- Email: [email protected]
Human bocavirus species 2 and 3 in Brazil
Journal of Clinical Virology 48 (2010) 127–130
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Short communication
Human bocavirus species 2 and 3 in Brazil Norma Santos a,∗ , Teresa C.T. Peret b , Charles D. Humphrey a,b , Maria Carolina M. Albuquerque a , Raquel Cirlene Silva a , Fabrício José Benati a , Xiaoyan Lu b , Dean D. Erdman b a b
Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, Brazil Center for Diseases Control and Prevention, Atlanta, GA 30333, United States
a r t i c l e
i n f o
Article history: Received 16 December 2009 Received in revised form 16 March 2010 Accepted 17 March 2010 Keywords: Human bocavirus Gastroenteritis Viral diagnostic
a b s t r a c t Background: The newly described human bocavirus (HBoV) species 2 and 3 have been repeatedly detected in stool strengthening the possibility that these viruses might present a tropism for the gastrointestinal tract and may be etiological agents of diarrhea. Objective: In this study we assessed the presence of HBoV2 and HBoV3 in stool specimens from Brazilians with acute gastroenteritis. Study design: Stool samples from Brazilian patients with acute diarrhea were analyzed for HBoV2 and HBoV3 by PCR assay. Full or partial genome sequences were obtained for selected isolates. Electron microscopy analysis was used to investigate virus morphology. Results: Electron microscopy confirmed the presence of virus-like particles in HBoV PCR-positive specimens, with morphology similar to other members of the Parvoviridae family. Five samples out of 807 (0.6%) were positive for HBoV3. Three of the HBoV3-positive patients were HIV/AIDS positive. A selected group of 144 samples was also tested for HBoV2 and 30 samples (20.8%) were positive, 11 of which were HIV/AIDS positive. Conclusion: This study reports the detection and genetic characterization of HBoV3 and HBoV2 in the stool of Brazilian patients with acute diarrhea. This is the first description of HBoV3 outside Australia, suggesting a wide global distribution of this virus. Further studies are needed to better understand the role of HBoV in gastrointestinal infections, particularly among patients with HIV/AIDS. © 2010 Elsevier B.V. All rights reserved.
1. Background
2. Objective
Human bocavirus (HBoV), a member of the family Parvoviridae, was proposed to be an etiologic agent of respiratory disease.1 HBoV has also been detected in the blood of some patients with respiratory illness2,3 and has become recognized as a possible agent of acute gastroenteritis.4–11 The HBoV genome possesses two major ORFs encoding a nonstructural protein (NS1) and at least two capsid proteins (VP1 and VP2). There is also a third ORF that encodes a nonstructural protein (NP1) with unknown function.1 Based on the sequences of the NS1 gene, there are three HBoVs species: HBoV1, HBoV2 and HBoV3.12,13
We report the full genomic sequences of novel strains of HBoV3 and the detection of HBoV2 and HBoV3 in stool specimens from Brazilians with acute gastroenteritis.
∗ Corresponding author at: Departamento de Virologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Cidade Universitária, CCS – Bl. I. Ilha do Fundão, Rio de Janeiro 21941-590, RJ, Brazil. Tel.: +55 21 2560 8344x165; fax: +55 21 2560 8028. E-mail address: [email protected] (N. Santos). 1386-6532/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2010.03.014
3. Study design 3.1. Stool specimens Eight hundred and seven stool samples from Brazilian patients with acute diarrhea were analyzed for HBoV. These samples were randomly selected based solely on the stool availability. The study protocol was approved by the Ethics Committees of the Instituto de Puericultura e Pediatria Martagão Gesteira and the Hospital Universitário Clementino Fraga Filho of the Federal University of Rio de Janeiro. 3.2. Virus strains The MC8 strain was detected in the stool sample from an 18month-old boy, and was the only pathogen detected in the stool.5
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N. Santos et al. / Journal of Clinical Virology 48 (2010) 127–130
The IM10 strain was detected in the stool from a HIV-positive, 9year-old boy, which was also positive for norovirus.5 3.3. DNA sequencing DNA extraction was performed using NucliSens Magnetic Extraction Reagent (Biomerieux, Durham, NC). Sequencing was performed using the BigDye® Terminator v3.1 Cycle Sequencing Kit and the 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA). Sequence editing and assembling were performed using Sequencher v4.7 (Gene Codes Corporation, Ann Arbor, MI). Sequence alignments were performed using Clustal W and phylogenetic trees were constructed using neighbor joining, maximum parsimony and maximum likelihood methods implemented in PAUP* 4.0.14 Full-genome sequences of HBoV3 strains MC8 and IM10 were deposited into GenBank (GQ867666 and GQ867667). 3.4. Negative stain EM One HBoV3-positive stool specimen was concentrated by sucrose cushion ultracentrifugation prior to negative stain electron microscopy. Specimens were viewed in an FEI Technai BioTwinR transmission electron microscope (FEI Company, Hillsboro, OR). 3.5. HBoV1/3 PCR assay A PCR assay for HBoV1 and HBoV3 was developed that targets the NS1 gene. Primer set P1 (CAT ATT ATA GTT GGG GGA GAA GG) and P2 (GGT AGT TTT TGA AGA AGC GAA GAG) amplifies a 286 bp fragment of both HBoV species; primer set P5 (TCA GAA GCA TCG GAA GTG GGT GTT) and P6 (ATG TGA GGC TTT ATG CTG GCT GAA) amplifies a 440 bp fragment of only HBoV3. The PCR reaction was performed using MgCl2 at a final concentration of 3 mM and each primer at a concentration of 0.4 M. Amplification consisted of 1 step of 95 ◦ C/15 min; 45 cycles of 94 ◦ C/20 s, 52 ◦ C/20 s, 72 ◦ C/40 s, followed by extension at 72 ◦ C/10 min. The PCR products were detected by agarose gel electrophoresis and ethidium bromide staining.
Fig. 1. Neighbor-joining trees of HBoV NS1 and NP1 (combined) nucleotide sequences. Bootstrap values (1000 replicates) are indicated at nodes of HBoV1, HBoV2 and HBoV3 clades. Trees were outgrouped with MVC sequence NC 00442 (not shown). GenBank accession numbers are shown with published HBoV strain names.
4.2. Electron microscopy HBoV3 PCR-positive specimens parvovirus-like particles (Fig. 2).
contained
22–24 nm
3.6. HBoV2 PCR assay HBoV2 was tested by using nested-PCR reactions previously described.12 The amplified DNAs of positive samples were sequenced. The nucleotide sequences obtained in this study were deposited in GenBank (GU256650–GU256655). 4. Results 4.1. Genome analysis The complete coding sequences of HBoV3 strains MC8 and IM10 were determined. Both strains were closely related, showing greater than 99% sequence identity. The predicted proteins for the NS1, NP1, VP1/VP2 open reading frames are similar to that described for HBoV3.13 Phylogenetic analyses confirmed that MC8 and IM10 form distinct clade with published HBoV3 strain W471 (Fig. 1). The genomes of these new viruses showed 79.7–80.6% identity with HBoV1; 79.2–80.7% identity with HBoV2; and 97.8–99.2% with published HBoV3. Pairwise analysis demonstrated that the nucleotide divergence of NS1 and NP1 was higher between the Brazilian strains and HBoV2 while divergence of the structural gene (VP1/VP2) was higher between the Brazilian strains and HBoV1.
Fig. 2. HBoV cluster as shown by negative stain electron microscopy. The stain used was 5% ammonium molybdate–1% trehalose in water, pH 6.9. The bar marker represents 100 nm.
N. Santos et al. / Journal of Clinical Virology 48 (2010) 127–130
Fig. 3. Phylogenetic analysis of a portion of the nucleotide sequences of the NS1 gene of HBoV2 enteric strains from Brazil. The dendrogram was constructed by the Clustal V algorithm of the MegAlign program in the Lasegene software package (DNASTAR, Madison, WI, USA). The length of each pair of branches represents the distance between sequence pairs, while the units at the bottom of the tree indicate the number of substitution events.
4.3. HBoV1/3 detection in diarrheic stool samples A PCR assay was used to screen the 807 stools for detection of HBoV1 and HBoV3; 195 of the specimens were from patients with some type of immunodeficiency: 10 (1.2%) were positive for HBoV1, of which none were immunocompromised. Five (0.6%) samples [including MC8 and IM10] were positive for HBoV3. The other three viruses were detected in the stool collected (i) from a 1-month-old boy also positive for rotavirus; (ii) a 37-year-old HIVpositive woman who was also positive for cytomegalovirus; and (iii) a 57-year-old HIV-positive woman also positive for Isospora belli. Co-infections with HBoV1 and HBoV3 were present in two samples. 4.4. HBoV2 detection in diarrheic stool samples A randomly selected group of 144 samples previously tested for HBoV1 and HBoV3 were screened for the presence of HBoV2: 93 of these specimens were from patients with some type of immunodeficiency. Thirty samples (20.8%) were positive for HBoV2 (Fig. 3). Co-infection with other viruses, bacteria or parasites was observed in 13 (43.3%) positive samples, but none with other HBoV species. Among the immunocompetent patients, 9 (17.6%; n = 51) were positive for HBoV2: 8 single infections and one co-infection with Escherichia coli. Twenty-two (22.6%; n = 93) immunocompromised patients were positive for HBoV2. Of those, 11 were HIV/AIDS patients (6 had single HBoV2 infections and 5 were co-infected with other pathogens such as cytomegalovirus, Strongyloides stercoralis and I. belli). Among the 10 patients with other types of immunodeficiency 3 had single HBoV2 infections and 7 had co-infections with cytomegalovirus or S. stercoralis. 5. Discussion HBoV3 found in stool samples of Australian children was recently reported.13 Like the Australian strains, the NS1 and NP1 gene region of the Brazilian HBoV3 show greater sequence similarity to HBoV1, whereas the VP1/VP2 gene are more like HBoV2, suggesting that these viruses occupy an intermediary evolutionary position between HBoV1 and HBoV2, possible as a result of a recombination event.13 While HBoV1 has been comprehensively characterized in respiratory samples collected world-wide, there have been no reports to
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date of detection of HBoV3 from the respiratory tract. Considering that (i) the primers most commonly used for screening respiratory specimens for HBoV target the nonstructural genes,1,5,9,15–21 and (ii) the nonstructural genes of HBoV1 and HBoV3 share a high level of genetic similarity, one would expect some level of primer cross-reactivity with these viruses. While failure to detect HBoV2 in respiratory specimens could be explained by its greater genetic divergence,12 HBoV3 would likely have been detected by now if it were a common respiratory pathogen. HBoV2 was first detected in the stool of healthy and diarrheic patients from Pakistan and the United Kingdom.12 Subsequently, the virus was detected in fecal samples of children collected from case–control pairs for an acute gastroenteritis study conducted in Australia.13 Later the virus was reported in children with gastroenteritis,8,22 or with respiratory tract infection23,24 in other countries. We analyzed 195 stool specimens from patients with some type of immunodeficiency for detection of HBoV1 and HBoV3, including 118 with HIV/AIDS. Three HIV/AIDS patients were infected with HBoV3, representing 60% (n = 5) of the HBoV3 strains detected in our study. In contrast, none of the patients infected with HBoV1 had any identified immunodeficiency. Ninety-three of these samples were also tested for HBoV2, of which 34 were HIV/AIDS patients. Eleven HIV/AIDS patients were positive of which six presented as single HBoV2 infections. Further studies are needed to better understand the role of HBoVs in gastroenteritis, particularly among patients with HIV/AIDS. Conflict of interest The authors of this work have no financial or personal relationship with other people or organization that could inappropriately influence their work. Acknowledgments We thank George Gallucci, Shifaq Kamili and Soluza dos Santos Gonc¸alves for the technical assistance. This study was supported in part by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES), and Fundac¸ão Carlos Chagas de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Brazil. References 1. Allander T, Tammi MT, Eriksson M, Bjerkner A, Tiveljung-Lindell A, Andersson B. Cloning of a human parvovirus by molecular screening of respiratory tract samples. Proc Natl Acad Sci USA 2005;102:12891–6. 2. Fry AM, Lu X, Chittaganpitch M, Peret T, Fischer J, Dowell SF, et al. Human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in Thailand. J Infect Dis 2007;195:1038–45. 3. Allander T, Jartti T, Gupta S, Niesters HGM, Lehtinen P, Österback R, et al. Human bocavirus and acute wheezing in children. Clin Infect Dis 2007;44:904–10. 4. Vicente D, Cilla G, Montes M, Perez-Yarza EG, Perez-Trallero E. Human bocavirus a respiratory and enteric virus. Emerg Infect Dis 2007;13:636–7. 5. Albuquerque MCM, Rocha LN, Benati FJ, Soares CC, Maranhão AG, Ramírez ML, et al. Human bocavirus infection in children with gastroenteritis, Brazil. Emerg Infect Dis 2007;13:1756–8. 6. Lee JI, Chung JY, Han TH, Song MO, Hwang ES. Detection of human bocavirus in children hospitalized because of acute gastroenteritis. J Infect Dis 2007;196:994–7. 7. Lau SKP, Yip CCY, Que TI, Lee RA, Au-Yeung RKH, Zhou B, et al. Clinical and molecular epidemiology of human bocavirus in respiratory and fecal samples from children in Hong Kong. J Infect Dis 2007;196:986–93. 8. Szomor KN, Kapusinzky B, Rigó Z, Kis Z, Rózsa M, Farkas Á, et al. Detection of human bocavirus from fecal samples of Hungarian children with acute gastroenteritis. Intervirology 2009;57:17–21. 9. Chieochansin T, Thongmee C, Vimolket L, Theamboonlers A, Poovorawan Y. Human bocavirus infection in children with acute gastroenteritis and healthy controls. Jpn J Infect Dis 2008;61:479–81.
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10. Huang Y, Mao P, Wang H. Detection of, and frequent co-infection with, human bocavirus in faecal specimens from children in Wuhan, China. Clin Microbiol Infect 2009, doi:10.1111/j.1469-0691.2009.02862.x [Epub ahead of print in 2009 June 22]. 11. Campe H, Hartberger C, Sing A. Role of human bocavirus infections in outbreaks of gastroenteritis. J Clin Virol 2008;43:340–2. 12. Kapoor A, Slikas E, Simmonds P, Chieochansin T, Naeem A, Shaukat S, et al. A newly identified bocavirus species in human stool. J Infect Dis 2009;199:196–200. 13. Arthur JL, Higgins GD, Davidson GP, Givney RC, Ratcliff RM. A novel bocavirus associated with acute gastroenteritis in Australian children. PLoS Pathog 2009;5(4):e1000391. 14. Swofford DL. PAUP*. Phylogenetic analysis using parsimony (*and other methods). Version 4. Sunderland, MA: Sinauer Associates; 2003. 15. Kesebir D, Vazquez M, Weibel C, Shapiro ED, Ferguson D, Landry, et al. Human bocavirus infection in young children in the United States: molecular. J Infect Dis 2006;194(November (9)):1276–82. 16. Sloots TP, Whiley DM, Lambert SB, Nissen MD. Emerging respiratory agents: new viruses for old diseases? J Clin Virol 2008;42:233–43.
17. Manning A, Russell V, Eastick K, Leadbetter GH, Hallam N, Templeton K, et al. Epidemiological profile and clinical associations of human bocavirus and other human parvoviruses. J Infect Dis 2006;194:1283–90. 18. Bastien N, Brandt K, Dust K, Ward D, Li Y. Human bocavirus infection, Canada. Emerg Infect Dis 2006;12:848–50. 19. Arden KE, McErlean P, Nissen MD, Sloots TP, Mackay IM. Frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections. J Med Virol 2006;78:1232–40. 20. Monteny M, Niesters HGM, Moll HA, Berger MY. Human bocavirus in febrile children, the Netherlands. Emerg Infect Dis 2007;13:180–1. 21. Qu XW, Duan ZJ, Qi ZY, Xie ZP, Gao HC, Liu WP, et al. Human bocavirus infection, People’s Republic of China. Emerg Infect Dis 2007;13:165–8. 22. Han TH, Kim CH, Park SH, Kim EJ, Chung JY, Hwang ES. Detection of human bocavirus-2 in children with acute Gastroenteritis in South Korea. Arch Virol 2009;154:1923–7. 23. Shan TL, Zhang W, Guo W, Cui L, Yuan CL, Dai XQ, et al. The first detection of human bocavirus 2 infections in China. J Clin Virol 2009;46:196–7. 24. Han TH, Chung JY, Hwang ES. Human bocavirus 2 in children, South Korea. Emerg Infect Dis 2009;15:1698–700.
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Short communication
Human bocavirus species 2 and 3 in Brazil Norma Santos a,∗ , Teresa C.T. Peret b , Charles D. Humphrey a,b , Maria Carolina M. Albuquerque a , Raquel Cirlene Silva a , Fabrício José Benati a , Xiaoyan Lu b , Dean D. Erdman b a b
Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, Brazil Center for Diseases Control and Prevention, Atlanta, GA 30333, United States
a r t i c l e
i n f o
Article history: Received 16 December 2009 Received in revised form 16 March 2010 Accepted 17 March 2010 Keywords: Human bocavirus Gastroenteritis Viral diagnostic
a b s t r a c t Background: The newly described human bocavirus (HBoV) species 2 and 3 have been repeatedly detected in stool strengthening the possibility that these viruses might present a tropism for the gastrointestinal tract and may be etiological agents of diarrhea. Objective: In this study we assessed the presence of HBoV2 and HBoV3 in stool specimens from Brazilians with acute gastroenteritis. Study design: Stool samples from Brazilian patients with acute diarrhea were analyzed for HBoV2 and HBoV3 by PCR assay. Full or partial genome sequences were obtained for selected isolates. Electron microscopy analysis was used to investigate virus morphology. Results: Electron microscopy confirmed the presence of virus-like particles in HBoV PCR-positive specimens, with morphology similar to other members of the Parvoviridae family. Five samples out of 807 (0.6%) were positive for HBoV3. Three of the HBoV3-positive patients were HIV/AIDS positive. A selected group of 144 samples was also tested for HBoV2 and 30 samples (20.8%) were positive, 11 of which were HIV/AIDS positive. Conclusion: This study reports the detection and genetic characterization of HBoV3 and HBoV2 in the stool of Brazilian patients with acute diarrhea. This is the first description of HBoV3 outside Australia, suggesting a wide global distribution of this virus. Further studies are needed to better understand the role of HBoV in gastrointestinal infections, particularly among patients with HIV/AIDS. © 2010 Elsevier B.V. All rights reserved.
1. Background
2. Objective
Human bocavirus (HBoV), a member of the family Parvoviridae, was proposed to be an etiologic agent of respiratory disease.1 HBoV has also been detected in the blood of some patients with respiratory illness2,3 and has become recognized as a possible agent of acute gastroenteritis.4–11 The HBoV genome possesses two major ORFs encoding a nonstructural protein (NS1) and at least two capsid proteins (VP1 and VP2). There is also a third ORF that encodes a nonstructural protein (NP1) with unknown function.1 Based on the sequences of the NS1 gene, there are three HBoVs species: HBoV1, HBoV2 and HBoV3.12,13
We report the full genomic sequences of novel strains of HBoV3 and the detection of HBoV2 and HBoV3 in stool specimens from Brazilians with acute gastroenteritis.
∗ Corresponding author at: Departamento de Virologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Cidade Universitária, CCS – Bl. I. Ilha do Fundão, Rio de Janeiro 21941-590, RJ, Brazil. Tel.: +55 21 2560 8344x165; fax: +55 21 2560 8028. E-mail address: [email protected] (N. Santos). 1386-6532/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2010.03.014
3. Study design 3.1. Stool specimens Eight hundred and seven stool samples from Brazilian patients with acute diarrhea were analyzed for HBoV. These samples were randomly selected based solely on the stool availability. The study protocol was approved by the Ethics Committees of the Instituto de Puericultura e Pediatria Martagão Gesteira and the Hospital Universitário Clementino Fraga Filho of the Federal University of Rio de Janeiro. 3.2. Virus strains The MC8 strain was detected in the stool sample from an 18month-old boy, and was the only pathogen detected in the stool.5
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N. Santos et al. / Journal of Clinical Virology 48 (2010) 127–130
The IM10 strain was detected in the stool from a HIV-positive, 9year-old boy, which was also positive for norovirus.5 3.3. DNA sequencing DNA extraction was performed using NucliSens Magnetic Extraction Reagent (Biomerieux, Durham, NC). Sequencing was performed using the BigDye® Terminator v3.1 Cycle Sequencing Kit and the 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA). Sequence editing and assembling were performed using Sequencher v4.7 (Gene Codes Corporation, Ann Arbor, MI). Sequence alignments were performed using Clustal W and phylogenetic trees were constructed using neighbor joining, maximum parsimony and maximum likelihood methods implemented in PAUP* 4.0.14 Full-genome sequences of HBoV3 strains MC8 and IM10 were deposited into GenBank (GQ867666 and GQ867667). 3.4. Negative stain EM One HBoV3-positive stool specimen was concentrated by sucrose cushion ultracentrifugation prior to negative stain electron microscopy. Specimens were viewed in an FEI Technai BioTwinR transmission electron microscope (FEI Company, Hillsboro, OR). 3.5. HBoV1/3 PCR assay A PCR assay for HBoV1 and HBoV3 was developed that targets the NS1 gene. Primer set P1 (CAT ATT ATA GTT GGG GGA GAA GG) and P2 (GGT AGT TTT TGA AGA AGC GAA GAG) amplifies a 286 bp fragment of both HBoV species; primer set P5 (TCA GAA GCA TCG GAA GTG GGT GTT) and P6 (ATG TGA GGC TTT ATG CTG GCT GAA) amplifies a 440 bp fragment of only HBoV3. The PCR reaction was performed using MgCl2 at a final concentration of 3 mM and each primer at a concentration of 0.4 M. Amplification consisted of 1 step of 95 ◦ C/15 min; 45 cycles of 94 ◦ C/20 s, 52 ◦ C/20 s, 72 ◦ C/40 s, followed by extension at 72 ◦ C/10 min. The PCR products were detected by agarose gel electrophoresis and ethidium bromide staining.
Fig. 1. Neighbor-joining trees of HBoV NS1 and NP1 (combined) nucleotide sequences. Bootstrap values (1000 replicates) are indicated at nodes of HBoV1, HBoV2 and HBoV3 clades. Trees were outgrouped with MVC sequence NC 00442 (not shown). GenBank accession numbers are shown with published HBoV strain names.
4.2. Electron microscopy HBoV3 PCR-positive specimens parvovirus-like particles (Fig. 2).
contained
22–24 nm
3.6. HBoV2 PCR assay HBoV2 was tested by using nested-PCR reactions previously described.12 The amplified DNAs of positive samples were sequenced. The nucleotide sequences obtained in this study were deposited in GenBank (GU256650–GU256655). 4. Results 4.1. Genome analysis The complete coding sequences of HBoV3 strains MC8 and IM10 were determined. Both strains were closely related, showing greater than 99% sequence identity. The predicted proteins for the NS1, NP1, VP1/VP2 open reading frames are similar to that described for HBoV3.13 Phylogenetic analyses confirmed that MC8 and IM10 form distinct clade with published HBoV3 strain W471 (Fig. 1). The genomes of these new viruses showed 79.7–80.6% identity with HBoV1; 79.2–80.7% identity with HBoV2; and 97.8–99.2% with published HBoV3. Pairwise analysis demonstrated that the nucleotide divergence of NS1 and NP1 was higher between the Brazilian strains and HBoV2 while divergence of the structural gene (VP1/VP2) was higher between the Brazilian strains and HBoV1.
Fig. 2. HBoV cluster as shown by negative stain electron microscopy. The stain used was 5% ammonium molybdate–1% trehalose in water, pH 6.9. The bar marker represents 100 nm.
N. Santos et al. / Journal of Clinical Virology 48 (2010) 127–130
Fig. 3. Phylogenetic analysis of a portion of the nucleotide sequences of the NS1 gene of HBoV2 enteric strains from Brazil. The dendrogram was constructed by the Clustal V algorithm of the MegAlign program in the Lasegene software package (DNASTAR, Madison, WI, USA). The length of each pair of branches represents the distance between sequence pairs, while the units at the bottom of the tree indicate the number of substitution events.
4.3. HBoV1/3 detection in diarrheic stool samples A PCR assay was used to screen the 807 stools for detection of HBoV1 and HBoV3; 195 of the specimens were from patients with some type of immunodeficiency: 10 (1.2%) were positive for HBoV1, of which none were immunocompromised. Five (0.6%) samples [including MC8 and IM10] were positive for HBoV3. The other three viruses were detected in the stool collected (i) from a 1-month-old boy also positive for rotavirus; (ii) a 37-year-old HIVpositive woman who was also positive for cytomegalovirus; and (iii) a 57-year-old HIV-positive woman also positive for Isospora belli. Co-infections with HBoV1 and HBoV3 were present in two samples. 4.4. HBoV2 detection in diarrheic stool samples A randomly selected group of 144 samples previously tested for HBoV1 and HBoV3 were screened for the presence of HBoV2: 93 of these specimens were from patients with some type of immunodeficiency. Thirty samples (20.8%) were positive for HBoV2 (Fig. 3). Co-infection with other viruses, bacteria or parasites was observed in 13 (43.3%) positive samples, but none with other HBoV species. Among the immunocompetent patients, 9 (17.6%; n = 51) were positive for HBoV2: 8 single infections and one co-infection with Escherichia coli. Twenty-two (22.6%; n = 93) immunocompromised patients were positive for HBoV2. Of those, 11 were HIV/AIDS patients (6 had single HBoV2 infections and 5 were co-infected with other pathogens such as cytomegalovirus, Strongyloides stercoralis and I. belli). Among the 10 patients with other types of immunodeficiency 3 had single HBoV2 infections and 7 had co-infections with cytomegalovirus or S. stercoralis. 5. Discussion HBoV3 found in stool samples of Australian children was recently reported.13 Like the Australian strains, the NS1 and NP1 gene region of the Brazilian HBoV3 show greater sequence similarity to HBoV1, whereas the VP1/VP2 gene are more like HBoV2, suggesting that these viruses occupy an intermediary evolutionary position between HBoV1 and HBoV2, possible as a result of a recombination event.13 While HBoV1 has been comprehensively characterized in respiratory samples collected world-wide, there have been no reports to
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date of detection of HBoV3 from the respiratory tract. Considering that (i) the primers most commonly used for screening respiratory specimens for HBoV target the nonstructural genes,1,5,9,15–21 and (ii) the nonstructural genes of HBoV1 and HBoV3 share a high level of genetic similarity, one would expect some level of primer cross-reactivity with these viruses. While failure to detect HBoV2 in respiratory specimens could be explained by its greater genetic divergence,12 HBoV3 would likely have been detected by now if it were a common respiratory pathogen. HBoV2 was first detected in the stool of healthy and diarrheic patients from Pakistan and the United Kingdom.12 Subsequently, the virus was detected in fecal samples of children collected from case–control pairs for an acute gastroenteritis study conducted in Australia.13 Later the virus was reported in children with gastroenteritis,8,22 or with respiratory tract infection23,24 in other countries. We analyzed 195 stool specimens from patients with some type of immunodeficiency for detection of HBoV1 and HBoV3, including 118 with HIV/AIDS. Three HIV/AIDS patients were infected with HBoV3, representing 60% (n = 5) of the HBoV3 strains detected in our study. In contrast, none of the patients infected with HBoV1 had any identified immunodeficiency. Ninety-three of these samples were also tested for HBoV2, of which 34 were HIV/AIDS patients. Eleven HIV/AIDS patients were positive of which six presented as single HBoV2 infections. Further studies are needed to better understand the role of HBoVs in gastroenteritis, particularly among patients with HIV/AIDS. Conflict of interest The authors of this work have no financial or personal relationship with other people or organization that could inappropriately influence their work. Acknowledgments We thank George Gallucci, Shifaq Kamili and Soluza dos Santos Gonc¸alves for the technical assistance. This study was supported in part by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES), and Fundac¸ão Carlos Chagas de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Brazil. References 1. Allander T, Tammi MT, Eriksson M, Bjerkner A, Tiveljung-Lindell A, Andersson B. Cloning of a human parvovirus by molecular screening of respiratory tract samples. Proc Natl Acad Sci USA 2005;102:12891–6. 2. Fry AM, Lu X, Chittaganpitch M, Peret T, Fischer J, Dowell SF, et al. Human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in Thailand. J Infect Dis 2007;195:1038–45. 3. Allander T, Jartti T, Gupta S, Niesters HGM, Lehtinen P, Österback R, et al. Human bocavirus and acute wheezing in children. Clin Infect Dis 2007;44:904–10. 4. Vicente D, Cilla G, Montes M, Perez-Yarza EG, Perez-Trallero E. Human bocavirus a respiratory and enteric virus. Emerg Infect Dis 2007;13:636–7. 5. Albuquerque MCM, Rocha LN, Benati FJ, Soares CC, Maranhão AG, Ramírez ML, et al. Human bocavirus infection in children with gastroenteritis, Brazil. Emerg Infect Dis 2007;13:1756–8. 6. Lee JI, Chung JY, Han TH, Song MO, Hwang ES. Detection of human bocavirus in children hospitalized because of acute gastroenteritis. J Infect Dis 2007;196:994–7. 7. Lau SKP, Yip CCY, Que TI, Lee RA, Au-Yeung RKH, Zhou B, et al. Clinical and molecular epidemiology of human bocavirus in respiratory and fecal samples from children in Hong Kong. J Infect Dis 2007;196:986–93. 8. Szomor KN, Kapusinzky B, Rigó Z, Kis Z, Rózsa M, Farkas Á, et al. Detection of human bocavirus from fecal samples of Hungarian children with acute gastroenteritis. Intervirology 2009;57:17–21. 9. Chieochansin T, Thongmee C, Vimolket L, Theamboonlers A, Poovorawan Y. Human bocavirus infection in children with acute gastroenteritis and healthy controls. Jpn J Infect Dis 2008;61:479–81.
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