LETTERS studies investigating the possibility of 6. Lierz M, Hafez HM, Honkavuori KS, Novel Astroviruses vertical transmission by ABV-infected Gruber AD, Olias P, Abdelwhab EM, et al. Anatomical distribution of avian in Children, Egypt pairs, especially to minimize the risk bornavirus in parrots, its occurrence in for such transmission to endangered clinically healthy birds and ABV-antibody To the Editor: Human astrovi- species with restricted breeding detection. Avian Pathol. 2009;38:491–6. ruses (HAstVs) are a common cause opportunities. doi:10.1080/03079450903349238 7. Gancz AY, Kistler AL, Greninger AL, of gastroenteritis in children, the el- Farnoushi Y, Mechani S, Perl S, et al. derly, and immunocompromised per- Acknowledgment Experimental induction of proventricular sons (1). Up to 10% of acute viral gas- We thank the Loro Parque Fundación, dilatation disease in cockatiels (Nymphi- cus hollandicus) inoculated with brain troenteritis in children and 0.5%–15% Tenerife, Spain, for fi nancial support homogenates containing avian bornavirus of diarrheal outbreaks are attributed (LPF-Project no.: PP-65-2010-1). 4. Virol J. 2009;6:100. doi:10.1186/1743- to astroviruses (2). Until 2008, eight 422X-6-100 classical astrovirus serotypes were 8. Gray P, Hoppes S, Suchodolski P, Mirhos- Michael Lierz, seini N, Payne S, Villanueva I, et al. Use known to cause human disease; in Anne Piepenbring, of avian bornavirus isolates to induce Egypt, HAstV-1 is the most frequent Christiane Herden, proventricular dilatation disease in con- astrovirus serotype detected (3). Re- Kirstin Oberhäuser, ures. Emerg Infect Dis. 2010;16:473–9. cently, 5 novel astroviruses have been doi:10.3201/eid1603.091257 Ursula Heffels-Redmann, 9. Raghav R, Taylor M, DeLay J, Ojkic D, discovered in human fecal samples and Dirk Enderlein Pearl DL, Kistler AL, et al. Avian borna- from patients with diarrhea or acute Author affi liations: Justus Liebig virus is present in many tissues of psitta- fl accid paralysis (4–7). Because the cine birds with histopathologic evidence University, Giessen, Germany (M. Lierz, prevalence of these virsues in the of proventricular dilatation disease. J A. Piepenbring, C. Herden, U. Heffels- Vet Diagn Invest. 2010;22:495–508. Middle East is unknown, we screened Redmann, D. Enderlein); and Loro Parque doi:10.1177/104063871002200402 fecal samples from children with di- Fundacion, Tenerife, Spain (K. Oberhauser) 10. Herzog S, Enderlein D, Heffels-Redmann arrhea residing in Egypt to ascertain U, Piepenbring A, Neumann D, Kaleta EF, the prevalence and diversity of these DOI: http://dx.doi.org/10.3201/eid1712.111317 et al. Indirect immunofl uorescence assay for intra vitam diagnosis of avian borna- novel astroviruses. Fecal samples were collected References virus infection in psittacine birds. J Clin Microbiol. 2010;48:2282–4. doi:10.1128/ from a cohort of 364 symptomatic chil- JCM.00145-10 1. Wyss S, Deb A, Watson R, Hammer S. dren <5 years of age who had diarrhea Radiographic measurements for PDD di- and were seeking medical care at Abu agnosis in Spix’s macaws (Cyanopsitta Address for correspondence: Michael Lierz, Homos Hospital in the Nile Delta of spixii) at Al Wabra Wildlife Preservation Clinic for Birds, Reptiles, Amphibians and Egypt from September 2006 through (AWWP), Qatar. In: Proceedings of the Fish, Faculty of Veterinary Medicine, Justus International Conference on Diseases of September 2007. RNA was extracted Zoo and Wild Animals. Zurich: University Liebig University Giessen, Frankfurter Str 91- from 10% fecal suspensions and re- of Zurich; 2009. p. 349–54. 93, 35392 Giessen, Germany; email: michael. verse transcription PCR for astrovirus 2. Lierz M, Herden C, Herzog S, Piepen- [email protected] bring A. Proventricular dilatation disease was performed as previously described and avian bornavirus as a possible cause. (4–6). Astrovirus consensus primer Tieraerztl Prax (K). 2010;38:87–94. pair SF0073/SF0076 amplifi ed an 3. Kistler AL, Gancz A, Clubb S, Skewes- ≈409-bp region of open reading frame Cox P, Fischer K, Sorber K, et al. Re- covery of divergent avian bornaviruses (ORF) 1b, encoding the RNA poly- from cases of proventricular dilatation merase gene. PCR-positive samples disease: identifi cation of a candidate were then tested by using primer sets etiologic agent. Virol J. 2008;5:88. Mon269–Mon270 (8) and SF0053– doi:10.1186/1743-422X-5-88 4. Honkavuori KS, Shivaprasad HL, Wil- SF0061, amplifying either a 449-bp liams BL, Quan PL, Hornig M, Street or a 402-bp product of the ORF2 cap- C, et al. Novel borna virus in psittacine sid gene from classical HAstVs (se- birds with proventricular dilatation dis- rotypes I–VIII), or astrovirus MLB1, ease. Emerg Infect Dis. 2008;14:1883–6. doi:10.3201/eid1412.080984 respectively. DNA sequences of PCR 5. Heffels-Redmann U, Enderlein D, Herzog products were determined by using S, Herden C, Piepenbring A, Neumann Big Dye Terminator Cycle technol- D, et al. Occurrence of avian bornavirus ogy (Applied Biosystems, Foster City, infection in captive psittacines in various European countries and its association CA, USA). Nucleotide sequences to proventricular dilation disease. Avian were compared with sequences ob- Pathol. 2011; 40:419–26. tained from GenBank. Phylogenies

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 12, December 2011 2391 LETTERS were constructed with the MEGA4 tive in 23 (6.3%) of 364 fecal samples. MLB1 on the basis of the partial ORF2 software (www.megasoftware.net) by Five common serotypes of classical sequence analysis. Egypt MLB1 sam- using the neighbor-joining method and HAstV were identifi ed constituting ples shared ≈99%-nt identity with a p-distance algorithm. Bootstrap resa- 16 (70%) of 23 positive samples; each other, and all grouped in 1 phylo- mpling was performed by using 2,000 HAstV type I was most prevalent (n genetic cluster (Figure, panel B) along replicates to demonstrate robustness of = 9). Alignment of the partial amino with a recently identifi ed MLB1 strain grouping. The nucleotide sequences acid sequences of the ORF2 capsid (GenBank accession no. HM450380 determined in this study were assigned region indicates that Egyptian HAstV [9]) isolated from a human fecal sam- GenBank accession nos. HQ674630– type I strains share 99%–100% iden- ple in Hong Kong. The Egypt MLB1 HQ674650. tity (Figure, panel A). strains shared ≈97%-nt identity with Consensus astrovirus reverse Five of the 7 remaining positive the prototype MLB1 Australia strain transcription PCR results were posi- samples were most closely related to but were more divergent from an iso- late recently described in the United States (92% nt identity, GenBank ac- cession no. FJ222451). However, all of the observed nucleotide differences represented silent mutations between Egypt MLB1 and Australia and US isolates; comparison of partial capsid protein sequences indicated no amino acid changes. The 2 remaining HAstV-positive samples were phylogenetically most similar to astrovirus VA2 (VA2) (Fig- ure, panel B). On the basis of the sequence of the amplicon from the ORF1b region, the Egyptian VA2 iso- lates shared 96.1%–100% aa identity to previously described VA2 and as- trovirus human/mink/ovine genomes in GenBank. Our study describes the detec- tion of the recently identifi ed viruses MLB1 and VA2 in a cohort of symp- tomatic children with diarrhea resid- ing in Egypt. This study expands the geographic range of these viruses to include northern Africa. The consen- sus primers used in our study were able to detect a higher percentage of positive HAstV serotypes I–VIII than the Mon340/Astman-2 primers (10) (data not shown), a fi nding that encourages use of these primers to screen humans with gastroenteritis for astroviruses. Increased understanding Figure. A) Phylogenetic analysis of the partial amino acid sequences of the open reading frame (ORF) 2 capsid region of human astrovirus (HAstV) types I–VIII (using Mon primer) of the genetic diversity within viral with other sequences from GenBank. B, C) Phylogenetic trees based on partial nucleotide families infecting humans will assist sequences of MLB1 ORF2 (B) and ORF1b (VA2) (C). Egyptian isolates are shown in in future studies of their pathogenicity boldface. Sequence alignment was performed by using ClustalW in the BioEdit software and the design of specifi c diagnostic package (www.clustal.org). Dendrograms were constructed by using the neighbor-joining assays. Further epidemiologic stud- method as in MEGA4 (www.megasoftware.net). Values on the branches represent the bootstrap values from 2,000 replicates. Scale bar in A indicates amino acid substitutions ies, including clinical cases and de- per site; scale bars in B and C indicate nucleotide substitutions per site. mographically matched healthy con-

2392 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 12, December 2011 LETTERS trols, are required to better defi ne their 6. Finkbeiner SR, Li Y, Ruone S, Conrardy Recent studies have reported iso- pathogenic potential. C, Gregoricus N, Toney D, et al. Identi- lation of M. tuberculosis in cattle with fi cation of a novel astrovirus (astrovirus This work was supported by the VA1) associated with an outbreak of acute prevalences of 4.7%–30.8% in Afri- Global Emerging Infections and Surveil- gastroenteritis. J Virol. 2009;83:10836–9. can and Asian countries (1–3). In cat- lance System, a Division of the Armed doi:10.1128/JVI.00998-09 tle, this infection occurs in countries Forces Health Surveillance Center and in 7. Kapoor A, Li L, Victoria J, Odernide B, with the highest incidence of human Mason C, Pandey P, et al. Multiple novel part by the National Institutes of Health astrovirus species in human stool. J Gen tuberculosis in the world. In Europe, under KL2 RR024994. The study protocol Virol. 2009;90:2965–72. doi:10.1099/ only 14 cases of M. tuberculosis infec- DOD NAMRU3.2000.0002 (IRB Protocol vir.0.014449-0 tion have been described in 3 eastern No. 096) was hospital-based surveillance 8. Noel JS, Lee TW, Kurtz JB, Glass RI, countries since implementation of Monroe SS. Typing of human astroviruses for enteric pathogens associated with se- from clinical isolates by enzyme immuno- eradication programs (4,5). The only vere diarrhea in Egyptian children. assay and nucleotide sequencimg. J Clin reported cases of M. tuberculosis in Microbiol. 1995;33:797–801. cattle in western Europe were de- 9. Chu DK, Chin AW, Smith GJ, Chan KH, scribed in Great Britain and date back Salwa F. Ahmed, Guan Y, Peiris JS, et al. Detection of Peter J. Sebeny, John D. Klena, novel astroviruses in urban brown rats to the 1950s (6). Guillermo Pimentel, and previously known astroviruses in During 2007–2009, three cases of Adel Mansour, Amel M. Naguib, humans. J Gen Virol. 2010;10:2457–62. tuberculosis caused by M. tuberculo- doi:10.1099/vir.0.022764-0 sis were detected in 3 unrelated cattle Jody Bruton, Sylvia Y.N. Young, 10. Rohayem J, Berger S, Juretzek T, Herch- Lori R. Holtz, and David Wang enroder O, Mogel M, Poppe M, et al. A farms, 2 of them free of tuberculosis Author affi liations: US Naval Medical Re- simple and rapid single-step m-RT-PCR (farms 1 and 2). As part of the surveil- to detect norovirus, astrovirus and ad- search Unit No.3, Cairo, Egypt (S.F. Ahmed, lance system of bovine tuberculosis, enovirus in clinical stool samples. J Virol a pool of tissue samples from each P.J. Sebeny, J.D. Klena, G. Pimentel, A. Methods. 2004;118:49–59. doi:10.1016/j. Mansour, J. Bruton, S.Y.N. Young); Ministry jviromet.2004.01.016 cow (respiratory lymph nodes and of Health, Cairo (A.M. Naguib); and Wash- lung) were homogenized with sterile ington University School of Medicine, St. Address for correspondence: Salwa F. Ahmed, distilled water, and culture was car- Louis, Missouri, USA (L.R. Holtz, D. Wang) US Naval Medical Research Unit No. 3 ried out by the BACTEC mycobacte- (NAMRU-3), PSC 452 Box 5000, FPO AE ria growth indicator tube 960 system DOI: http://dx.doi.org/10.3201/eid1712.110909 09835, USA; email: salwa.fouad.eg@med. (Beckton Dickinson, Madrid, Spain). navy.mil Members of the M. tuberculosis com- References plex were identifi ed and genotyped 1. Klein EJ, Boster DR, Stapp JR, Wells by direct variable repeat spacer olig- JG, Qin X, Clausen CR, et al. Diarrhea nucleotide typing and mycobacterial etiology in a children’s hospital emer- interspersed repetitive unit–variable gency department: a prospective cohort number tandem repeat (MIRU-VNTR) study. Clin Infect Dis. 2006;43:807–13. doi:10.1086/507335 typing (7). 2. Lyman WH, Walsh JF, Kotch JB, Weber The 3 M. tuberculosis–infected DJ, Gunn E, Vinje J. Prospective study Humans as Source animals were <9 months of age (Table). of etiologic agents of acute gastroen- As described (6), the possibility of in- teritis outbreaks in child care centers. J of Mycobacterium fection in young animals could be more Pediatr. 2009;154:253–7. doi:10.1016/j. tuberculosis jpeds.2008.07.057 probable than infection in older cows. 3. Nafi cy AB, Rao MR, Holmes JL, Abu- Infection in M. tuberculosis–infected animals Elyazeed R, Savarino SJ, Wierzba TF, from farms 1 and 3 were detected by et al. Astrovirus diarrhea in Egyptian Cattle, Spain children. J Infect Dis. 2000;182:685–90. the intradermal tuberculin test (Table). doi:10.1086/315763 To the Editor: Mycobacterium The animal without immunologic re- 4. Finkbeiner SR, Kirkwood CD, Wang D. tuberculosis is the main causative sponse (farm 2) was detected because Complete genome sequence of a highly agent of tuberculosis in humans. How- an M. bovis infection was confi rmed divergent astrovirus isolated from a child ever, little attention has been paid to its with acute diarrhea. Virol J. 2008;5:117. in the herd, and all animals were 5. Finkbeiner SR, Holtz LR, Jiang Y, Ra- transmission from humans to animals. slaughtered. Confi rmation of infec- jendran P, Franz CJ, Zhao G, et al. Hu- We report M. tuberculosis infections tion by culture without immunologic man stool contains a previously unrec- in 3 cattle farms in Spain. The epide- response is rare, although the high ognized diversity of novel astroviruses. miologic investigation traced humans Virol J. 2009;6:161. doi:10.1186/1743- sensitivity of the mycobacteria growth 422X-6-161 as the source of infection, with 1 of the indicator tube system could detect a strains showing multidrug resistance. low bacterial load in the initial stages

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