emerging pathogens or viral agents of bioterrorism...... 72 Samples from pediatric patients...... 72 Samples from seriously ill pediatric patients ...... 72 When adenovirus is the viral suspect ...... 72 Viral Testing of Vesicular Lesions ...... 72 Exceptions in Viral Testing of Vesicular Lesions...... 72 Specimens from vesicles suspicious for variola virus...... 72 Specimens from vesicles suspicious for monkey pox or other poxvirus infections ...... 72 Viral Testing of CSF...... 72 Enteroviruses in CSF ...... 72 Herpesviruses in CSF...... 73 JC virus in CSF ...... 73 Arboviruses in CSF...... 73 Viral Testing of Other Types of Samples...... 73 Fecal samples for detection of enteric viruses...... 73 Peripheral blood samples ...... 73 Urine samples...... 73 General Suggestions for Viral Diagnostic Laboratories...... 73 CONCLUSION...... 74 ACKNOWLEDGMENTS ...... 74 REFERENCES ...... 74

INTRODUCTION ination with antibiotics and clean-air equipment, and helped decrease the use of experimental animals (55). Viruses reach The discovery in the early 1900s that human cells could be high titers when grown within susceptible cells, and culture propagated in vitro provided virologists with an alternative to tubes are convenient to manipulate. embryonated eggs and laboratory animals for in vitro isolation Although virus isolation in cell cultures was employed by of viruses. Cell cultures, which are derived from dispersed cells research laboratories by the early 1960s, diagnostic services taken from original tissue and disaggregated by enzymatic, were very limited, varying from laboratory to laboratory and mechanical, or chemical means, provided large numbers of often not available at all, except in major medical centers. cells suitable for virus isolation, facilitated control of contam- However, by the early 1970s, diagnostic virology expanded dramatically, largely because of the availability of highly puri- ﬁed reagents and commercially prepared cell lines (71). The * Corresponding author. Mailing address: Department of Pathology types of cells that can be grown in vitro in ﬂasks and test tubes and Laboratory Medicine, Clarian Pathology Laboratory, Room 6027F, 350 W. 11th Street, Indianapolis, IN 46202. Phone: (317) 491- are many, providing living hosts that many human viruses can 6646. Fax: (317) 491-6649. E-mail: [email protected]. infect. Cell cultures are more convenient and less expensive

49 50 LELAND AND GINOCCHIO CLIN.MICROBIOL.REV. than eggs and animals, are convenient to examine microscop- human epidermoid carcinoma cells (HEp-2), human lung car- ically for evidence of viral proliferation, and, for many years, cinoma cells (A549), and others. The number and types of cell have provided a desirable environment for the detection and culture tubes inoculated for each clinical specimen depend on identification of many human viral pathogens. Virus isolation the specimen source and the viruses suspected of causing a in cell cultures has long served as the “gold standard” for virus given disease. The cost of cell culture tubes ranges from $1.50 detection, and it is the method to which all others have been to $6.50 per tube, depending on the cell line; primary cells are compared (71). However, in recent years, technological ad- more expensive than nonprimary cells (Table 1). The cost per vances, ranging from the development of monoclonal antibod- tube also depends on numbers of tubes purchased, shipping ies to the introduction of molecular diagnostics, have provided specifications, etc. powerful tools to use in attempting to detect the presence of The appropriate selection, collection, transport, and pro- viral infections. Molecular detection of viral DNAs and RNAs cessing of clinical samples are important for successful virus and molecular amplification by PCR and other techniques are isolation. Collection of samples that contain the highest titer of now becoming more widely available in diagnostic laborato- virus is most desirable. Preservation of the viral titer and viral ries. Sensitive and highly specific viral identification can be infectivity until cell cultures can be inoculated is essential. obtained with these techniques. Molecular methods, as well as Body sites and collection methods vary according to the type of others such as viral antigen detection, do not require the infection and viral etiology. In general, clinical samples col- lengthy incubation period needed for viral isolation in cell lected from body sites such as skin and the genital tract, which cultures, may involve less technical expertise, and are useful for are usually contaminated with microbial flora, are collected viruses that do not proliferate in standard cell cultures. with a Dacron or polyester swab and placed in viral transport At this point, it is provocative to ask, “Is virus isolation in medium (VTM), most types of which contain antibiotics, a cell cultures still a useful approach in viral diagnostics?” and buffered salt solution, a proteinaceous substance (such as “What does the future hold for this approach in the diagnostic albumin, gelatin, or serum), and a pH indicator. Respiratory virology laboratory?” The purpose of this review is to present tract samples include sputum, bronchial alveolar lavage spec- the current status of viral detection in cell cultures, describe imens, nasopharyngeal (NP) washes, NP aspirates (NPA), NP developments in the field, and critically analyze situations in swabs (NPS) in VTM, oropharyngeal swabs in VTM or a com- viral diagnosis to indicate when viral isolation methods are bination of NP and oropharyngeal swabs in a single VTM tube. likely to yield the most desirable outcome. Specimens such as cerebrospinal fluid (CSF) and body fluids, which are expected to be free of microbial contamination, are VIRUS ISOLATION IN TRADITIONAL CELL CULTURES collected in sterile containers and are not placed in transport medium. Keeping the samples cool (2 to 8°C or on wet ice) As early as 1913 vaccinia virus (152) was grown in cell cul- until cell culture inoculation helps preserve viral infectivity and tures, and in the 1930s both smallpox virus (133) and yellow increases the virus recovery rate, particularly for labile viruses fever virus (94) were propagated in cell cultures for the pur- such as respiratory syncytial virus (RSV). Information concern- pose of vaccine production. However, it was not until the 1950s ing selecting, collecting, and transporting clinical samples for that the interest in using cell cultures for virus isolation ex- viral culture is provided in several reference texts (52, 146). panded, largely due to the discovery that polioviruses would Although specimen processing guidelines differ from labo- proliferate in cell cultures that were not of neural origin (43, ratory to laboratory, many laboratories clarify certain sample 134). The use of cultured cells to isolate viruses was advanced types (e.g., respiratory samples) as follows prior to inoculation further by the addition of antibiotics to cell culture media, the into cell cultures. The transport medium tube is vortexed, the development of chemically defined culture media, and the use swab is discarded, the liquid medium is centrifuged, and the of cell-dispensing equipment for preparing replicate cultures supernatant fluid is used to inoculate the cell cultures. Thus, (142). Although, initially, flasks and tubes of cells for use in bacteria, fungi, cells, blood, mucus, fibers, etc., are pelleted diagnostic laboratories were prepared in the laboratory, bio- into the bottom of the spun tube, while the viruses, which will logical supply houses soon began to mass produce various cell not be spun down by the g-force generated by most general strains and lines which could be purchased and delivered ready laboratory centrifuges, remain dispersed throughout the liquid. to use. Although many diagnostic virology laboratories pur- The sample pellet can be used for various antigen detection chase all of their cell cultures, some laboratories still prepare assays. Extensively contaminated clinical material such as stool cell cultures in-house. may be liquefied in antibiotic-containing medium and filtered Although cell cultures can be purchased or prepared in a through a 0.45-␮m filter prior to inoculation into cell cultures. variety of containers, the 16- by 125-mm glass or plastic round- Samples from sites expected to be free of microbial contami- bottom screw-cap tube is standard, with the cell monolayer nation may be used for cell culture inoculation without any adhering from the midpoint to the bottom of one side of the treatment or processing. Suggestions for processing clinical tube. Typically, several different cell lines are inoculated with samples for viral culturing are available in several reference each clinical sample in an attempt to provide a suitable host for texts (52, 146). whichever virus might be present in the sample. Cell cultures of As with specimen collection and processing, procedures for primary, diploid, and heteroploid cells are kept on hand in the inoculation of cell cultures and the number and types of cell virology laboratory. Examples of well-known cell types that are cultures inoculated for each specimen may vary among labo- standard for most virology laboratories are primary rhesus ratories and according to specimen type, virus suspected, and monkey kidney (RhMK) cells, primary rabbit kidney cells, hu- patient population. The processed inoculum may be added to man lung fibroblasts (MRC-5), human foreskin fibroblasts, the cell culture tube, either by simply adding 0.2 ml or 0.3 ml VOL. 20, 2007 ROLE OF CELL CULTURE IN VIRUS DETECTION 51

TABLE 1. Cost, turnaround time, advantages, and disadvantages of various virus detection approaches

Method Costa/avg turnaround time Advantages Disadvantages Cell culturesb Traditional tubes $1.50–$4.00 per tube Isolate wide variety of viruses (including Technical expertise needed to read CPE; for nonprimary cells unanticipated agents, mixed cultures); long incubation period for some and $2.15–$6.15 per provide isolate for additional studies: viruses, need for tube for primary antiviral susceptibility testing, purchasing/maintaining a variety of cells; use 2 to 6 tubes serotyping, and epidemiologic studies; cell culture types in-house per culture/5–10 days increased sensitivity over rapid antigen tests Shell vials with Same as comparable Short turnaround time for detection; Not as sensitive as traditional cultures centrifugation/ cell culture tubes; use take up less space than tubes; some for culturing blood samples for CMV; pre-CPE stain at least 2 vials of available as cryopreserved cells; may reading stained preparations is time- each cell line/24–48 h isolate viruses that replicate poorly or consuming and labor-intensive; not at all in standard tube cell unanticipated agents may be missed cultures; require less technical when pre-CPE staining targets only expertise than tube cultures if one or a few viruses; isolates not pre-CPE staining is used available from fixed/stained vials Cocultivated cells Approx. $1.25 more per Same as for shell vials plus decreased Same as for shell vials vial than standard need for maintaining wide variety of shell vials; use 3 vials cell cultures, support growth of a for each culture/ wider range of viruses, most results 24–48 h finalized in 2–3 days when pre-CPE staining is used, may be more sensitive than tube cultures for some viruses Transgenic cells $2.35–$3.00 more per Same as for shell vials plus detection by Targeted for detection of only a single (ELVIS) vial than standard color change rather than application virus group (HSV) shell vials; use 2 vials of MAbs, simplify identification for each culture/ because of specificity for a single 24–48 h virus, can be used to type HSV-1 and HSV-2

Nonculturec Antigen detection by $2–$7.00 for MAbs for Generally good sensitivity (which varies Generally not as sensitive as cell IF each sample/40 min with virus detected); excellent cultures; requires expertise in reading; specificity; CMV antigenemia is more not useful for all viruses; adenovirus sensitive than traditional or shell vial sensitivity especially poor cultures for CMV in blood Antigen detection, $10–$22 for each Generally good specificity for RSV and Generally poor sensitivity compared to cell non-IF sample/30 min influenza A and B viruses; no special culture; currently available for RSV and technical expertise required; results influenza A and B viruses only; available very rapidly; most cleared additional testing of negative samples by for point-of-care testing cell culture is recommended Nucleic acid detection $35–$125 for each Excellent sensitivity and specificity; FDA-cleared kits and standardized (molecular) sample tested with short turnaround with real-time PCR; protocols not widely available for most ASR or FDA-cleared useful for viruses that cannot be viruses; technical expertise required kits; $10–$35 for each cultured in traditional cell cultures in-house for developing and in-house developed standardizing methods; expensive due assay (may require to costs of instrumentation (especially patent royalties)/2 h for low-vol testing); probes and for real-time PCR; primers extremely specific (may miss 8 h for traditional mutated virus); detects only viruses PCR sought (will miss unanticipated agents and mixed infections in most cases); many assays available at reference/ research laboratories only

a Cost includes reagents only. b All types of cell cultures require viable virus in order to produce a positive result. This allows these methods to differentiate viable from nonviable virus. Because viable virus is required, specimens must be handled carefully to preserve viral infectivity. c None of the nonculture methods requires viable virus in order to produce a positive result; therefore, these methods cannot differentiate viable and nonviable virus. Because viability is not required, specimen handling is not as critical. No viral isolate is available upon completion of testing.

of the sample to each tube or by adsorption inoculation. Ad- to 37°C, excess inoculum is discarded and fresh cell culture sorption inoculation involves decanting the cell culture me- medium is added (90). Adsorption inoculation is thought to dium from the cell culture monolayer and applying the inocu- allow more efficient adsorption of viral particles to the cells lum directly to the monolayer. After a 30- to 90-min (88) and to enhance rates of recovery of some viruses (52, 88, 146). incubation of the inoculated tube in a horizontal position at 35 Inoculated cell culture tubes can be incubated in stationary 52 LELAND AND GINOCCHIO CLIN.MICROBIOL.REV. slanted racks or, alternatively, in rotating/rolling racks at 35 to commonly used techniques in diagnostic virology laboratories 37°C, which may enhance the speed and sensitivity of virus today. recovery (102). Viral tube cultures are incubated for days to Confirmatory testing of virus cultures positive by CPE or weeks depending on the specimen source and the suspected HAD has traditionally been based on the reaction of antibod- virus(es). Cell monolayers are screened by microscopic exam- ies of known specificity with viral antigens expressed in the ination daily for the first week of incubation to maximize the infected cells. Most of this testing is accomplished at present detection of viral growth and on alternate days for the remain- through immunofluorescence (IF) techniques that use fluores- der of the incubation period (88). The microscopic examina- cein isothiocyanate (FITC)-labeled monoclonal antibodies tion involves placing the tube on the stage of a standard light (MAbs). The cells are scraped from the infected monolayer microscope and viewing the cells through the glass wall of the and placed on a microscope slide. The preparation is fixed in tube with the low-power (10ϫ) objective. acetone and then flooded with FITC-labeled MAbs of known The microscopic examination of the unstained cell culture specificity. Binding of MAbs to viral proteins is signaled by the monolayer has long been the standard approach for detecting presence of fluorescence when the preparation is viewed using viral proliferation. Degenerative changes in monolayer cells the fluorescence microscope. The type of fluorescence (e.g., provide evidence of viral presence. The spectrum of change is speckled versus confluent) and the location of the fluorescence broad, ranging from swelling, shrinking, and rounding of cells in the cell (e.g., nuclear versus cytoplasmic) are also useful in to clustering, syncytium formation, and, in some cases, com- differentiating certain viruses. This process takes only 1 to 2 h plete destruction of the monolayer. These changes are collec- and overall gives a sensitive and specific viral identification. tively called the cytopathogenic or cytopathic effect (CPE) of Unfortunately, IF staining cannot be used to definitively the virus. The typical CPEs of several common viruses are identify all viruses. Examples of this are the coxsackieviruses, shown in Fig. 1. Dramatic CPE may be easily detected, but the polioviruses, and echoviruses of the “enterovirus” group, which subtle CPE of many viruses, early CPE, or CPE that is not are closely related and have numerous serotypes. In some typical may go unrecognized unless the observer has consider- cases, these may be identified as to their family by IF. How- ever, the MAbs for enteroviral identification have been shown able expertise. Although herpes simplex virus (HSV) may pro- to lack sensitivity (77), cross-react with rhinoviruses (77), and duce easily visible CPE within the first 24 h of incubation, most lack reactivity with enterovirus 71 (155). Identification of en- viruses demonstrate CPE only after 5 to 10 days of incubation, teroviral serotypes within the families requires confirmation by

with some, such as cytomegalovirus (CMV), averaging 10 to 30 the neutralization method (132). In neutralization testing, the days for CPE production (Table 2). The experienced observer virus-infected cells are incubated with antibodies of known may be able to predict which virus is present based on the viral specificity; an aliquot of the mixture is then inoculated characteristics of the CPE, the cell line involved, the length of into susceptible cell cultures, and the cell cultures are observed incubation, and the type of clinical specimen, but confirmatory for evidence of viral proliferation. CPE production indicates testing is needed to make a definitive viral identification. that the antibodies did not bind, inactivate, or neutralize the An alternative approach for detecting viruses that produce virus. Conversely, a lack of CPE production indicates that the CPE slowly or not at all in primary culture of clinical specimens antibodies bound to the virus and inactivated or neutralized it, is to perform a hemadsorption (HAD) test. HAD is useful only allowing the identity of the virus to be established according to for viruses such as influenza virus, parainfluenza virus, and the specificity of the antibody used. This is a cumbersome mumps virus that express their hemagglutinating proteins on procedure that requires determining the titer of the virus the plasma membranes of virus-infected cells. These proteins prior to the start of the procedure and a lengthy incubation are not visible with the light microscope but can be detected by after inoculation of cell culture tubes with the mixture of their affinity for erythrocytes. HAD testing is routinely per- antibody and virus-infected cells. Although neutralization formed at the end of the incubation period for cell cultures testing may be used in identifying all types of viruses, it is that fail to produce CPE or earlier in the incubation period, at used only when less cumbersome, more rapid methods are days 3 and 7 of incubation (105). Hemadsorbing foci have been not available. Neutralization testing is not routinely per- found in human fetal lung diploid cell cultures within 12 h after formed in most clinical laboratories and is generally re- inoculation with influenza viruses A and B (122). In HAD served for reference laboratories. testing the cell culture medium is removed and replaced with a The main advantage of the traditional cell culture approach dilute suspension of erythrocytes, usually guinea pig erythro- (Table 1) is the capacity to isolate a wide variety of viruses. At cytes, and the cell culture tubes are incubated at 4°C for 30 min this writing, the following familiar viral pathogens are the ones (90). Tubes are then examined microscopically. If a hemad- that can be isolated in traditional tube cultures: adenovirus, sorbing virus is present, erythrocytes will adhere in clumps to CMV, many of the enteroviruses (i.e., polioviruses, coxsackie- the infected areas of the cell monolayer (Fig. 2). Erythrocytes viruses, and echoviruses), HSV, influenza A and B viruses, will not adhere to uninfected cells or to cells infected by non- measles virus, mumps virus, parainfluenza virus types 1 to 4, hemadsorbing viruses. Nonadherent erythrocytes float free RSV, rhinoviruses, and varicella-zoster virus (VZV). Other when the cell culture tube is tapped or rotated. Although only viruses, such as Ebola virus, severe acute respiratory syndrome a few human viral pathogens produce a positive HAD result, coronavirus (SARS-CoV), and human metapneumovirus confirmatory testing of all HAD-positive cell cultures is re- (hMPV), also proliferate in traditional tube cultures. By inoc- quired to differentiate among the hemadsorbing viruses. De- ulating clinical samples into several types of cell cultures, a spite the availability of alternative methods for detecting viral suitable environment is provided for most of these viruses. presence in infected cell cultures, CPE and HAD are the most Using a broad range of cultured cells may allow the detection VOL. 20, 2007 ROLE OF CELL CULTURE IN VIRUS DETECTION 53

FIG. 1. Uninfected cell cultures and cell cultures showing CPE of viruses commonly isolated. (A) Uninfected A549 cells; (B) HSV-2 in A549 cells; (C) adenovirus in A549 cells; (D) uninfected MRC-5 fibroblasts; (E) CMV in MRC-5 fibroblasts; (F) rhinovirus in MRC-5 fibroblasts; (G) uninfected RhMK cells; (H) enterovirus in RhMk cells; (I) influenza A virus in RhMk cells; (J) uninfected HEp-2 cells; (K) RSV in HEp-2 cells; (L) monkey virus contaminant in RhMk cells. Magnification, ϫ85. of unanticipated agents, rather than focusing on the detection of immunocompetent patients in an average of 5 to 10% of of only one or a few specific viruses. This approach also facil- cultures. The medical significance of this is not established, but itates detection of more than one virus from the same sample. it has been suggested that dual infections, particularly in young In a review of mixed viral infections, Waner (158) noted that children, may increase the severity of respiratory disease (11, mixed infections are reported to occur in respiratory samples 54). An increased incidence of mixed infections was seen in 54 LELAND AND GINOCCHIO CLIN.MICROBIOL.REV.

TABLE 2. Cytopathogenic effect in standard cell cultures of human viral pathogens common in the United Statesa

Cytopathogenic effect in: Final identification of Virus Fibroblasts A549 cellsb RhMK cells Otherc isolates Adenovirus Some produce clusters Grape-like clusters Some produce clusters HNK: grape-like IF for group, or “lacy” pattern; clusters; 5–7 days neutralization 5–8 days for type CMV Foci of contiguous rounded None None Use shell vials for CPE alonee cells; 10–30 days rapid detection Enteroviruses Some produce CPE, same as Infrequent, Small, round cells with IF for groups, in RhMK cells; 2–5 days degenerative cytoplasmic tails; neutralization 2–5 days for type HSV Rounded large cells; 2–6 Rounded large Some produce CPE, RK or HNK: rounded IF days cells; 1–4 days same as in A549 large cells; 1–4 days cells, 4–8 days Influenza virus None None Undifferentiated CPE, HAD-positive with GP IF cellular granulation; 4–8 days Parainfluenza None None Rounded cells, some HAD-positive with GP IF virus syncytia; 4–8 days Rhinovirus Degeneration, rounding; None None Incubate fibroblasts at CPE onlyf (difficult 7–10 days 33°C to differentiate from enteroviruses) RSV Infrequent, granular Infrequent Syncytia; 4–10 days HEp-2d: syncytia; 4–10 IF degeneration days VZV Some CPE; small, round Small, round cells; None HNK: small, round IF cells; 6–8 days 6–8 days cells; 6–8 days

a Measles, mumps, and rubella viruses are seldom encountered in the United States at present. Measles virus produces large syncytia in RhMK cells in 7 to 10 days and is hemadsorption positive with Rh cells. Virus identification may be confirmed by IF. Mumps virus produces rounded cells with large syncytia in RhMK cells in 6 to 8 days and is hemadsorption positive with guinea pig erythrocytes, and its identification may be confirmed by IF. Rubella virus requires special cultures such as African green monkey kidney, rabbit kidney, or BSC-1 cells and does not produce CPE; special detection by interference challenge or another method is needed. b Human lung carcinoma. c GP, guinea pig erythrocytes; HAD, hemadsorption; HNK, human neonatal kidney cells, RK, rabbit kidney cells. d Human laryngeal carcinoma. e Some laboratories may base final identification of CMV on characteristic CPE alone. Others may inoculate shell vials and stain for CMV early antigen to confirm the identification. f Some laboratories may base final identification of rhinovirus on CPE, which is similar to that of the enteroviruses but appears in fibroblast lines rather than RhMk cells. The term “rhino-like virus” is sometimes used for reporting when the identification is based on CPE alone. Others may test for acid lability to differentiate rhinoviruses from the enteroviruses.

immunocompromised patients, especially with latent viruses for additional studies such as antiviral susceptibility testing, such as HSV and CMV. Accurate detection of all viruses serotyping, and epidemiologic evaluations. Viral proliferation present is especially important with these patients to ensure also confirms the viability of the virus and differentiates viable timely treatment with proper antiviral therapy. Replication of from nonviable virus, a differentiation that is not made by viruses in cell cultures also provides an isolate that can be used antigen and most nucleic acid detection methods. This information may be important for medical decision-making, such as in differentiating disease from latent infection (92); deciding when to implement, discontinue, or change antiviral therapy (93, 97); or making other decisions concerning patient management. The cell culture approach also offers increased sensitivity over most rapid antigen detection methods. However, the need for technical expertise in evaluating cell culture monolayers microscopically, the generally long incubation period required for some viruses to produce CPE, the inability of some viruses to proliferate in traditional cell cultures, the expense involved in purchasing and maintaining cell cultures, and the availability and constant improvement of alternate technologies are all factors to consider when evaluating the long-standing gold standard of virus isolation in cell culture. Other advanced technologies, such as nucleic acid amplification tests (NAATs), have been applied to speed viral detection and decrease the level of technical expertise required FIG. 2. Positive hemadsorption result in parainfluenza virus-infor the process of viral isolation and identification. However, fected RhMk cells. Magnification, ϫ100. cell culture technology has also experienced innovative modi- VOL. 20, 2007 ROLE OF CELL CULTURE IN VIRUS DETECTION 55

fications, allowing virus isolation in cell culture to continue to with the actual transport process involving a variety of delivery make a significant contribution in viral disease diagnosis. services. Regardless of the delivery service used, including “express” couriers, the cell cultures may be stressed during trans- NEWER CELL CULTURE FORMATS port by extremes in temperature; may be mishandled as they are packed, stacked, and loaded; or may be compromised by The traditional 16- by 125-mm screw-cap cell culture tube, delays in delivery due to bad weather, holiday closures, and which has been the standard for many years, is now only one of many other uncontrollable circumstances. All of these factors several different configurations in which cell cultures are used may contribute to suboptimal performance of the cell cultures in viral diagnostic laboratories. A small vial, called a 1-dram (74). In addition, the virology laboratory must determine in vial or a shell vial, has become popular for containment of cell advance how many culture tubes will be needed. If a circum- culture monolayers. In this configuration, the cell monolayer is stance such as an outbreak of a viral illness in the hospital or grown on a coverslip that resides in the bottom of the vial. The community occurs, the virology laboratory may not have suf- small vials fit easily into a centrifuge for use in viral detection ficient numbers of culture tubes on hand to deal with the assays that involve centrifugation-enhanced inoculation of the increased specimen volume. monolayer. These assays, which facilitate rapid detection of The use of cryopreserved cells may help to minimize some of viruses (especially those that grow slowly in traditional tube the issues involved with the in-house preparation of shell vials, cultures) is described in “Centrifugation-Enhanced Inocula- tubes, or cluster plates. Virology laboratories that prepare cell tion and Pre-CPE Detection of Viruses in Cell Cultures” be- culture material in-house could prepare cryopreserved cells, if low. The inoculated vials take up much less space in incubators desired. However, many clinical virology laboratories today rely and are not usually incubated in rotating or rolling racks, as is solely on commercial vendors for all their cell culture products. In done with traditional cell culture tubes. The cost of cell cul- response to the need for cryopreseved cells, Diagnostic Hybrids, tures in shell vials is the same as that of cell cultures prepared Inc. (Athens, OH) offers frozen preparations of a number of types in traditional cell culture tubes (Table 1). of cells. These are named Frozen FreshCells. The suspensions Cell monolayers are also available in microwell plates, some- for clinical virology applications are produced with cells at times called “cluster plates.” The plates feature flat-bottomed densities suitable for making monolayers that grow to conflu- wells. The number of wells in a cluster plate may vary, ranging ence within 4 days from planting. The virology laboratory from 24 to 96, depending on the purpose for which it is used. maintains these cell mixtures in the frozen state; the cells have A 24-well configuration is very popular, but even with the an extended shelf life when stored according to the manufac- 24-well configuration, the user can determine how many of the turer’s instructions. When there is a need for additional cell wells will actually contain cell monolayers; anywhere between cultures or for a type of cell culture that is not routinely kept 6 and 24 wells may actually contain cells. The microwell plate on hand in the laboratory, the frozen cell suspensions can be arrangement is convenient to use because the entire plate may thawed and aliquoted into culture tubes or shell vials. Instruc- be positioned on the stage of the microscope for the purpose of tions and feeding medium are provided along with the frozen observing the stained cell preparations in the wells. In addition, cell mixtures. the microwell plate format facilitates centrifugation if desired. Both the shell vial and cluster plate configurations have taken Another application of cryopreservation technology has on a greater role in the laboratory with the advent of the made the routine use and maintenance of prepared cell lines centrifugation-enhanced inoculation and pre-CPE assays as even easier. This technology involves cryopreserved ready-to- well as the development of transgenic and cocultivated cell use cell monolayers grown in shell vials that are shipped on dry Ϫ lines (see “CENTRIFUGATION-ENHANCED INOCULA- ice and stored at 70°C (ReadyCells; Diagnostic Hybrids, Inc.). TION AND PRE-CPE DETECTION OF VIRUSES IN Prior to using these cells, the desired number of frozen vials is CELL CULTURES,” “VIRUS ISOLATION IN COCUL- removed from the freezer and incubated in a 35 to 37°C water TURED CELLS,” and “VIRUS ISOLATION IN TRANS- bath for 4 min. The freeze medium is removed and replaced GENIC CELL LINES” below). The extensive use of cell cul- with cell culture medium supplied by the manufacturer. The tures contained in shell vials and microwell plates allows some clinical sample is then added. Currently, cryopreserved mono- virologists to boast of “totally tubeless” virus isolation. Cross layers of cells that are highly susceptible to chlamydiae (McCoy contamination of cultures is rare but can occur when using ReadyCells), HSV and CMV (Hs27 ReadyCells), and the var- cluster plates (via splashing from well to well) and with shell ious viral respiratory pathogens (R-Mix ReadyCells) are avail- vials (from cap to cap) (140). Therefore, technical procedures able. In comparison studies, these frozen monolayers per- should be monitored and enforced to prevent carryover during formed with sensitivity comparable to that of standard cell both sample inoculation and detection manipulation. cultures for the detection of HSV and influenza A and B viruses (74). In addition, cryopreserved cells were shown to have the following benefits (74): (i) they retain the same level CRYOPRESERVED CELL CULTURES of sensitivity and are stable for up to 4 months when stored at Although some diagnostic laboratories continue to prepare Ϫ70°C under proper storage conditions, (ii) they are always on their own cell cultures in-house, many buy all cell cultures from hand for variable volumes of test requested, (iii) the purchase commercial sources. Multiple cell lines are kept on hand in the of a large number at the same time reduces interlot variations virology laboratory to accommodate isolation of the common in testing and eliminates the costs and stresses involved with human viral pathogens. Purchased cell cultures are routinely shipping, (iv) a new lot of cells can be subjected to quality shipped to the virology laboratory once or twice each week, control procedures prior to being put into use, and (v) tighter 56 LELAND AND GINOCCHIO CLIN.MICROBIOL.REV. inventory control is possible because cell cultures are used only that involved low-speed centrifugation at the time of inocula- when needed. tion of the vials and ended, after a brief incubation period, with detection of viral antigen in the monolayer cells by staining of CENTRIFUGATION-ENHANCED INOCULATION AND the monolayer with MAbs to early CMV proteins. They re- PRE-CPE DETECTION OF VIRUSES IN ported detection in 16 to 24 h of 90% of CMV-positive urine CELL CULTURES cultures. Others further investigated the technique for CMV detection in various types of specimens (91, 120, 129) and for Incubation of tube cell cultures in rotating or rolling racks CMV quantitation in peripheral blood granulocytes (19), gen- has been shown to enhance viral replication (102). HSV tube erally showing significantly more positive samples detected in cultures rolled at 2 rpm or 96 rpm showed CPE faster than shell vials than in cell cultures. Sensitive and rapid CMV de- tube cultures incubated in stationary racks. Tube cultures tection in shell vials was seen in specimens from most body rolled at 2 rpm had a 2.4-fold increase in HSV foci over sites; however, of concern was the 25% of positive blood sam- stationary tube cultures, and those rolled at 96 rpm had a ples detected by traditional tube culture alone (120). This 6.8-fold increase in foci over stationary tube cultures (102). study and others have prompted investigators to suggest that if The knowledge that movement enhances viral proliferation culture alone is to be used, both shell vial and conventional may have spurred interest in investigating the use of centrifu- tube culture systems must be used for optimal CMV recovery gation to enhance infectivity in cell culture systems. (103, 104, 120). Originally used to enhance the isolation of Chlamydia Centrifugation inoculation and pre-CPE detection by IF trachomatis, a technique featuring cell monolayers grown on staining were rapidly adapted for use with viruses other than 12-mm round coverslips in 1-dram shell vials was adapted for CMV. By changing the cell line grown on the coverslip and the use in virus isolation. Inoculation of a shell vial involves de- specificity of the MAbs used for staining, detection of other canting the medium and placing the processed clinical sample viruses was easily facilitated. HSV (63), influenza virus (45, directly on the monolayer. The entire inoculated vial is spun in 130), mumps virus (56), various respiratory viruses (108, 117, the centrifuge at low speed (700 ϫ g) for an hour, fresh culture 126), enteroviruses (155), adenoviruses (155), dengue virus medium is then added, and the vials are incubated at 35 to (136), and VZV (15, 161) have been isolated in shell vials. 37°C for the desired time period in an upright position with the Recent studies by Landry et al. (85) demonstrated the ability cell monolayer covered by the cell culture medium Although to detect hMPV by day 2 postinoculation in A549, HEp-2, the shell vial monolayer may be examined microscopically and LLC-MK2 shell vials when stained with a MAb (MAb for CPE with the inverted microscope, detection of viral 8510; Chemicon International, Temecula, CA) to hMPV infection is usually performed at a designated time interval, matrix protein. and the detection method does not rely on CPE production Human diploid foreskin fibroblast cells inoculated with cen- by the virus. This pre-CPE detection routinely involves trifugation-enhanced inoculation, incubated overnight, and staining of the infected monolayer with horseradish peroxi- stained with HRP-labeled MAbs against HSV type 1 (HSV-1) dase (HRP)- or FITC-labeled MAbs of the desired specificity and HSV-2 produced stained plaques of infected cells that to detect viral antigen in the infected cells. The coverslip may were large enough to be detected with the naked eye (168). be removed from the vial and stained (32), or staining may be This method of detecting HSV-positive results was as sensitive carried out while the coverslip remains in the vial (90). Stained as that of observing for CPE for 10 days and typing by enzyme coverslips are mounted on a microscope slide. Examination immunoassay (EIA). The HRP staining method often yielded with the light (for HRP-labeled MAbs) or fluorescence (for results within less than 24 h of inoculation, whereas standard FITC-labeled MAbs) microscope follows. This system typically isolation based on CPE required an average of 3 to 4 days after speeds virus detection dramatically, compared with virus iso- inoculation for detection of a positive result. Centrifugation lation in the traditional tube cell culture system. The exact cultures of MRC-5 and primary rabbit kidney cells stained mechanism by which centrifugation enhances the rate of viral after 16 to 24 h of incubation with a direct IF stain or an infectivity is not known. Although it was initially assumed that indirect HRP stain were compared with standard tube cell accelerated infectivity resulted from forcing bits of virus-in- cultures of MRC-5 and primary rabbit kidney incubated for 7 fected material against the monolayer, it is reported that the days for detection of HSV (121). The tube cell cultures had the stressing of the monolayer cells by centrifugal force is the best isolation rate, detecting more positive samples than any of important factor (75). This has been shown to increase cell the centrifugation culture-stain combinations. However, the proliferation, decrease cell generation times, activate genes, primary rabbit kidney cells stained with HRP stain were the alter cell metabolism, and increase cell longevity. most sensitive combination, and the indirect HRP stain was The shell vial system was initially adapted for use in virology more sensitive than the direct IF stain for both types of the in an attempt to speed CMV isolation. In traditional tube cell centrifugation-enhanced cell cultures. culture tubes, CMV is slow to produce CPE, typically requiring Through the use of centrifugation inoculation and pre-CPE 10 to 30 days of incubation before CPE is detectable. This detection methods, virus isolation in cell cultures has been extremely slow proliferation of CMV in cell cultures, coupled accelerated and enhanced. Some advantages of this cell culture with the increasing interest in CMV disease in the rapidly approach include the following: (i) viruses that replicate poorly growing population of immunocompromised patients, was the and may require subsequent passages in tube cell cultures catalyst that spurred investigation of approaches to speed before detection can be definitely identified at the end of the CMV detection. Gleaves and colleagues (62) used MRC-5 initial incubation period in the shell vial, (ii) the time to pos- cells grown on coverslips in shell vials and pioneered a method itivity is shorter than that required for most traditional tube VOL. 20, 2007 ROLE OF CELL CULTURE IN VIRUS DETECTION 57

FIG. 3. Immunofluorescence detection of respiratory pathogens in R-Mix cells. (A) Uninoculated R-Mix cells; (B) adenovirus; (C) influenza A; (D) influenza B virus; (E) parainfluenza virus type 1; (F) parainfluenza virus type 2; (G) parainfluenza virus type 3; (H) RSV. Magnification, ϫ170. Photos courtesy of Diagnostic Hybrids, Inc. cultures, (iii) this approach may detect viruses that would not UV filter. This assay produced sensitivities of 93.8% for ade- replicate in tube cell cultures, and (iv) the assays are relatively novirus, 88.9% for CMV, and 100% for HSV compared to easy to perform and less subjective (require less expertise than individual tube cell cultures performed in parallel (17). reading for CPE). Currently, several commercially produced cocultivated cell As with other cell culture systems, the shell vial system is lines are available (Diagnostic Hybrids, Inc.) for the rapid effective in virus isolation only when specimens are collected, identification of a variety of viruses. The R-Mix rapid cell transported, and stored properly to maintain the viability of culture technique uses patented cell monolayers of mixed cells the viruses. Although processing and reading of shell vials are selected for their ability to isolate a variety of viruses that cause time-consuming and labor-intensive, results for the detection respiratory infections. R-Mix is comprised of A549 and mink of the most common viruses are available within 24 to 48 h. lung (Mv1Lu) cells and is available as ready-to-use fresh cells Hence, this adaptation of cell culture technology has enabled in shell vials or cluster plates or as frozen cell suspensions that viral isolation in cell culture to provide a timely diagnosis that can be aliquoted by the laboratory (R-Mix Frozen FreshCells) in many cases is useful for effective patient management. This or purchased frozen as monolayers in shell vials (R-Mix Ready- approach has been applied with cocultured cells (see “VIRUS Cells) (see “CRYOPRESERVED CELL CULTURES” above). ISOLATION IN COCULTURED CELLS” below) and with Three R-Mix vials are inoculated for each clinical specimen in transgenic cell lines (see “VIRUS ISOLATION IN TRANS- combination with a proprietary Refeed medium (Diagnostic GENIC CELL LINES” below). Because centrifugation cul- Hybrids, Inc.). The vials are then centrifuged and incubated at tures are routinely blind stained for a specific virus or viruses at 37°C in 5% CO . After 18 to 24 h of incubation, one R-Mix a designated time interval rather than evaluated for CPE, only 2 monolayer is stained for the presence of viral antigens by using the viruses sought will be detected, and unanticipated viruses a pool of fluorescein-labeled MAbs directed against influenza will be missed. A virus; influenza B virus; parainfluenza virus types 1, 2, and 3; adenovirus; and RSV (Fig. 3). If a specimen is positive, a VIRUS ISOLATION IN COCULTURED CELLS second R-Mix monolayer is scrapped and cell spots made. The Techniques involving combinations of different cell types cell s