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Circulating of human bocavirus 1, 2, 3, and 4 in pediatric patients with acute gastroenteritis in Thailand
Infection, Genetics and Evolution 12 (2012) 565–569
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Circulating of human bocavirus 1, 2, 3, and 4 in pediatric patients with acute gastroenteritis in Thailand Pattara Khamrin a, Rungnapa Malasao a, Natthawan Chaimongkol a, Nuthapong Ukarapol b, Tipachan Kongsricharoern c, Shoko Okitsu d, Satoshi Hayakawa d, Hiroshi Ushijima d, Niwat Maneekarn a,⇑ a
Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand Department of Pediatrics, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand Pediatric Hematology Unit, Nakornping Hospital, Chiang Mai, Thailand d Division of Microbiology, Department of Pathology and Microbiology, Nihon University School of Medicine, Tokyo, Japan b c
a r t i c l e
i n f o
Article history: Received 13 December 2011 Received in revised form 13 January 2012 Accepted 29 January 2012 Available online 8 February 2012 Keywords: Bocavirus Diarrhea Children Thailand
a b s t r a c t Human bocavirus (HBoV) is a newly identified human parvovirus that associated with respiratory and gastrointestinal diseases. Epidemiological surveillance of HBoV was conducted on fecal specimens collected from hospitalized children with diarrhea in Chiang Mai, Thailand in 2011. Among a total of 222 fecal specimens tested, 17 (7.7%) were positive for HBoV by PCR. Of the 17 HBoV positive samples, double- or triple-infections together with other enteric viruses were found in 10 (58.8%) pediatric patients, while monoinfection with HBoV alone was detected in seven (41.2%) cases. Mixed infection among HBoV with norovirus GII was frequently observed in this population. The partial VP1 nucleotide sequences of all 17 HBoV strains demonstrated that all four species of HBoV were found in the specimens tested. Eleven strains were HBoV1. Other three strains showed the sequence identity with HBoV2, which were most closely related to the HBoV2A. In addition, other two HBoV strains showed the highest level of nucleotide sequence identity with the HBoV3. It was surprisingly to observe that one Thai HBoV strain showed a unique characteristic similar to the HBoV4, a rare species of HBoV found in acute gastroenteritis patients. In summary, this study presents the genetic background information of HBoV circulated in acute gastroenteritis children in Chiang Mai, Thailand and it was clearly demonstrated that HBoVs circulated in this area were genetically diverse as all four species of HBoVs (HBoV1–4) were detected in the fecal specimens collected from pediatric patients admitted to the hospitals in this area. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Gastroenteritis is one of the most common causes of morbidity and mortality in children and accounts for two to three million deaths per year which are most occurring in infants and young children world wide (Kosek et al., 2003). The majority of acute gastroenteritis is caused by virus infections. Among these, rotavirus, calicivirus (norovirus and sapovirus), enteric adenovirus, and astrovirus have been reported as the most important etiologic viral agents (Clark and McKendrick, 2004; Dennehy, 2011). Recently, there are several reports of newly discovered enteric viruses, which are potentially associated with acute gastroenteritis including human bocavirus (HBoV). HBoV is a newly identified human parvovirus that was first described in 2005 in nasopharyngeal aspi⇑ Corresponding author. Address: Department of Microbiology, Faculty of Medicine, Chiang Mai University, 110 Inthawarorose, Sripoom, Muang, Chiang Mai 50200, Thailand. Tel.: +66 53 945332; fax: +66 53 217144. E-mail address: [email protected] (N. Maneekarn). 1567-1348/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2012.01.025
rate of children with respiratory tract infection (Allander et al., 2005). Several articles have confirmed recently that HBoV has also been detected in fecal samples (Allander et al., 2005; Lee et al., 2007; Chieochansin et al., 2008; Yu et al., 2008; Huang et al., 2010; Jartti et al., 2011; Jin et al., 2011; Pham et al., 2011; Wang et al., 2011). HBoV is classified in the family Parvoviridae, which includes small non-enveloped, icosahedral viruses with singlestranded DNA genome of about 5.3 kb. The genome encodes two forms of nonstructural proteins, NS1 and NP1, as well as two major structural proteins, VP1 and VP2 (Berns and Parrish, 2007; Chen et al., 2010; Kapoor et al., 2010). Currently, HBoVs have been classified into four species, HBoV1, HBoV2, HBoV3, and HBoV4. The HBoV2 can be further divided into two groups, 2A and 2B (Kapoor et al., 2010). HBoV1 is predominantly found to be a respiratory pathogen. In addition, HBoV2 and HBoV1 have also been found mainly in stool samples, whereas HBoV3 and HBoV4 are occasionally be detected (Allander et al., 2007; Allander, 2008; Han et al., 2009; Kapoor et al., 2010; Jartti et al., 2011; Jin et al., 2011). This statement was supported by
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seroepidemiological study of HBoV1–4 in adults and children with respiratory tract infection. The data demonstrated that antibody titer to HBoV1 was higher than those to HBoV2–4 (Kantola et al., 2011). In addition, a report from the US revealed that HBoV1, 2, and 3 were detected in untreated sewage water throughout the country (Blinkova et al., 2009). In Thailand, few molecular epidemiological studies of HBoV have been conducted. The studies from three different epidemiological areas (Bangkok, Chiang Mai, and Buriram) revealed that HBoV detection rates in acute gastroenteritis patients were varied from 0.9–2.3%. Based on the nucleotide sequence analysis, HBoV identified in this country mainly belonged to HBoV1 and HBoV2 (Chieochansin et al., 2008; Pham et al., 2011). The previous study of HBoV in Chiang Mai, Thailand in 2002–2004 revealed that only one specimen was positive for HBoV1 (Pham et al., 2011). In order to gain the overview genetic backgrounds of HBoV diversity and distribution in Thailand, we conducted the epidemiological study of HBoV in children with diarrhea in Thailand in 2011. Sequence and phylogenetic analyses of HBoVs detected in the present study were further characterized for their genetic evolutionary relationships with the viruses circulating in this area and HBoV reference strains. 2. Materials and methods 2.1. Specimen collection A total of 222 stool specimens were collected from children with diarrhea attending two hospitals in Chiang Mai city, Thailand. Only the pediatric patients who had a clinical diagnosis of acute gastroenteritis with watery diarrhea have been included in this study. The study period was from January to August, 2011. The ages of the patients enrolled in this study ranged from neonate up to 5 years old. All the collected specimens were also screened for other diarrheal viruses including group A, B, C rotaviruses, adenovirus, norovirus GI and GII, sapovirus, astrovirus, Aichi virus, human parechovirus and enterovirus based on the protocols described previously (Yan et al., 2003, 2004; Pham et al., 2010). The study was conducted with the approval of the ethical committee for human rights related to human experimentation, Faculty of Medicine, Chiang Mai University (No. 181/2554). 2.2. Bocavirus detection and genome characterization The viral genomes were first extracted from 10% fecal suspension supernatant using the QIAamp viral RNA Mini Kit (Qiagen, Germany). The presence of bocavirus in stool specimens was detected by polymerase chain reaction (PCR) using pan-bocavirus PCR primers described previously (Kapoor et al., 2010). All of the detected viruses were analyzed for their species by nucleotide sequence and phylogenetic analyses of the partial sequence encoding the VP1 region. Briefly, a forward primer AK-VP-F1 (50 -CGCCGT GGCTCCTGCTCT-30 ) was used in combination with the reverse primer AK-VP-R1 (50 -TGTTCGCCATCACAAAAGATGTG-30 ) for the first PCR. The nested-PCR was performed by using AK-VP-F2 (50 -GGCTC CTGCTCTAGGAAATAAAGAG-30 ) and AK-VP-R2 (50 -CCTGCTGTTAGG TCGTTGTTGTATGT-30 ) primers which targeted the partial VP1 region of HBoV. The PCR amplicons were purified with a Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI) and sequenced using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) on an automated sequencer (ABI 3100; Applied Biosystems, Foster City, CA). The sequences were compared with those of reference strains available in the NCBI GenBank database using BLAST server (http://www.ncbi.nlm.nih.gov/blast). Phylogenetic and molecular evolutionary relationships were evaluated by using MEGA 5 (Tamura et al., 2011).
2.3. Nucleotide sequence accession numbers The partial nucleotide sequences of HBoV VP1 gene described in the present study were deposited in the GenBank database. The accession numbers are JQ267773–JQ267789. 3. Results Of a total of 222 fecal specimen tested, 17 (7.7%) were positive for HBoV by PCR. Among 17 HBoV positive samples, double- or triple-infections together with other enteric viruses were found in 10 (58.8%) pediatric patients, while monoinfection with HBoV alone was detected in seven (41.2%) cases. Mixed infection among HBoV with norovirus GII was frequently observed in this population (Table 1). The partial VP1 nucleotide sequences (457 nucleotides long) of all 17 HBoV strains were determined and compared with those of four established HBoV species, including HBoV1, HBoV2 (2A and 2B), HBoV3, HBoV4 (Kapoor et al., 2010). The sequence comparison shown in Table 1 indicates that the partial VP1 nucleotide sequences of all 17 HBoV strains were divided into four species. Eleven strains (CMH-N006-11, CMH-N007-11, CMH-N009-11, CMH-N010-11, CMH-N062-11, CMH-N077-11, CMH-N079-11, CMH-N081-11, CMH-N096-11, CMH-N098-11, and CMH-S03111) were closely related to the HBoV1 reference strain (TU-A110-08) at 98.2–99.5%. Other three HBoVs (CMH-N118-11, CMH-N129-11, and CMH-N133-11) showed high sequence identity with HBoV2 ranging from 93.4% to 98.0%. It was, however, observed that within HBoV2 species, all three Thai HBoV strains showed the highest level of nucleotide sequence identity with the HBoV2A (TU-A-146-06) at 98.0% while exhibited lower sequence identity with the HBoV2B (US-MN-510-05) at only 93.4%. In addition, the partial nucleotide sequences of other two HBoV strains (CMH-N005-11 and CMH-N008-11) found in this study showed highest level of nucleotide sequence identity with the HBoV3 reference strain (US-MN-647-06) at 99.1%. It was surprisingly to observe that one Thai HBoV strain (CMH-S011-11) showed a unique characteristic of nucleotide sequence identity that most closely related to the HBoV4 reference strain (USMN-964-05), a rare species of HBoV found in acute gastroenteritis patients, at 98.2%. A phylogenetic tree constructed from the partial nucleotide sequences of the four HBoV reference strains and those of 17 HBoV Thai strains is shown in Fig. 1. It was clearly confirmed that HBoVs detected in Thailand were genetically diversified and all four species were circulating in children hospitalized with diarrhea. HBoV1 was the most common species as 11 strains clustered exclusively in the same branch with HBoV1 reference strains. HBoV2, which has been reported previously as a major HBoV species related to diarrhea also found in our study. Three HBoV2 clustered closely with the HBoV2A than 2B reference strains and appeared to be most closely related to the Thai (CU47TH) and Chinese (LZ53819) HBoV2A strains. In addition, this phylogenetic analysis also provided a molecular basis of genetic relationship of the two HBoV strains found in this study with that of HBoV3 Chinese strain BCH1978. Moreover, the only one Thai HBoV4 (CMH-S011-01) formed the closest genetic cluster with HBoV4 reference strains and two major lineages were found among HBoV4 species reported so far. One lineage included the majority of HBoV4 strains identified from several parts of the world, including the US, Nigeria, and Tunisia, while another lineage was formed exclusively by the US-MN-964-05 (reported previously in the US) and CMH-S011-11 (Thai HBoV found in this study). This finding implies that there might be at least two separate variants of HBoV4 circulating in nature, as previously proposed by Kapoor et al. (2010).
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Table 1 Co-infected viruses and the partial VP1 nucleotide sequence identities between HBoV strains found in this study and the HBoV reference strains. The nucleotide sequence identities over 90% between the HBoV strains detected in the present study and the reference strains are presented in boldface. Sample code
CMH-N006-11 CMH-N007-11 CMH-N009-11 CMH-N010-11 CMH-N062-11 CMH-N077-11 CMH-N079-11 CMH-N081-11 CMH-N096-11 CMH-N098-11 CMH-S031-11 CMH-N118-11 CMH-N129-11 CMH-N133-11 CMH-N005-11 CMH-N008-11 CMH-S011-11
Co-infection
Norovirus GII Norovirus GII None None Rotavirus A Rotavirus A None None Norovirus GII, adenovirus Norovirus GII None Norovirus GII Human parechovirus None Norovirus GII Norovirus GII, rotavirus A None
Nucleotide sequence identity (%) HBoV1 TU-A-110-08
HBoV2A TU-A-146-06
HBoV2B US-MN-510-05
HBoV3 US-MN-647-06
HBoV4 US-MN-964-05
98.6 99.5 98.6 98.8 98.8 98.6 98.2 98.6 98.6 99.3 98.6 73.7 73.7 73.7 73.0 73.0 71.9
73.9 73.7 73.9 73.5 73.5 73.9 73.9 73.7 73.9 73.0 73.5 98.0 98.0 98.0 86.6 86.6 83.8
74.8 74.8 74.8 74.3 74.3 74.8 74.8 74.6 74.8 73.9 74.3 93.4 93.4 93.4 88.1 88.1 85.5
73.0 72.6 73.0 73.0 73.0 73.0 72.6 73.0 73.0 72.4 72.8 85.9 85.9 85.9 99.1 99.1 87.7
73.0 72.4 73.0 72.8 72.8 73.0 72.6 72.8 73.0 72.2 72.6 85.1 85.1 85.1 89.0 89.0 98.2
4. Discussion HBoV is a newly identified virus that was first described in 2005 in nasopharyngeal aspirate of children with respiratory tract infection (Allander et al., 2005). Recently, several articles reported that HBoV were also found in fecal samples (Allander et al., 2005; Lee et al., 2007; Chieochansin et al., 2008; Yu et al., 2008; Huang et al., 2010; Jartti et al., 2011; Jin et al., 2011; Pham et al., 2011; Wang et al., 2011). Data for molecular surveillance of HBoV in Chiang Mai, Thailand, in 2002–2004 demonstrated that only one (1.2%) case of HBoV was found from a total of 82 specimens tested (Pham et al., 2011), and in 2011 (present study) the prevalence increases up to 7.7%. The discrepancy between the present and the previous studies is the primers used for PCR screening for HBoV and the specimens tested. The present study used the consensus primers that have been proved to be specific for all four HBoV species (Kapoor et al., 2010), while the primers specific for HBoV1 was used in the previous study (Pham et al., 2011). Additionally, the stool samples included in the previous study, collected in 2002– 2004 were only tested negative for other enteric viruses. Numerous studied reported that HBoV is commonly co-infected with some other diarrheal viruses, with the rates of co-infection ranging from 21% to 80%. The report from Brazil demonstrated that 21.4% of HBoV was co-infected with rotavirus, norovirus, or adenovirus (Albuquerque et al., 2007). A report from China also demonstrated that about 80% (35 out of 44) of HBoVs isolated from acute gastroenteritis patients were co-infected with other viral pathogens (Wang et al., 2011). Likewise, the present study demonstrated that double- and triple-infections were found in 10 out of 17 (58.8%) stool specimens and the majority of HBoV positive samples were found to be co-infected with norovirus GII. This finding is again confirmed that co-infection among HBoV and other viral pathogens is not uncommon for HBoV. Sequence alignment of the HBoV strains revealed that four species of HBoVs (HBoV1-4) have been documented (Kapoor et al., 2010). The accumulated epidemiological studies of HBoV detected both in respiratory secretion and stool samples worldwide indicate that HBoV1 is predominantly found to be a respiratory pathogen. In addition, HBoV2 and HBoV1 have been found mainly in stool samples, whereas HBoV3 and HBoV4 are occasionally detected (Allander et al., 2007; Allander, 2008; Han et al., 2009; Kapoor et al., 2010; Jartti et al., 2011; Jin et al., 2011). Two recent epidemiological studies of HBoV from acute gastroenteritis children in
China reported that HBoV2 was found to be the most common type (9.0–20.4%), followed by HBoV1 (2.5–4.3%) and HBoV3 (0.5–0.9%), while HBoV4 was not detected (Jin et al., 2011; Wang et al., 2011). The high prevalence of HBoV2 in acute gastroenteritis children was confirmed by the surveillance in Australia and South Korea (Arthur et al., 2009; Han et al., 2009). Our study demonstrated that panbocavirus primer set is a consensus primer which specific for multiple species of HBoV. It was observed that HBoVs detected in Thailand were genetically variable and all four species were found in the specimens collected in 2011 from children hospitalized with diarrhea. Among these, the prevalence of HBoV1 was obviously higher than those of HBoV2 and HBoV3. Interestingly, HBoV4, a candidate novel species of HBoV, had also been found in one clinical specimen in this study. This HBoV4 strain showed a close genetic relationship with that of HBoV4 US-MN-964-05, previously reported in the US. This is the first report of HBoV4 infecting pediatric patient with acute gastroenteritis in Asia, suggesting that HBoV4 has a worldwide distribution. Nevertheless, full-genome sequence analysis may need to be conducted to see whether these HBoVs circulating in Thailand are recombinant strains or belong to the new HBoV variants. A recent review of HBoV demonstrated that variable spectrum of symptoms cause by HBoV infections have been described to associate mainly to respiratory diseases and diarrhea. However, the differences in severity of clinical symptoms cause by HBoV1, HBoV2, HBoV3, and HBoV4 with the diseases are still unclear (Jartti et al., 2011). In this study, unfortunately, analysis and comparison of the clinical significance between single-, double-, and tripleinfections, and the correlation with the clinical symptoms among different HBoV species could not be evaluated due to the lack of availability of clinical data. In summary, this study presents the genetic background information of HBoV in pediatric patients with acute gastroenteritis in Chiang Mai, Thailand and reveals a great genetic diversity of HBoVs circulated in this area although a short period of the surveillance study (8 months) was conducted. Therefore, the continue comprehensive screening as well as genetic molecular characterization of other genes among HBoV strains circulating in this area need to be further performed in order to monitor the incidence and genetic diversity in each HBoV species, and also its clinical significance in patients with diarrhea. The data obtained will be helpful for confirmation of genetic diversity and association of this viral agent with acute gastroenteritis in humans.
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Fig. 1. Construction of the phylogenetic analysis of the partial nucleotide sequence encoding the VP1 region of HBoVs. The tree was generated using the neighbor-joining algorithm, in MEGA 5. Scale bar indicates nucleotide substitutions per site and bootstrap values (>80) are indicated for the corresponding nodes. The HBoV strains detected in the present study are presented in boldface.
Acknowledgement This research was supported by a grant for Medical Research, Faculty of Medicine, Chiang Mai University, Thailand. References Albuquerque, M.C., Rocha, L.N., Benati, F.J., Soares, C.C., Maranhão, A.G., Ramírez, M.L., Erdman, D., Santos, N., 2007. Human bocavirus infection in children with gastroenteritis, Brazil. Emerg. Infect. Dis. 13, 1756–1758.
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Contents lists available at SciVerse ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Circulating of human bocavirus 1, 2, 3, and 4 in pediatric patients with acute gastroenteritis in Thailand Pattara Khamrin a, Rungnapa Malasao a, Natthawan Chaimongkol a, Nuthapong Ukarapol b, Tipachan Kongsricharoern c, Shoko Okitsu d, Satoshi Hayakawa d, Hiroshi Ushijima d, Niwat Maneekarn a,⇑ a
Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand Department of Pediatrics, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand Pediatric Hematology Unit, Nakornping Hospital, Chiang Mai, Thailand d Division of Microbiology, Department of Pathology and Microbiology, Nihon University School of Medicine, Tokyo, Japan b c
a r t i c l e
i n f o
Article history: Received 13 December 2011 Received in revised form 13 January 2012 Accepted 29 January 2012 Available online 8 February 2012 Keywords: Bocavirus Diarrhea Children Thailand
a b s t r a c t Human bocavirus (HBoV) is a newly identified human parvovirus that associated with respiratory and gastrointestinal diseases. Epidemiological surveillance of HBoV was conducted on fecal specimens collected from hospitalized children with diarrhea in Chiang Mai, Thailand in 2011. Among a total of 222 fecal specimens tested, 17 (7.7%) were positive for HBoV by PCR. Of the 17 HBoV positive samples, double- or triple-infections together with other enteric viruses were found in 10 (58.8%) pediatric patients, while monoinfection with HBoV alone was detected in seven (41.2%) cases. Mixed infection among HBoV with norovirus GII was frequently observed in this population. The partial VP1 nucleotide sequences of all 17 HBoV strains demonstrated that all four species of HBoV were found in the specimens tested. Eleven strains were HBoV1. Other three strains showed the sequence identity with HBoV2, which were most closely related to the HBoV2A. In addition, other two HBoV strains showed the highest level of nucleotide sequence identity with the HBoV3. It was surprisingly to observe that one Thai HBoV strain showed a unique characteristic similar to the HBoV4, a rare species of HBoV found in acute gastroenteritis patients. In summary, this study presents the genetic background information of HBoV circulated in acute gastroenteritis children in Chiang Mai, Thailand and it was clearly demonstrated that HBoVs circulated in this area were genetically diverse as all four species of HBoVs (HBoV1–4) were detected in the fecal specimens collected from pediatric patients admitted to the hospitals in this area. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Gastroenteritis is one of the most common causes of morbidity and mortality in children and accounts for two to three million deaths per year which are most occurring in infants and young children world wide (Kosek et al., 2003). The majority of acute gastroenteritis is caused by virus infections. Among these, rotavirus, calicivirus (norovirus and sapovirus), enteric adenovirus, and astrovirus have been reported as the most important etiologic viral agents (Clark and McKendrick, 2004; Dennehy, 2011). Recently, there are several reports of newly discovered enteric viruses, which are potentially associated with acute gastroenteritis including human bocavirus (HBoV). HBoV is a newly identified human parvovirus that was first described in 2005 in nasopharyngeal aspi⇑ Corresponding author. Address: Department of Microbiology, Faculty of Medicine, Chiang Mai University, 110 Inthawarorose, Sripoom, Muang, Chiang Mai 50200, Thailand. Tel.: +66 53 945332; fax: +66 53 217144. E-mail address: [email protected] (N. Maneekarn). 1567-1348/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2012.01.025
rate of children with respiratory tract infection (Allander et al., 2005). Several articles have confirmed recently that HBoV has also been detected in fecal samples (Allander et al., 2005; Lee et al., 2007; Chieochansin et al., 2008; Yu et al., 2008; Huang et al., 2010; Jartti et al., 2011; Jin et al., 2011; Pham et al., 2011; Wang et al., 2011). HBoV is classified in the family Parvoviridae, which includes small non-enveloped, icosahedral viruses with singlestranded DNA genome of about 5.3 kb. The genome encodes two forms of nonstructural proteins, NS1 and NP1, as well as two major structural proteins, VP1 and VP2 (Berns and Parrish, 2007; Chen et al., 2010; Kapoor et al., 2010). Currently, HBoVs have been classified into four species, HBoV1, HBoV2, HBoV3, and HBoV4. The HBoV2 can be further divided into two groups, 2A and 2B (Kapoor et al., 2010). HBoV1 is predominantly found to be a respiratory pathogen. In addition, HBoV2 and HBoV1 have also been found mainly in stool samples, whereas HBoV3 and HBoV4 are occasionally be detected (Allander et al., 2007; Allander, 2008; Han et al., 2009; Kapoor et al., 2010; Jartti et al., 2011; Jin et al., 2011). This statement was supported by
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seroepidemiological study of HBoV1–4 in adults and children with respiratory tract infection. The data demonstrated that antibody titer to HBoV1 was higher than those to HBoV2–4 (Kantola et al., 2011). In addition, a report from the US revealed that HBoV1, 2, and 3 were detected in untreated sewage water throughout the country (Blinkova et al., 2009). In Thailand, few molecular epidemiological studies of HBoV have been conducted. The studies from three different epidemiological areas (Bangkok, Chiang Mai, and Buriram) revealed that HBoV detection rates in acute gastroenteritis patients were varied from 0.9–2.3%. Based on the nucleotide sequence analysis, HBoV identified in this country mainly belonged to HBoV1 and HBoV2 (Chieochansin et al., 2008; Pham et al., 2011). The previous study of HBoV in Chiang Mai, Thailand in 2002–2004 revealed that only one specimen was positive for HBoV1 (Pham et al., 2011). In order to gain the overview genetic backgrounds of HBoV diversity and distribution in Thailand, we conducted the epidemiological study of HBoV in children with diarrhea in Thailand in 2011. Sequence and phylogenetic analyses of HBoVs detected in the present study were further characterized for their genetic evolutionary relationships with the viruses circulating in this area and HBoV reference strains. 2. Materials and methods 2.1. Specimen collection A total of 222 stool specimens were collected from children with diarrhea attending two hospitals in Chiang Mai city, Thailand. Only the pediatric patients who had a clinical diagnosis of acute gastroenteritis with watery diarrhea have been included in this study. The study period was from January to August, 2011. The ages of the patients enrolled in this study ranged from neonate up to 5 years old. All the collected specimens were also screened for other diarrheal viruses including group A, B, C rotaviruses, adenovirus, norovirus GI and GII, sapovirus, astrovirus, Aichi virus, human parechovirus and enterovirus based on the protocols described previously (Yan et al., 2003, 2004; Pham et al., 2010). The study was conducted with the approval of the ethical committee for human rights related to human experimentation, Faculty of Medicine, Chiang Mai University (No. 181/2554). 2.2. Bocavirus detection and genome characterization The viral genomes were first extracted from 10% fecal suspension supernatant using the QIAamp viral RNA Mini Kit (Qiagen, Germany). The presence of bocavirus in stool specimens was detected by polymerase chain reaction (PCR) using pan-bocavirus PCR primers described previously (Kapoor et al., 2010). All of the detected viruses were analyzed for their species by nucleotide sequence and phylogenetic analyses of the partial sequence encoding the VP1 region. Briefly, a forward primer AK-VP-F1 (50 -CGCCGT GGCTCCTGCTCT-30 ) was used in combination with the reverse primer AK-VP-R1 (50 -TGTTCGCCATCACAAAAGATGTG-30 ) for the first PCR. The nested-PCR was performed by using AK-VP-F2 (50 -GGCTC CTGCTCTAGGAAATAAAGAG-30 ) and AK-VP-R2 (50 -CCTGCTGTTAGG TCGTTGTTGTATGT-30 ) primers which targeted the partial VP1 region of HBoV. The PCR amplicons were purified with a Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI) and sequenced using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) on an automated sequencer (ABI 3100; Applied Biosystems, Foster City, CA). The sequences were compared with those of reference strains available in the NCBI GenBank database using BLAST server (http://www.ncbi.nlm.nih.gov/blast). Phylogenetic and molecular evolutionary relationships were evaluated by using MEGA 5 (Tamura et al., 2011).
2.3. Nucleotide sequence accession numbers The partial nucleotide sequences of HBoV VP1 gene described in the present study were deposited in the GenBank database. The accession numbers are JQ267773–JQ267789. 3. Results Of a total of 222 fecal specimen tested, 17 (7.7%) were positive for HBoV by PCR. Among 17 HBoV positive samples, double- or triple-infections together with other enteric viruses were found in 10 (58.8%) pediatric patients, while monoinfection with HBoV alone was detected in seven (41.2%) cases. Mixed infection among HBoV with norovirus GII was frequently observed in this population (Table 1). The partial VP1 nucleotide sequences (457 nucleotides long) of all 17 HBoV strains were determined and compared with those of four established HBoV species, including HBoV1, HBoV2 (2A and 2B), HBoV3, HBoV4 (Kapoor et al., 2010). The sequence comparison shown in Table 1 indicates that the partial VP1 nucleotide sequences of all 17 HBoV strains were divided into four species. Eleven strains (CMH-N006-11, CMH-N007-11, CMH-N009-11, CMH-N010-11, CMH-N062-11, CMH-N077-11, CMH-N079-11, CMH-N081-11, CMH-N096-11, CMH-N098-11, and CMH-S03111) were closely related to the HBoV1 reference strain (TU-A110-08) at 98.2–99.5%. Other three HBoVs (CMH-N118-11, CMH-N129-11, and CMH-N133-11) showed high sequence identity with HBoV2 ranging from 93.4% to 98.0%. It was, however, observed that within HBoV2 species, all three Thai HBoV strains showed the highest level of nucleotide sequence identity with the HBoV2A (TU-A-146-06) at 98.0% while exhibited lower sequence identity with the HBoV2B (US-MN-510-05) at only 93.4%. In addition, the partial nucleotide sequences of other two HBoV strains (CMH-N005-11 and CMH-N008-11) found in this study showed highest level of nucleotide sequence identity with the HBoV3 reference strain (US-MN-647-06) at 99.1%. It was surprisingly to observe that one Thai HBoV strain (CMH-S011-11) showed a unique characteristic of nucleotide sequence identity that most closely related to the HBoV4 reference strain (USMN-964-05), a rare species of HBoV found in acute gastroenteritis patients, at 98.2%. A phylogenetic tree constructed from the partial nucleotide sequences of the four HBoV reference strains and those of 17 HBoV Thai strains is shown in Fig. 1. It was clearly confirmed that HBoVs detected in Thailand were genetically diversified and all four species were circulating in children hospitalized with diarrhea. HBoV1 was the most common species as 11 strains clustered exclusively in the same branch with HBoV1 reference strains. HBoV2, which has been reported previously as a major HBoV species related to diarrhea also found in our study. Three HBoV2 clustered closely with the HBoV2A than 2B reference strains and appeared to be most closely related to the Thai (CU47TH) and Chinese (LZ53819) HBoV2A strains. In addition, this phylogenetic analysis also provided a molecular basis of genetic relationship of the two HBoV strains found in this study with that of HBoV3 Chinese strain BCH1978. Moreover, the only one Thai HBoV4 (CMH-S011-01) formed the closest genetic cluster with HBoV4 reference strains and two major lineages were found among HBoV4 species reported so far. One lineage included the majority of HBoV4 strains identified from several parts of the world, including the US, Nigeria, and Tunisia, while another lineage was formed exclusively by the US-MN-964-05 (reported previously in the US) and CMH-S011-11 (Thai HBoV found in this study). This finding implies that there might be at least two separate variants of HBoV4 circulating in nature, as previously proposed by Kapoor et al. (2010).
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Table 1 Co-infected viruses and the partial VP1 nucleotide sequence identities between HBoV strains found in this study and the HBoV reference strains. The nucleotide sequence identities over 90% between the HBoV strains detected in the present study and the reference strains are presented in boldface. Sample code
CMH-N006-11 CMH-N007-11 CMH-N009-11 CMH-N010-11 CMH-N062-11 CMH-N077-11 CMH-N079-11 CMH-N081-11 CMH-N096-11 CMH-N098-11 CMH-S031-11 CMH-N118-11 CMH-N129-11 CMH-N133-11 CMH-N005-11 CMH-N008-11 CMH-S011-11
Co-infection
Norovirus GII Norovirus GII None None Rotavirus A Rotavirus A None None Norovirus GII, adenovirus Norovirus GII None Norovirus GII Human parechovirus None Norovirus GII Norovirus GII, rotavirus A None
Nucleotide sequence identity (%) HBoV1 TU-A-110-08
HBoV2A TU-A-146-06
HBoV2B US-MN-510-05
HBoV3 US-MN-647-06
HBoV4 US-MN-964-05
98.6 99.5 98.6 98.8 98.8 98.6 98.2 98.6 98.6 99.3 98.6 73.7 73.7 73.7 73.0 73.0 71.9
73.9 73.7 73.9 73.5 73.5 73.9 73.9 73.7 73.9 73.0 73.5 98.0 98.0 98.0 86.6 86.6 83.8
74.8 74.8 74.8 74.3 74.3 74.8 74.8 74.6 74.8 73.9 74.3 93.4 93.4 93.4 88.1 88.1 85.5
73.0 72.6 73.0 73.0 73.0 73.0 72.6 73.0 73.0 72.4 72.8 85.9 85.9 85.9 99.1 99.1 87.7
73.0 72.4 73.0 72.8 72.8 73.0 72.6 72.8 73.0 72.2 72.6 85.1 85.1 85.1 89.0 89.0 98.2
4. Discussion HBoV is a newly identified virus that was first described in 2005 in nasopharyngeal aspirate of children with respiratory tract infection (Allander et al., 2005). Recently, several articles reported that HBoV were also found in fecal samples (Allander et al., 2005; Lee et al., 2007; Chieochansin et al., 2008; Yu et al., 2008; Huang et al., 2010; Jartti et al., 2011; Jin et al., 2011; Pham et al., 2011; Wang et al., 2011). Data for molecular surveillance of HBoV in Chiang Mai, Thailand, in 2002–2004 demonstrated that only one (1.2%) case of HBoV was found from a total of 82 specimens tested (Pham et al., 2011), and in 2011 (present study) the prevalence increases up to 7.7%. The discrepancy between the present and the previous studies is the primers used for PCR screening for HBoV and the specimens tested. The present study used the consensus primers that have been proved to be specific for all four HBoV species (Kapoor et al., 2010), while the primers specific for HBoV1 was used in the previous study (Pham et al., 2011). Additionally, the stool samples included in the previous study, collected in 2002– 2004 were only tested negative for other enteric viruses. Numerous studied reported that HBoV is commonly co-infected with some other diarrheal viruses, with the rates of co-infection ranging from 21% to 80%. The report from Brazil demonstrated that 21.4% of HBoV was co-infected with rotavirus, norovirus, or adenovirus (Albuquerque et al., 2007). A report from China also demonstrated that about 80% (35 out of 44) of HBoVs isolated from acute gastroenteritis patients were co-infected with other viral pathogens (Wang et al., 2011). Likewise, the present study demonstrated that double- and triple-infections were found in 10 out of 17 (58.8%) stool specimens and the majority of HBoV positive samples were found to be co-infected with norovirus GII. This finding is again confirmed that co-infection among HBoV and other viral pathogens is not uncommon for HBoV. Sequence alignment of the HBoV strains revealed that four species of HBoVs (HBoV1-4) have been documented (Kapoor et al., 2010). The accumulated epidemiological studies of HBoV detected both in respiratory secretion and stool samples worldwide indicate that HBoV1 is predominantly found to be a respiratory pathogen. In addition, HBoV2 and HBoV1 have been found mainly in stool samples, whereas HBoV3 and HBoV4 are occasionally detected (Allander et al., 2007; Allander, 2008; Han et al., 2009; Kapoor et al., 2010; Jartti et al., 2011; Jin et al., 2011). Two recent epidemiological studies of HBoV from acute gastroenteritis children in
China reported that HBoV2 was found to be the most common type (9.0–20.4%), followed by HBoV1 (2.5–4.3%) and HBoV3 (0.5–0.9%), while HBoV4 was not detected (Jin et al., 2011; Wang et al., 2011). The high prevalence of HBoV2 in acute gastroenteritis children was confirmed by the surveillance in Australia and South Korea (Arthur et al., 2009; Han et al., 2009). Our study demonstrated that panbocavirus primer set is a consensus primer which specific for multiple species of HBoV. It was observed that HBoVs detected in Thailand were genetically variable and all four species were found in the specimens collected in 2011 from children hospitalized with diarrhea. Among these, the prevalence of HBoV1 was obviously higher than those of HBoV2 and HBoV3. Interestingly, HBoV4, a candidate novel species of HBoV, had also been found in one clinical specimen in this study. This HBoV4 strain showed a close genetic relationship with that of HBoV4 US-MN-964-05, previously reported in the US. This is the first report of HBoV4 infecting pediatric patient with acute gastroenteritis in Asia, suggesting that HBoV4 has a worldwide distribution. Nevertheless, full-genome sequence analysis may need to be conducted to see whether these HBoVs circulating in Thailand are recombinant strains or belong to the new HBoV variants. A recent review of HBoV demonstrated that variable spectrum of symptoms cause by HBoV infections have been described to associate mainly to respiratory diseases and diarrhea. However, the differences in severity of clinical symptoms cause by HBoV1, HBoV2, HBoV3, and HBoV4 with the diseases are still unclear (Jartti et al., 2011). In this study, unfortunately, analysis and comparison of the clinical significance between single-, double-, and tripleinfections, and the correlation with the clinical symptoms among different HBoV species could not be evaluated due to the lack of availability of clinical data. In summary, this study presents the genetic background information of HBoV in pediatric patients with acute gastroenteritis in Chiang Mai, Thailand and reveals a great genetic diversity of HBoVs circulated in this area although a short period of the surveillance study (8 months) was conducted. Therefore, the continue comprehensive screening as well as genetic molecular characterization of other genes among HBoV strains circulating in this area need to be further performed in order to monitor the incidence and genetic diversity in each HBoV species, and also its clinical significance in patients with diarrhea. The data obtained will be helpful for confirmation of genetic diversity and association of this viral agent with acute gastroenteritis in humans.
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Fig. 1. Construction of the phylogenetic analysis of the partial nucleotide sequence encoding the VP1 region of HBoVs. The tree was generated using the neighbor-joining algorithm, in MEGA 5. Scale bar indicates nucleotide substitutions per site and bootstrap values (>80) are indicated for the corresponding nodes. The HBoV strains detected in the present study are presented in boldface.
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