Virology 277, 316–329 (2000) doi:10.1006/viro.2000.0594, available online at http://www.idealibrary.com on

Cloning of Human Genomic Segments and Development of an RT-PCR Detection Assay

Blair I. Rosen,*,†,1 Zhao-Yin Fang,‡ Roger I. Glass,* and Stephan S. Monroe*,2

*Viral Section, Respiratory and Enteric Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333; †Department of Veterans Affairs, Atlanta Research and Education Foundation, Decatur, Georgia 30033; and ‡Enteric Branch, Institute of Virology, Chinese Academy of Preventive Medicine, Beijing, China 100052 Received May 1, 2000; returned to author for revision June 15, 2000; accepted August 4, 2000

Nearly full-length genomic segments 2 and a partial-length genomic segment 1 of human picobirnavirus were cloned and sequenced. The clones were derived from viruses obtained from human immunodeficiency virus (HIV)-infected patients in Atlanta, Georgia (strains 3-GA-91 and 4-GA-91) and a nonHIV-infected person from China (strain 1-CHN-97). The picobirna- virus genomic segments lacked sequence similarities with other viral sequences in GenBank and EMBL. Comparison of genomic segment 1 from a human and a rabbit picobirnavirus identified a region of 127 nucleotides with 54.7% identity. The genomic segments 2 of the 4-GA-91 and 1-CHN-97 strains had 41.4% nucleic acid identity and 30.0% amino acid similarity and contained amino acid motifs typical of RNA-dependent RNA polymerase genes. Reverse transcription-PCR detection assays were developed with primers targeted to the genomic segments 2 of strains 4-GA-91 or 1-CHN-97. related to the China strain were the predominant viruses detected in stool samples from people in four countries on three continents. Picobirnaviruses were detected in samples from two outbreaks of gastroenteritis in long-term elder care facilities but were not determined to be the primary pathogen. Our findings support the view that picobirnaviruses constitute a distinct family of viruses. © 2000 Academic Press

INTRODUCTION ens (Leite et al., 1990; Alfieri et al., 1988), pigs (Chasey, 1990; Gatti et al., 1989; Ludert et al., 1991; Pongsuwanna Picobirnavirus is the name ascribed to a recently re- et al., 1996), calves (Vanopdenbosch and Wellemans, ported group of unclassified viruses that are relatively small, 35 nm in diameter, and have a nondescript virion 1990), and foals (Browning et al., 1991). surface when visualized by electron microscopy. The Methods for detecting the virus are limited to electron buoyant density of the virus in cesium chloride has been microscopy and detection of viral RNA by PAGE and reported in the range of 1.39 to 1.41 g/ml. The picobirna- silver staining. The virus has primarily been detected in virus consists of bisegmented, double-stranded stool samples as coincidental discoveries in surveys for RNA (dsRNA). The estimated size of the segments . Rotavirus is a gastrointestinal pathogen char- ranges from 2.3 to 2.6 and 1.5 to 1.9 kb. acterized by a segmented dsRNA genome with 11 RNA Picobirnavirus was initially discovered by the finding segments. The electrophoretic migration of picobirnavi- of two nucleic acid segments by polyacrylamide gel rus RNA falls within the migration range of rotavirus electrophoresis (PAGE) extracted from a small proportion genomic segments. of specimens during investigations of gastroenteritis in The role of picobirnavirus as a cause of gastroenteritis children (Pereira et al., 1988a). The virus has subse- has not been clearly established. In animals, investiga- quently been detected in fecal samples from domestic tions of the association of picobirnavirus with disease and wild animal species, including rats (Pereira et al., have primarily been reported in studies with swine. In 1988b), giant anteaters (Haga et al., 1999), hamsters surveys for gastroenteritis in swine, picobirnavirus was (Pereira et al., 1988a), guinea pigs (Pereira et al., 1989), detected in 11.6% (Gatti et al., 1989) and 11.1% (Ludert et rabbits (Gallimore et al., 1993; Ludert et al., 1995), chick- al., 1991) of samples analyzed by PAGE. Gatti et al. (1989) reported picobirnavirus detection in 15.3% of animals with diarrhea and 9.6% of animals without diarrhea. In 1 Current address: Wadsworth Center, New York State Department of contrast, Ludert et al. (1991) detected the virus at equal Health, Albany, NY 12201. 2 frequencies among animals with and without diarrhea. To whom correspondence and reprint requests should be ad- The frequency of detection of picobirnavirus in studies dressed at Viral Gastroenteritis Section (MSG04), Centers for Disease Control and Prevention, 1600 Clifton Road NE, Atlanta, GA 30333. Fax: of gastroenteritis in humans has varied in different in- (404) 639-3645. E-mail: [email protected]. vestigations. Pereira et al. (1988a), in the first study de-

0042-6822/00 $35.00 Copyright © 2000 by Academic Press 316 All rights of reproduction in any form reserved. HUMAN PICOBIRNAVIRUS SEQUENCE AND RT-PCR DETECTION 317 scribing the detection of picobirnavirus, reported a fre- care facilities for the elderly in which picobirnavirus was quency of up to 20% in outbreaks of human gastroenter- detected. itis. In a study by Gallimore et al. (1995a), picobirnavirus was detected in 9 to 13% of patients with and without RESULTS diarrhea in various age groups. A lower frequency of detection of picobirnavirus was reported in studies of Cloning and confirmation of picobirnavirus cDNA gastroenteritis in children in Brazil (Pereira et al., 1993) Picobirnavirus RNA was extracted and isolated di- and Italy (Cascio et al., 1996), with a frequency of 0.5 and rectly from stool samples because it has not been pos- 0.43%, respectively. sible to propagate the virus in animal models or cell Picobirnavirus has been detected in several studies of culture. The picobirnavirus-positive stool samples used gastroenteritis in human immunodeficiency virus (HIV)- as a source of viral RNA for cloning were selected on the infected patients. Grohmann et al. (1993) reported the basis of the intensity of the viral RNA in silver-stained detection of several viruses, including , pico- PAGE gels, which provided an indirect measure of the birnavirus, calicivirus, and adenovirus, in stool speci- virus titer. The volume of the stool samples from any one mens from HIV-infected patients. Picobirnavirus was de- person was limited, and only 1.5 to 2 ml was available for tected in 9% of patients with diarrhea and in 2% of extracting template RNA for cloning and for further anal- patients without diarrhea and was the second most com- yses. monly detected virus after astrovirus. In studies in Ar- To confirm the identity of the cloned picobirnavirus gentina, Giordano et al. (1998, 1999) detected picobirna- cDNA products, riboprobes prepared from the recombi- virus at a frequency of up to 14.6% in HIV-infected pa- nant plasmids were hybridized to nucleic acid extracted tients with diarrhea and none in HIV-infected patients from the original picobirnavirus stool samples by RNA without diarrhea or in nonHIV-infected patient groups. In blot or to recombinant plasmids confirmed to contain contrast, in a study in Venezuela, Gonzalez et al. (1998) picobirnavirus genomic segments (data not shown). The detected picobirnavirus in only 2.3% of samples from hybridization reactions confirmed the production of a HIV-infected patients without diarrhea. single cDNA clone from the larger picobirnavirus RNA Although picobirnavirus has been implicated in gas- segment (genomic segment 1) from strain 3-GA-91. Sev- troenteritis in animals and humans, information on the eral independent clones of the smaller picobirnavirus prevalence of the virus and its association with disease RNA segment (genomic segment 2) were produced from has been restricted to research studies in which PAGE both the 4-GA-91 and the 1-CHN-97 picobirnavirus assays were used for analysis of clinical samples. The strains. sensitivity of this assay for the detection of picobirnavi- rus, which has been reported to occur at low titers in Analysis of genomic segment 1 of picobirnavirus many clinical samples, is limited and varies depending A single partial-length clone was obtained from pico- on the procedure used for RNA extraction (Gallimore et birnavirus genomic segment 1 from strain 3-GA-91. The al., 1995a; Giordano et al., 1998). Additional assays with clone was derived from viral RNA extracted from a stool greater sensitivity are needed for detection of the virus in sample of an HIV-infected patient in Atlanta. The full- clinical samples. Although the sequence for the larger length RNA segment was estimated to be 2300 bp on the picobirnavirus RNA segment from a rabbit has been basis of its electrophoretic mobility in a PAGE gel in published (Green et al., 1999), no sequence information comparison with the Wa strain of human rotavirus. The is available for the smaller picobirnavirus RNA segment partial-length clone was 1572 bp and was assigned Ac- or for any strains of picobirnavirus from humans. The cession No. AF246941. Comparisons of the nucleic acid limited sequence information available for this virus has and predicted amino acid sequence with rabbit picobir- prevented the development of alternative assays and has navirus genomic segment 1 (Green et al., 1999) revealed restricted efforts to ascertain the relationship of this virus limited regions of identity. The greatest region of nucleic to established families. In this study, the sequences of a acid identity, 54.7%, was observed between nucleotides partial-length cDNA clone of the larger RNA segment of 670 to 797 and nucleotides 1523 to 1650 of the human one strain of human picobirnavirus and nearly full-length strain 3-GA-91 and rabbit picobirnavirus, respectively. clones of the smaller RNA segment from two different Comparisons of the predicted amino acids revealed an strains of human picobirnavirus are reported. Primers for identity of 29.9% (42.9% similarity) between amino acids reverse transcription-PCR (RT-PCR) detection of the virus 19 to 172 and amino acids 310 to 465 of the human strain were developed and applied for the detection of picobir- 3-GA-91 and rabbit picobirnavirus, respectively. The nu- navirus from HIV-infected and nonHIV-infected patients. cleic acid and deduced amino acid sequences of the In addition, information pertaining to the association of genomic segment 1 of strain 3-GA-91 did not have sig- picobirnavirus with disease was provided by the inves- nificant similarities to any nucleic acid and protein se- tigation of two outbreaks of gastroenteritis in long-term quences in the EMBL and GenBank databases. The 318 ROSEN ET AL. partial nature of the cloned genomic segment 1 pre- vented definitive analysis of the encoded ORFs. One potential ORF extended from nucleotides 1 to 725 at the 5Ј end.

Analysis of genomic segment 2 of picobirnavirus Nearly full-length consensus sequences of genomic segments 2 were derived from five clones of the 4-GA-91 FIG. 1. Alignment of predicted amino acid sequences containing strain of picobirnavirus and four clones of the 1-CHN-97 specific motifs characteristic of RDRP-coding genes identified in the strain of picobirnavirus. The genomic segment 2 clones genomic segments 2 of the 4-GA-91 and 1-CHN-97 picobirnavirus were prepared from picobirnavirus RNA templates iso- strains. The motifs are aligned with the RDRP-coding gene of other dsRNA and ssRNA viruses. The viruses compared were Rota, rotavirus; lated from stool of HIV-infected (for the 4-GA-91 strain) IBDV, infectious bursal disease virus; Polio, poliovirus; FMDV, foot and and nonHIV-infected (for the 1-CHN-97 strain) persons. mouth disease virus; PBV 1-CHN-97, a picobirnavirus strain from China; The consensus sequences of strain 4-GA-91 (1674 bp) and PBV 4-GA-91, a picobirnavirus strain from Atlanta, Georgia. and strain 1-CHN-97 (1696 bp) were in close agreement with the estimated lengths of 1640 and 1730 bp, respec- tively, of RNA segments 2 that were determined by com- respectively. The GDD amino acid sequence that com- parison of viral RNA segment migration in a PAGE gel prised motif 3 was observed between amino acids 378 to with the Wa strain of rotavirus. The nucleotide and de- 382 and amino acids 359 to 363 of the 4-GA-91 and duced amino acid sequences of 4-GA-91 and 1-CHN-97 1-CHN-97 picobirnavirus strains, respectively. were assigned Accession Nos. AF246940 and AF246939, Although picobirnavirus has been detected in stool respectively. The genomic segment 2 of picobirnavirus samples, the ability of picobirnavirus to replicate in the from strain 4-GA-91 contained a single open reading cells of vertebrate hosts has not been confirmed. An frame from nucleotides 90 to 1643 and coded for a atypical picobirnavirus with a similar bisegmented ge- predicted protein of 517 amino acids, with a molecular nome profile, but with smaller genome segments, was mass of 59.6 kDa. The strain 1-CHN-97 genomic seg- reported to be associated with Cryptosporidium (Galli- ment 2 contained a single open reading frame from more et al., 1995b) and raised speculation concerning nucleotides 58 to 1650 and coded for a predicted protein the host of picobirnaviruses. A subsequent study, how- of 530 amino acids, with a molecular mass of 60.3 kDa. ever, comparing the sequence of genomic segment 1 of The 5Ј noncoding regions of both virus strains were AT rabbit picobirnavirus with atypical picobirnaviruses de- rich, and A and T nucleotide residues made up 82.9 and termined these viruses to be unrelated (Green et al., 74.1% of the nucleotides in the 5Ј noncoding region of 1999). The genomic segment 2 sequences of the 4-GA-91 4-GA-91 and 1-CHN-97 strains, respectively. In addition, and 1-CHN-97 strains were compared with those of the a distinctive nucleotide sequence consisting of the RDRP-coding genes of a cluster of noninfectious dsRNA bases TTTCTT was observed in both strains and was viruses of lower eukaryotes and that of a plant virus with located at nucleotides 16 to 21 and 13 to 18 of the a related RDRP-coding gene. The viruses selected for 4-GA-91 and 1-CHN-97 strains, respectively. The comparison included the pathogenic fungus virus Usti- genomic segments 2 of the picobirnavirus strains had lago maydis virus, the yeast virus Saccharomyces cer- 41.4% nucleic acid identity and 30.0% amino acid similar- evisiae virus, the protozoan virus Trichomonas vaginalis ity. virus, and the plant virus beet cryptic virus. This group of Comparison of the nucleic acid and deduced amino viruses has been described as a distinct monophyletic acid sequences of the viral genomic segments with se- group and, although they consist of viruses from different quences in the EMBL and GenBank databases did not phylogenic families, possess eight conserved motifs in reveal extensive homologies with other known organ- their RDRP-coding gene that are similar and that are isms. However, three motifs and a conserved residue unrelated to the RDRP-coding genes of other dsRNA common among the RNA-dependent RNA polymerases viruses of vertebrates. Analysis of the 4-GA-91 and (RDRPs) of dsRNA and some single-stranded RNA vi- 1-CHN-97 gene 2 sequences by several alignment meth- ruses were identified (Fig. 1) (Bruenn, 1991; Cohen et al., ods did not reveal any significant conservation of RDRP- 1989). Motif 1 was observed between amino acid resi- specific motifs between picobirnavirus and this mono- dues 259 and 269 of both strains of picobirnavirus. The phyletic group (data not shown). amino acid sequence SG—T characteristic of motif 2, Development of an RT-PCR assay and screening of which has been reported to be the most highly con- samples from HIV-infected patients served region of the RDRP (Bruenn, 1991), was observed between amino acids 318 to 332 and amino acids 320 to The strategy used for the selection of primer pairs was 334 of the 4-GA-91 and 1-CHN-97 picobirnavirus strains, based initially on identifying similarities between the HUMAN PICOBIRNAVIRUS SEQUENCE AND RT-PCR DETECTION 319

TABLE 1 Sequence of Primers Used for Reverse Transcription-PCR Detection of Picobirnavirus

Primer Sample derived from Polarity Nucleotides Sequence

PicoB23 4-GA-91 ϩ 685–699 CGG TAT GGA TGT TTC PicoB24 4-GA-91 Ϫ 1039–1053 AAG CGA GCC CAT GTA PicoB25 1-CHN-97 ϩ 665–679 TGG TGT GGA TGT TTC PicoB43 1-CHN-97 Ϫ 850–865 A(GA)T G(CT)T GGT CGA ACT T

genomic segments 2 of the 4-GA-91 and 1-CHN-97 virus Investigation of picobirnavirus in elder care facilities strains on the basis of the predicted amino acid se- Picobirnavirus was identified in stool samples from quence. The primer design was modified after additional outbreaks of gastroenteritis in two elder care facilities in sequence information was acquired from other strains of Florida. The samples were identified during the develop- picobirnavirus. The primers selected were PicoB23 and ment of the RT-PCR assay by the inclusion of nucleic acid PicoB24 for the 4-GA-91 strain and PicoB25 and PicoB43 from outbreak samples for use as controls. The outbreak for the 1-CHN-97 strain (Table 1). samples were previously submitted to our laboratory for The primers from the 1-CHN-97 strain had the broad- screening and characterization of other enteric viral est reactivity. Amplicons of the expected size were pro- pathogens as part of our department’s routine diagnostic duced from picobirnavirus-positive stool samples of the investigations of diarrheal outbreaks in response to re- nonHIV-infected person from China from whom the clone quests by State health departments and other agencies. was derived, and HIV-infected patients in Atlanta (two The samples were analyzed by RT-PCR assay with prim- individuals other than the patient from whom the strain ers derived from the 4-GA-91 and 1-CHN-97 picobirnavi- 4-GA-91 was derived), one individual from Argentina, and rus strains (Fig. 4). Nucleic acid bands of the expected two individuals from Venezuela (Fig. 2A). An RT-PCR size were observed only with primers derived from the product was also produced from a synthetic China pico- 1-CHN-97 picobirnavirus strain and were detected in one birnavirus genomic segment 2. The RT-PCR bands were of five samples in Outbreak 451 and four of seven sam- confirmed to be of picobirnavirus origin by sequence ples in Outbreak 453. Prior to this study, the outbreak analysis (Fig. 3). There was 100% sequence identity be- samples were assayed for Norwalk-like viruses by an tween the RT-PCR products produced from the stool RT-PCR assay and genogrouped as previously described specimen from China and the synthetic China genomic (Fankhauser et al., 1998). In Outbreak 451, three of five segment 2. In addition, the sequences of both RT-PCR samples were positive for Norwalk-like virus and in Out- products had 100% sequence identity with the consen- break 453, six of seven samples were positive for Nor- sus sequence of the China picobirnavirus genomic seg- walk-like virus. In both outbreaks, samples positive for ment 2. picobirnavirus were also positive for Norwalk-like virus The primers derived from the 4-GA-91 strain pro- (Table 2). duced RT-PCR products of the expected size with viral Sequence analysis was done for the RT-PCR products nucleic acid extracted from three stool samples (4-GA- produced with the picobirnavirus-specific primers (Fig. 91, 5-GA-91, and 6-GA-91) of an Atlanta patient from 3). Picobirnavirus was confirmed in one sample in Out- whom the clone was derived and a homologous syn- break 451 and in three of four samples in Outbreak 453. thetic genomic segment 2 (Fig. 2B). The three samples The three samples in Outbreak 453 differed in nucleic were collected from one patient separated by a 3- or acid sequence by 24 to 38%. The predicted phylogenetic 4-month interval, as reported in the study of Grohmann relationships of picobirnaviruses were determined on et al. (1993). Sequence analysis of the PCR products the basis of nine partial-length genomic segment 2 se- did not detect any sequence changes during the pro- quences for picobirnaviruses from Outbreaks 451 and longed shedding of this virus (data not shown). The 453 from the elder care facilities in Florida with picobir- sequence of the RT-PCR products from all three sam- navirus strains from HIV-infected persons from the ples had 100% sequence identity with the sequence of United States and Argentina and a nonHIV-infected per- the cloned genomic segment 2 of 4-GA-91. Amplicons son from China (Fig. 5). No picobirnaviruses of one se- larger or smaller than expected were observed with quence type were observed to infect HIV-infected per- some clinical samples, e.g., 2-VEN-96 and primers sons as opposed to persons not infected with HIV. The PicoB23 and PicoB24. Sequence analysis of these picobirnavirus strain 2-GA-91 collected from an HIV-in- products confirmed them to be of bacterial or other fected patient in Atlanta was determined to be more heterologous origin. closely related to a picobirnavirus strain from Argentina FIG. 2. Detection of picobirnavirus RT-PCR products produced from clinical samples collected from people from four countries. All of the clinical samples were obtained from HIV-infected persons with the exception of the samples from China and Florida. The same set of samples was reacted with separate pairs of primers in A and B. (A) Results using primers PicoB25 and PicoB43 derived from the 1-CHN-97 strain of picobirnavirus. Samples 4-GA-91, 5-GA-91, and 6-GA-91 were collected from the same patient several months apart. Sample 4-GA-91 was the source of the cloned picobirnavirus genomic segment 2 from an HIV-infected patient from Atlanta. M 123 indicates 123-bp DNA markers (Gibco BRL). Samples 1-CHN-SYN, a synthetic China picobirnavirus genomic segment 2, and 4-GA-SYN, a synthetic Atlanta picobirnavirus genomic segment 2, were used as positive controls for primers derived from the China strain and Atlanta strain, respectively. Samples 101-FL-97 and 301-MO-98 were used as negative controls. (B) Results using primers PicoB23 and PicoB24 derived from the 4-GA-91 strain of picobirnavirus. The samples tested were identical to the samples in A. HUMAN PICOBIRNAVIRUS SEQUENCE AND RT-PCR DETECTION 321

FIG. 3. Sequence of selected RT-PCR products produced from picobirnavirus-positive stool samples. The samples were reverse-transcribed and PCR-amplified using primers PicoB25 and PicoB43 derived from the 1-CHN-97 strain of picobirnavirus. The sequence of the RT-PCR products produced from the samples from Venezuela (1-VEN-97 and 2-VEN-97) were not included because of multiple nucleic acid sequences contained within the samples. The sequence of the cloned genomic segment 2 of the 4-GA-91 strain was included for comparison and contained an insert not found in the other strains. No other gaps, other than that needed for alignment with the 4-GA-91 insert, were inserted into the sequences of the RT-PCR products produced with primers PicoB25 and PicoB43. A gap 6 nucleotides in length, between residues 161 and 168, was inserted into the sequence of 4-GA-91 to maintain alignment with the other strains based on the deduced amino acid sequences. than to strain 1-GA-91, which was collected in the same sequence by 3 to 44% (3% difference observed between geographic area as strain 2-GA-91. The eight picobirna- 2-GA-91 and 1-ARG-97; 44% difference observed be- virus strains related to 1-CHN-97 differed in nucleic acid tween 2-GA-91 and 202-FL-97). The predicted amino acid 322 ROSEN ET AL.

FIG. 4. Analysis in an agarose gel of RT-PCR products produced from samples from two outbreaks of gastroenteritis in long-term elder care facilities in Florida. The RT-PCR products were produced with primers PicoB25 and PicoB43 derived from the 1-CHN-97 strain of picobirnavirus. M 123 and 1-CHN-SYN indicate 123-bp DNA markers (Gibco BRL) and a synthetic China picobirnavirus genomic segment 2 used as a positive control, respectively. sequence of the eight picobirnavirus strains differed by 2 mans. Current methods for detection have been limited to 46% (a 2% difference was observed between 2-GA-91 to extraction of viral RNA from stool samples and frac- and 1-ARG-97; a 46% difference was observed between tionation and staining in PAGE gels. Although the phys- 1-CHN-97 and strains 104-FL-97 and 202-FL-97). Strain ical properties of the virus have been described, efforts 4-GA-91 was determined to be phylogenetically distinct to further characterize the viral genome have been hin- from other picobirnavirus strains. This observation was dered because of the inability to propagate the virus in supported by the difference in nucleic acid sequence cell culture or in animals and because of the generally between the cloned genomic segments 2 of 4-GA-91 and low titers of the virus in stool samples. In this study, a 1-CHN-97 and the restricted reactivity of RT-PCR primers procedure was developed for the direct isolation of indi- derived from strain 4-GA-91. The nucleic acid sequence vidual picobirnavirus RNA segments from stool samples of picobirnavirus strain 4-GA-91 differed from that of all in a PAGE gel. Polyacrylamide gels were selected be- other picobirnavirus strains by 60 to 63% and its pre- cause of their ability to sufficiently fractionate the viral dicted amino acid sequence differed from that of all other RNA from other nucleic acids in the stool samples and picobirnavirus strains by 72 to 83%. the greater clarity of the gel matrix compared with aga- Relationship with other virus families rose gels for visualization of faint nucleic acid bands. Ligation, reverse transcription, and PCR amplification of Comparisons of the physical characteristics and RDRP the viral RNA were performed by a modification of a gene coding assignments of picobirnavirus with other protocol previously described by Lambden et al. (1992). families of 1- to 3-segmented dsRNA viruses indicates This procedure enabled the production of cDNA from that picobirnaviruses are distinct (Table 3). very small amounts of picobirnavirus RNA template. The clones prepared in our study included a single DISCUSSION partial-length genomic segment 1 of one strain of human Picobirnavirus has been detected in many epidemio- picobirnavirus (3-GA-91) and four clones (1-CHN-97) and logic investigations of gastroenteritis in animals and hu- five clones (4-GA-91) of the genomic segment 2 of two HUMAN PICOBIRNAVIRUS SEQUENCE AND RT-PCR DETECTION 323

TABLE 2 Stool Samples Used as a Source of Picobirnavirus RNA for Cloning or Analyzed by RT-PCR Assay for Picobirnavirus

Reaction with primerse Year IDa Country isolated HIVb Copathogenc PAGEd CHN GA

1-GA-91 USA 1991 ϩ Cryp, Micro ϩϩϪ 2-GA-91 USA 1991 ϩ Cryp, Micro ϩϩϪ 3-GA-91 USA 1991 ϩ Cryp, Micro ϩϪϪ 4-GA-91 USA 1991 ϩ Cryp ϩϪϩ 5-GA-91 USA 1991 ϩ Cryp, Micro ϩϪϩ 6-GA-91 USA 1991 ϩ Cryp ϩϪϩ 1-ARG-97 Argentina 1997 ϩ — ϩϩϪ 1-CHN-97 China 1997 Ϫ — ϩϩϪ 1-VEN-96 Venezuela 1996 ϩ Cryp ϩϩϪ 2-VEN-96 Venezuela 1996 ϩ Cryp ϩϩϪ 3-VEN-96 Venezuela 1996 ϩ Cryp ϩϪϪ 101-FL-97 USA 1997 Ϫ NLV ND ϪϪ 102-FL-97 USA 1997 Ϫ NLV ND ϪϪ 103-FL-97 USA 1997 Ϫ —NDϪϪ 104-FL-97 USA 1997 Ϫ NLV ϪϩϪ 105-FL-97 USA 1997 Ϫ —NDϪϪ 201-FL-97 USA 1997 Ϫ NLV ND ϪϪ 202-FL-97 USA 1997 Ϫ NLV ND ϩϪ 203-FL-97 USA 1997 Ϫ NLV ϩϩϪ 204-FL-97 USA 1997 Ϫ — Ϫ (ϩ)f Ϫ 205-FL-97 USA 1997 Ϫ NLV ND ϪϪ 206-FL-97 USA 1997 Ϫ NLV ND ϪϪ 207-FL-97 USA 1997 Ϫ NLV ϪϩϪ 301-MO-98 USA 1998 Ϫ NLV ND ϪϪ

a The last four or five characters of each sample identification indicate the state or country and the year of collection. b Indicates the HIV infection status of persons from whom the stool was obtained. (ϩ) indicates infected with HIV. (Ϫ) indicates not infected with HIV. c Pathogens detected in the stool samples other than picobirnavirus. Cryp, cryptosporidia. Micro, microsporidia. NLV, Norwalk-like viruses. d Observation of picobirnavirus RNA segments in silver-stained polyacrylamide gels (PAGE). (ϩ) indicates RNA segments were observed. (Ϫ) indicates RNA segments were not observed. (ND) indicates electrophoresis analysis was not done. e Production (ϩ) or absence (Ϫ) of RT-PCR products of the expected size with primers derived from CHN, the 1-CHN-97 strain of picobirnavirus, and GA, the 4-GA-91 strain of picobirnavirus. f Sequence analysis did not confirm this RT-PCR band to be of picobirnavirus origin. other strains of human picobirnavirus. As with other despite the theoretical potentials of the technique. Fur- cloning techniques, limitations in the ability to clone ther optimization of the procedure may enable the clon- relatively large intact nucleic acid molecules, including ing of nucleic acid segments of similar or greater sizes. picobirnavirus genomic segment 1, were encountered It should be noted that the protocol used in our study,

FIG. 5. Dendrogram of predicted phylogenetic relationship among picobirnavirus strains based on 191 nucleotides of the genomic segment 2 sequence. The scale bar represents 5 substitutions per 100 nucleotide residues. 324 ROSEN ET AL.

TABLE 3 Comparison of RDRP Genomic Coding Assignment and Physical Characteristics of Picobirnaviruses with Other Known One-, Two-, and Three-Segmented dsRNA Viruses

Genomic segment coding for RNA- dependent RNA Virion density Enveloped Number of polymerase Virus diameter in CsCl virus genomic Genomic (multisegmented Virus Hosts infected (nm) (g/ml) particles segments segment length viruses)

Birnaviridae Vertebrates; 60 1.32–1.35 No 2 3,100–3,200 bp Smaller genomic insects; 2,750–2,850 bp segment molluscs Fungi; protozoa 30–40 1.40–1.43 No 1ϩa 4,600–7,000 bp NA Fungi; plants 30–40 1.34–1.39 No 2ϩb 1,400–3,000 bp; Larger genomic the 2 segments segment of individual viruses are usually of similar size Hypoviridae Fungi 50–80 pleomorphic 1.27–1.3 Yes 1ϩa 10,000–13,000 bp NA vesicles (no true ) Cystoviridae Bacteria 86 1.27 Yes 3 6,374 bp Larger genomic 4,057 bp segment 2,948 bp Unclassified Protozoa RNA exists naked, no Unknown Unknown 2 1,786 bp Larger genomic dsRNA virus of virus particles have 1,374 bp segment Cryptosporidium been associated parvum with the nucleic acid Unclassified Vertebrates 32–35 1.4 No 3 2,900 bp Unknown Picotrirnavirus 2,400 bp 900–1,320 bp Unclassified Vertebrates 35–41 1.39–1.40 No 2 2,300–2,600 bp Smaller genomic Picobirnavirus 1,500–1,900 bp segment

Note. NA, not applicable. a Virus particles may contain additional defective dsRNAs. Satellite virions also occur. b The virus genome consists of two unrelated linear dsRNA segments that are separately encapsidated. Some virus isolates contain additional (three to four total) segments of dsRNA and are tentatively considered satellite or defective dsRNAs. which was an adaptation of the protocol of Lambden et (Leite et al., 1990; Pereira, 1991). The sequence of al. (1992), was originally used to clone the smaller human genomic segment 1 of a rabbit strain of picobirnavirus group C rotavirus genomic segment 10, a molecule of was recently reported by Green et al. (1999). The lack of 728 bp. The consensus sequences of the cloned identifiable motifs led to the postulation that genomic genomic segments 2 of both picobirnavirus strains were segment 1 of picobirnavirus contains the gene coding for similar in length to the estimated size of the correspond- the capsid protein. Our studies on human picobirnavirus ing viral RNA segments 2 as determined in PAGE gels. indirectly corroborate these findings based on the iden- However, the small volumes of clinical samples available tification of RDRP motifs in genomic segment 2. Compar- prevented further studies aimed at determining the se- ison of the partial-length genomic segment 1 of human quences at the precise ends of the viral genomic seg- picobirnavirus strain 3-GA-91 and rabbit picobirnavirus ments 2. determined a limited region of 127 nucleotides with Comparisons of the nucleic acid and predicted amino 54.7% identity. The genetic diversity between picobirna- acid sequences of the human picobirnavirus strain viruses within a single animal species and viruses in- 3-GA-91 genomic segment 1 with sequences in EMBL fecting different species is currently unknown. The re- and GenBank did not reveal any significant similarities. gion of nucleic acid similarity identified in this study may The lack of sequence similarities was not unexpected possibly code for a common antigenic region on the because of the unique characteristics of the picobirna- picobirnavirus capsid. Other regions of the genome not virus genome pertaining to the number of RNA seg- contained in our partial-length clone may also be in- ments, dsRNA composition, and RNA segment size volved in the formation of a common antigen. HUMAN PICOBIRNAVIRUS SEQUENCE AND RT-PCR DETECTION 325

In this study, genomic segments 2 were cloned from tovirus and Betacryptovirus within the Partitiviridae. picobirnavirus strains from an HIV-infected patient from However, viruses of the Alphacryptovirus and Betacryp- Atlanta and a nonHIV-infected person from China. Three tovirus are known to only infect the plant Trifolium re- motifs, characteristic of RDRP-coding genes of single- pens, and the RDRP is coded by the larger genomic stranded RNA and dsRNA viruses, were identified in the segment. The differences in the physical characteristics, genomic segment 2 of both picobirnavirus strains. De- RDRP gene coding assignments, and nucleic acid se- spite the presence of the RDRP functional domains, the quences shown in our study lend support to suggestions RNA segments did not have significant similarities with by other researchers that picobirnaviruses are represen- other sequences in EMBL and GenBank. The RDRP- tative of a distinct virus family. coding genes of dsRNA viruses have been reported to be The RT-PCR assay developed in this study provided variable in regions outside of the conserved motifs greater sensitivity in the detection of picobirnavirus in (Bruenn, 1993). clinical samples compared with analysis of viral RNA in Although picobirnaviruses are generally considered to PAGE gels and silver staining. Direct comparisons be viruses of vertebrates, the pathogenic host of picobir- showed RT-PCR detection to be at least 100 times more naviruses has not been confirmed. Investigations by sensitive than detection by PAGE. Several samples that Green et al. (1999) reported few sequence similarities were negative for picobirnavirus by PAGE analysis (e.g., between genomic segment 1 of a rabbit picobirnavirus 104-FL-97, 204-FL-97, and 207-FL-97 listed in Table 2) and a dsRNA virus of Cryptosporidium. In our study, few were confirmed to be picobirnavirus-positive by the RT- similarities were noted in comparisons of RDRP-specific PCR assay and sequence analysis. The increased sen- motifs of the genomic segment 2 of human picobirnavi- sitivity of the assay was useful in overcoming the prob- ruses with a unique group of closely related dsRNA lem of low virus titers in clinical samples that has been viruses of lower eukaryotes that included viruses of observed in our study and reported by other research yeast, fungi, and protozoa (Bruenn, 1993). Evidence for groups (Gallimore et al., 1995a; Grohmann et al., 1993). picobirnavirus infection of vertebrates has come from Picobirnaviruses related to the 1-CHN-97 strain were several studies. Picobirnaviruses have been detected in the predominant viruses detected in RT-PCR assays of the stools of suckling pigs less than 3 weeks old (Ludert stool samples collected from people in four countries on et al., 1991) and guinea pigs (Pereira et al., 1989), sug- three continents. Primers derived from the 4-GA-91 strain gesting that passive ingestion with food and excretion of produced RT-PCR products only from stool samples col- the virus do not occur and virus replication occurs in lected from a single patient who shed the virus from animals hosts. Although picobirnaviruses have not been which the clone was derived. The strain-specific reactiv- serially propagated in cell culture, picobirnavirus strains ity of the primers, and the nucleic and amino acid se- from guinea pigs have been detected after first-passage quence differences observed between the gene 2 seg- attempts in several different cell types (Pereira et al., ments of the 4-GA-91 and 1-CHN-97 picobirnavirus 1989). Immune responses to picobirnavirus have been strains, suggests that these picobirnavirus strains may reported in studies of infection in pigs and rabbits (Gal- be representative of two distinct genogroups. An unex- limore et al., 1993; Ludert et al., 1991). In an epidemio- pected finding was the extent of genetic diversity within logic survey for enteric viruses in swine, immunoelectron a genogroup, as evidenced by up to 38% sequence microscopy of one stool sample showed evidence of difference between strains amplified by a single primer picobirnavirus immune complexes mediated by IgA (Lud- set. Nucleic acid extracted from two stool samples (e.g., ert et al., 1991). In studies with rabbits, seroconversion of 3-GA-91 and 3-VEN-96 listed in Table 2) determined to be rabbits experimentally infected with picobirnavirus was picobirnavirus-positive by PAGE analysis failed to pro- observed in two rabbits, and two other rabbits were duce RT-PCR bands when amplified with primers derived determined to have antibody titers to picobirnavirus prior from the Atlanta or China strains. This result is sugges- to infection (Gallimore et al., 1993). Further studies using tive of the presence of picobirnaviruses with other gene molecular reagents based on the sequence of picobir- 2 sequence variations. The possibility, however, of dam- navirus are needed to confirm the host and location of age to the integrity of the viral nucleic acid upon storage virus replication. of the virus prior to analysis cannot be ruled out. In this study, the sequences of human picobirnavirus Picobirnaviruses have been associated with gastroen- genomic segments 1 and 2 were compared with those of teritis in several studies of HIV-infected patients (Groh- other known viruses and were determined to be distinct. mann et al., 1993; Gonzalez et al., 1998; Giordano et al., Additional comparisons relating to differences in the 1998, 1999) and in nonHIV-infected people (Pereira et al., physical characteristics and RDRP gene coding assign- 1993; Gallimore et al., 1995a; Cascio et al., 1996). In our ments were made with other known 1- to 3-segmented study, picobirnavirus was detected in stool samples from dsRNA virus families. Viruses that share characteristics several individuals infected with HIV. Sequence analysis with picobirnavirus related to virion particle size and of RT-PCR products from picobirnavirus shed over an genome segment length occur in the genera Alphacryp- interval of several months from one HIV-infected individ- 326 ROSEN ET AL. ual from the study of Grohmann et al. (1993) did not ing Group study (Grohmann et al., 1993; Navin et al., detect any nucleic acid changes. Previous studies have 1999). The patients were part of a cohort study followed reported gene alterations in virus infections of people at three hospitals in Atlanta. Two samples, which were who are immunocompromised (Hundley et al., 1987; Ei- the source for virus strains 3-GA-91 and 4-GA-91, were den et al., 1985). During the course of our study, picobir- confirmed to contain picobirnavirus by PAGE analysis navirus was detected in stool samples from two out- and were selected for use in cloning. A third stool sam- breaks of gastroenteritis at long-term care facilities for ple that was the source for picobirnavirus strain the elderly in Florida. Picobirnavirus-specific products 1-CHN-97 from China was also used as a source of were obtained by RT-PCR only with primers derived from picobirnavirus for cloning. The Chinese sample was ob- the Chinese strain of picobirnavirus. The RT-PCR prod- tained from an HIV-negative adult with gastroenteritis ucts obtained from three individuals in Outbreak 453 all from Lulong County, China, in 1997 and was confirmed to differed in nucleic acid sequence, indicating the absence contain picobirnavirus by PAGE analysis. of a point source for infection. The detection of Norwalk- Viral RNA preparations used in RT-PCR assays were like viruses of one particular genotype from the same extracted from the following samples: (1) stool samples outbreak samples implicated this virus as the principal from HIV-infected patients from the study of Grohmann et pathogen, with picobirnavirus playing a role as either a al. (1993) that were the source of virus strains 1- to secondary opportunistic pathogen or an innocuous virus 6-GA-91; (2) a stool sample from China containing strain in the intestine. The role of picobirnavirus as a primary 1-CHN-97; (3) a stool sample from an HIV-infected per- pathogen has yet to be established. It is of interest to son from Argentina containing strain 1-ARG-97, submit- note, however, that picobirnavirus isolated from a non- ted by Sylvia V. Nates, Institute of Virology, Faculty of HIV-infected person in China with gastroenteritis in this Medical Sciences, National University of Cordoba, Cor- study was detected at very high titers based on the doba, Argentina; (4) three stool samples, 1- to 3-VEN-96, staining intensity of picobirnavirus RNA in PAGE gels, from HIV-infected persons from Venezuela, submitted by indicating a possible primary association of the virus Juan E. Ludert, Center of Microbiology and Cellular Biol- with disease. ogy, Venezuelan Institute of Scientific Investigations, Ca- The picobirnavirus strains detected in this study were all isolated from adults (26 years of age or older). No racas, Venezuela; (5) stool from Outbreak 451, samples samples were examined from children. Previous surveys 101- to 105-FL-97, collected from residents at a long-term for picobirnavirus by PAGE analyses detected relatively care facility during an outbreak of gastroenteritis in 1997 low percentages in children (Pereira et al., 1993; Cascio in Port Saint Joe, Florida, submitted to the Centers for et al., 1996). Further studies using nucleic acid primers Disease Control and Prevention (CDC); (6) stool from developed in this study along with additional molecular Outbreak 453, samples 201- to 207-FL-97, collected from reagents based on the sequence information presented residents at a long-term care facility during an outbreak are needed to clarify the role of picobirnavirus with of gastroenteritis in 1997 in Sarasota, Florida, submitted disease and its association with people of different age to the CDC; and (7) a stool sample, 301-MO-98, collected groups. from an outbreak of gastroenteritis in 1998 at a nursing home in Versailles, Missouri, and submitted to the CDC. MATERIALS AND METHODS The Wa strain of human rotavirus was propagated in MA-104 cells. The viral RNA was extracted as described Virus samples for picobirnavirus and used for development and optimi- The virus samples investigated in this study were zation of protocols for the production of cDNA and as collected from people in three states and four countries molecular size markers in PAGE and agarose gels. and in four different years (Table 2). The HIV-infection status of the patients from whom the samples were Nucleic acid extraction from stool samples collected and the presence of other enteric pathogens in the stool samples from these patients are indicated. Nine Nucleic acid was extracted from stool samples by a HIV-infected patients were coinfected with cryptospo- modification of the methods of Boom et al. (1990) and ridia and 10 HIV-negative patients were coinfected with Ando et al. (1995). Briefly, 50 ␮l of each stool sample was Norwalk-like viruses. The sample identifications listed in added to 1.5-ml microcentrifuge tubes and mixed with Table 2 were also used as designations for picobirnavi- 450 ␮l of lysis buffer containing 4.7 M guanidine thiocy- rus strains in those samples in which picobirnavirus was anate, 20 mM EDTA, and 1.2% (W/V) Triton X-100. The detected. The results of PAGE analyses and RT-PCR nucleic acid samples were extracted with phenol–chlo- amplifications with primers derived from the cloned pi- roform and incubated with silica. The silica-bound nu- cobirnavirus strains are also shown. cleic acid was washed twice with 0.5 to 1.0 ml of wash Stool samples were obtained from HIV-infected pa- buffer containing 5.3 M guanidine thiocyanate and 0.1 M tients as part of an Enteric Opportunistic Infections Work- Tris, pH 6.4. The pellets were then sequentially washed HUMAN PICOBIRNAVIRUS SEQUENCE AND RT-PCR DETECTION 327 once each with 0.5 to 1.0 ml of 70% ethanol and acetone Sigma Chemical Co., St. Louis, MO). Excess primer was and dried at room temperature for 30 min. The nucleic removed by size-fractionation chromatography with Link- acid was eluted by two sequential incubations with 100 ers 6 Quick Spin Columns (Boehringer Mannheim Corp.) ␮l of DEPC-treated water at 65°C for 15 min. The eluted and ethanol precipitated. The RNA was suspended in 5 nucleic acid from individual samples was pooled, etha- ␮lof5%DMSOand1.5␮M primer PicoB04 (5Ј-TAAGAAT- nol precipitated, suspended in DEPC-treated water, and TCTCTAGAGTCGACGGG-3Ј). The RNA was denatured at stored at Ϫ70°C. 94°C for 5 min and then cooled in ice. Reverse transcrip- tion was performed at 42°C for1hinafinal volume of 25 PAGE for screening stool samples for picobirnavirus ␮l and contained 10 mM DTT, a 500 ␮M concentration of RNA patterns each dNTP, 500 U of MMuLV Superscript II (Gibco BRL, Electrophoresis was performed in PAGE gels by the Grand Island, NY) and the supplied RT buffer diluted to method described by Laemmli (1970) without sodium 1ϫ, 40 U of RNase Inhibitor (Boehringer Mannheim dodecyl sulfate. The samples were resolved in 7.5% poly- Corp.), and the denatured RNA mix. The reaction was acrylamide gels, 8.4 ϫ 7.3 cm by 0.75 mm in a Mini inactivated by incubation at 70°C for 15 min and the RNA Protean II Dual Slab Cell (Bio-Rad, Hercules, CA). A template was digested with RNase H (Boehringer Mann- constant voltage of 150 V was applied for1h45minat heim Corp.). room temperature. Nucleic acid from 100 ␮l of stool The DNA was denatured at 94°C for 5 min and the sample was used for detection of picobirnavirus RNA. plus and minus strands of cDNA were annealed by The gels were stained with silver by the method de- incubation at 65°C for 16 h. The ends of the cDNA were scribed by Bassam et al. (1991). repaired by incubation in a PCR mix containing 10ϫ Buffer II without magnesium (Perkin–Elmer Corp., Foster Isolation of picobirnavirus RNA for cloning City, CA) diluted to a final concentration of 1ϫ, a 200 ␮M concentration of each dNTP, 0.1% Triton X-100, 4 mM Nucleic acid was extracted from 150 to 500 ␮l of stool MgCl , and 2.6 U of Expand High Fidelity Taq (Boehringer for isolation of picobirnavirus RNA. An electrophoresis 2 Mannheim Corp.) at 72°C for 7 min. Primer PicoB04 was tank, central core unit, glass plates, and all accessories for a Mini Protean II Dual Slab Cell were cleaned with added (520 nM final concentration) and PCR was per- abSOLVE (Dupont NEN Research Products, Boston, MA) formed by an initial denaturation step at 94°C for 2 min to minimize RNase contamination prior to use for viral and 40 PCR cycles with reaction conditions as described RNA isolation. Nucleic acid samples were electropho- by Lambden et al. (1992) with the exception of an 8-s resed in 6% polyacrylamide gels without a stacking gel at cycle extension during each elongation step. 150Vfor1h30min. The polyacrylamide gels were prepared with TAE to give a 1ϫ final concentration (1ϫ Cloning ϫ TAEis40mMTrisand1mMEDTA),and1 TAE was The ligation-RT-PCR products were reamplified by used as the electrophoresis buffer. PCR with the reagents and cycling conditions described The polyacrylamide gels were stained with ethidium above. The purified PCR products were digested with bromide and gel fragments containing the picobirnavirus XbaI and ligated into a XbaI-digested pBluescript II RNA gene segments were excised. The gel slices were KS(Ϫ) Phagemid vector (Stratagene, La Jolla, CA). The macerated and incubated in 350 ␮l of elution buffer (10 recombinant plasmids were transfected into Epicurian mM Tris, pH 7.3, 1 mM EDTA, 0.1% SDS) for6hor Coli Sure Competent Cells (Stratagene) by electropora- overnight. The eluted RNA was extracted with phenol: tion. The clones were screened by agarose electro- chloroform:isoamyl alcohol 25:24:1 and ethanol precipi- phoresis for cDNA inserts of the expected size. tated.

Production of picobirnavirus cDNA Confirmation of the identity of the cloned ligation–RT-PCR products The protocol of Lambden et al. (1992) was used for the production of cDNA from dsRNA with several modifica- Nucleic acid was extracted from 100 ␮l of the original tions. The primer PicoB03 (5Ј-CCCGTCGACTCTA- picobirnavirus-positive stool samples that were used as GAGAATTCTTA-3Ј) (9.5 pmol) containing an XbaI restric- a source of viral RNA for cloning. Recombinant plasmids tion site was ligated to the 3Ј ends of the viral RNA, containing picobirnavirus cDNA inserts were digested which ranged from an estimated 10 to 120 ng. The primer with XbaI for use as controls. The nucleic acids were was 5Ј phosphorylated and labeled with digoxigenin at electrophoresed in 7.5% PAGE gels as described above. the 3Ј end. Ligation was performed for 16 h at 22°C in a The gels were stained with ethidium bromide and pho- reaction mixture containing 10 U of T4 RNA ligase tographed adjacent to a fluorescent ruler (Research (Boehringer Mannheim Corp., Indianapolis, IN) and a Products International Corp., Mount Prospect, IL). The final concentration of 25% PEG (polyethylene glycol 8000, nucleic acid was denatured as previously described 328 ROSEN ET AL.

(Tanaka et al., 1988) and electroblotted to nylon mem- RT-PCR assay for picobirnavirus detection in stool branes (Boehringer Mannheim Corp.) at 20 V for 6 h. samples Riboprobes labeled with DIG-11–UTP (Boehringer Mann- Nucleic acid extracted from stool samples by the gua- heim Corp.) were prepared from the cloned picobirnavi- nidine thiocyanate–silica extraction protocol was sus- rus cDNA. Hybridization signals of the riboprobes with pended in a volume of DEPC-treated H2O equivalent to the blotted nucleic acid samples were detected by incu- the volume of stool extracted. The preparations of syn- bation of the blots with anti-digoxigenin-AP Fab frag- thetic picobirnavirus dsRNA were used at a dilution of ments (Boehringer Mannheim Corp.) and chemilumines- 1:2500. One to 3 ␮l of nucleic acid from each sample was cent detection with CSPD (Boehringer Mannheim Corp.). dispensed into 500-␮l siliconized microcentrifuge tubes and dried in a Savant DNA Speed Vac (Savant Instru- Sequencing ments, Inc., Holbrook, NY). The RNA was suspended in 5 ␮l of a primer mix that contained a final concentration of Nucleotide sequencing of both strands of the cloned 5% DMSO and a 1.5 ␮M concentration of each primer. picobirnavirus gene segments and RT-PCR products pro- The nucleic acid primers were designed on the basis of duced with picobirnavirus-specific primers was per- regions of nucleic acid sequence similarity between the formed using an ABI Prism BigDye Terminator Cycle smaller RNA segment (gene 2) of two picobirnavirus Sequencing Ready Reaction Kit (Perkin–Elmer Corp.) and strains (Table 1). an automated sequencer (Applied Biosystems Model Prior to reverse transcription, the RNA was denatured 377, Applied Biosystems, Foster City, CA). Primers com- at 94°C for 5 min and then cooled on ice. Reverse plementary to the T3 and T7 promoters of the pBluescript transcription was performed at 42°C for 1 h in a final II KS(Ϫ) Phagemid vector were used to obtain initial volume of 25 ␮l and contained 10ϫ Buffer II without sequence data of the cloned picobirnavirus gene seg- magnesium (Perkin–Elmer Corp.) diluted to a final con- ments. Subsequent primers for sequencing were based centration of 1ϫ, a 500 ␮M concentration of each dNTP,

on sequence data from the preceding analysis. The prim- 10 mM DTT, 0.1% Triton X-100, 2 mM MgCl2,12.5Uof ers used to sequence RT-PCR products were developed RNase Inhibitor (Boehringer Mannheim Corp.), 6.8 U of to initially produce the products. A consensus sequence AMV Reverse Transcriptase (Boehringer Mannheim from each clone or RT-PCR product was generated using Corp.), and the denatured RNA mix. The reverse tran- the Sequencher program (Gene Codes Corp., Ann Arbor, scription reaction mix was incubated at 70°C for 15 min MI). Sequence analyses and comparisons were per- prior to PCR. formed using the programs contained in the Wisconsin The reagents for PCR were added directly to the mi- Package Version 10.0 (Genetics Computer Group, Mad- crocentrifuge tubes in which reverse transcription was ison, WI). Alignment of pairs or multiple sequences were performed. The reagents added were a PCR mix contain- ␮ ␮ produced using GAP or PILEUP, respectively, and con- ing 25 l of 0.3% Triton X-100, 2 mM MgCl2, 2.6 l each sensus sequences were determined by analysis with of primers PicoB23 (10 ␮M) and PicoB24 (10 ␮M), 2.5 U PRETTY. The diversity of the strains was estimated using of AmpliTaq (Perkin–Elmer Corp.), and 50 ␮l of Master- ϫ the DISTANCES program with the Jukes Cantor method. Amp 2 Premix G (Epicentre Technologies, Madison, A dendrogram was created using the GROWTREE pro- WI). In PCRs using primers PicoB25 and PicoB43, the ␮ gram. PCR mix was modified to contain 2.6 l primer PicoB25 (10 ␮M) and 8.0 ␮l of primer PicoB43 (10 ␮M). The conditions used for PCR amplification were an Preparation of synthetic picobirnavirus dsRNA genes initial denaturation step at 94°C for 2 min followed by 40 Recombinant plasmids containing picobirnavirus cycles of denaturation at 94°C for 1 min, annealing at cDNA were linearized by digestion with SpeIorNotI 49°C for 2 min, and elongation at 72°C for 3 min followed restriction enzymes. The linearized plasmids were ex- by a single incubation at 72°C for 7 min. tracted with phenol:chloroform:isoamyl alcohol and precipitated in ethanol. Single-stranded RNA tran- ACKNOWLEDGMENTS scripts were prepared using T7 or T3 RNA polymerase (Boehringer Mannheim Corp.), digested with DNase B. I. Rosen is a postdoctoral fellow supported by the Atlanta Re- search and Education Foundation, Department of Veterans Affairs, RNase-free (Boehringer Mannheim Corp.), and purified VAMC, Atlanta, Georgia. This research project was supported by funds separately by size-exclusion chromatography with from the Enteric Opportunistic Infections Working Group. We thank CENTRI-SEP columns (Princeton Separations, Inc., Ad- David Brown and co-workers for providing their manuscript and se- elphia, NJ). The purified T7 and T3 RNA transcripts quence prior to publication. We also thank Sylvia V. Nates, Institute of prepared from identical recombinant plasmids were Virology, Faculty of Medical Sciences, National University of Cordoba, Cordoba, Argentina, and Juan E. Ludert, Center of Microbiology and pooled, denatured at 94°C for 6 min, and annealed for Cellular Biology, Venezuelan Institute of Scientific Investigations, Cara- 16 h at 65°C. cas, Venezuela, for the valuable clinical samples they contributed. HUMAN PICOBIRNAVIRUS SEQUENCE AND RT-PCR DETECTION 329

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