key: cord-0940475-x8t8a2r4 authors: Lin, Jih-Hui; Chiu, Shu-Chun; Lin, Yung-Cheng; Chen, Hsiu-Lin; Lin, Kuei-Hsiang; Shan, Kuo-Hao; Wu, Ho-Sheng; Liu, Hsin-Fu title: Clinical and genetic analysis of Human Bocavirus in children with lower respiratory tract infection in Taiwan date: 2009-03-31 journal: Journal of Clinical Virology DOI: 10.1016/j.jcv.2008.12.018 sha: ec19eeac8c7143dd23b95e00afa811002687b8ff doc_id: 940475 cord_uid: x8t8a2r4 Abstract Background Human Bocavirus (HBoV) is a likely etiologic agent of acute respiratory disease in children. The prevalence of this virus has been studied in several sites worldwide. We conducted the first clinical and molecular study of HBoV in Taiwan at the Centers for Diseases Control, Taiwan. Objectives To investigate the genomic and epidemiologic profiles of HBoV infection in Taiwan. Study design Throat swabs or nasopharyngeal aspirates were obtained from hospitalized pediatric patients with acute lower respiratory tract infections. Specimens negative for other respiratory viruses by molecular screening were examined for HBoV. Results HBoV was the only virus detected in 30 (5.6%) of 531 samples. Of these positive cases, 56.7% were from children less than 2 years old. Two groups of HBoV co-circulated in Taiwan during the study. Results of evolutionary networks evaluation suggest that HBoV might have had an opportunity for interbreeding of viruses and genetic recombinations among the different genes. Conclusion HBoV may have circulated in Taiwan for some time and it appears to be one of the etiological agents responsible for lower respiratory tract infection in children. Human Bocavirus (HBoV), currently classified in the genus Bocavirus within the family Parvoviridae, was first described in 2005 after large-scale molecular screening of respiratory specimens for virus genome sequences led to its discovery. 1 The etiologic role of HBoV in the causation of respiratory illness is not clearly defined due to lack of viral propagation techniques in cell culture or animal models. 1 However, many studies have reported finding this virus associated with respiratory tract infections, especially in infants and young children. [1] [2] [3] [4] [5] . The virus has been reported worldwide, [6] [7] [8] [9] [10] [11] with incidence rates determined by nucleic acid amplification ranging from 3 to 19%. 7, [12] [13] [14] [15] Recently, the virus has also been identified in blood and faecal samples. 12, [16] [17] [18] [19] Epidemiological data reported in most previous studies have been generated using the NP1 gene for HBoV detection. However, the complete coding sequence of this virus in the database is incomplete, and thus, the true prevalence of the virus may be slightly underestimated. 1 In order to better understand the genomic and epidemiological profiles of HBoV infection in Taiwan, we examined specimens from pediatric patients with lower respiratory tract infections (LRTIs). The genes of HBoV were determined using a newly designed set of conserved primers to both amplify and sequence. All experiments involving human material were approved by an ethical commission from the local partner university hospitals. Clinical specimens were obtained from pediatric patients hospitalized in Taiwan Viral DNA was extracted using either the QIAamp DNA Mini Kit (Qiagen, Santa Clara, CA) or an automated MagNA Pure LC instrument using the MagNA Pure LC Total Nucleic Acid Isolation kit TW2739 06 S TW2835 06 S TW2888 06 S TW2953 06 S Ic TW830 06 K S T TW2836 06 S T TW3003 06 S T TW3023 06 S T TW925 07 S T TW125 07 S T TW141 07 S T a Deduced amino acid difference in the HBoV, only locations where differences from the sequence of ST2 were observed are indicated. The PCR product was purified using QIA quick spin columns (Qiagen, Valencia, CA, USA). Purified amplicons were cycle sequenced by using BigDye 3.1 Terminator cycle sequencing reagents, with reaction products resolved on an ABI Prism 3130XL DNA Analyzer (Applied Biosystems, Forest City, CA, USA). For phylogenetic analysis, regions of the NS1, NP1, and VP1/VP2 genes were amplified and sequenced. Four primers were used to amplify and sequence the NS1 gene, and two primers were used to amplify and sequence the NP1 gene. Primer sequences are shown in Table 1 . In addition, primers from a previous publication 22 were used to amplify and sequence VP1/VP2 genes. Complete coding sequences were determined according to a previous report 23 . Nucleotide sequence data were analyzed using Version 10.3 of the sequence analysis software package of the University of Wisconsin Genetic Group. Phylogeny construction and evaluation were performed with the maximum likelihood (ML) and neighbor-joining (NJ) methods in the Phylip software package (Version 3.66, University of Washington, Seattle, WA, USA) 24 and Bayesian analysis in the Mr. Bayes software (Version 3.1). 25, 26 Empirical transition/transversion ratio was estimated by the TREE-PUZZEL software (Version 5.2) 27 to calculate evolutionary distances. The robustness of the NJ tree was statistically evaluated by bootstrap analysis with 1000 bootstrap samples. Since the ML method is already a statistically validated method (with a statistical evaluation of the branch length), no bootstrapping was done for this method. For Bayesian analysis, nucleotide substitution model was estimated by MrModeltest (Version 2.2). 28 Split decomposition in the Splits tree (Version 4.8, www.splitstree.org) 29 and BootScan in the SimPlot (Version 3.5.1, SCRoftware) 30 were used to detect evolutionary networks. The nucleotide sequences obtained in this study have been submitted to GenBank and have been assigned accession numbers EU984231-EU984245. The age range of all 531 patients studied was 1 month to 16 years. HBoV was the only virus isolated in 30 (5.6%) of the 531 cases. The medical records of the 30 patients with samples positive only for HBoV were reviewed. All 30 HBoV-positive cases were less than 8 years of age, and 17 (57%) were less than 2 years of age. Mean age (±S.D.) was 4.32 ± 2.98 years. The youngest HBoVpositive patient was 40 days of age at the time the respiratory specimen was obtained. The male:female ratio was 1.18:1. Twentyfive of the 30 HBoV-positive cases had pneumonia and 5 had acute bronchitis/bronchiolitis. Fever, cough and rhinorrhea were the most common symptoms. Days of fever ranged from 1 to 8 days (Table 2) . HBoV isolates obtained in this study were very similar, with highly conserved sequences among the different isolates. The 5.2 kb genome of HBoV contains 3 ORFs (open reading frames) encoding NS1, NP1, and VP1/VP2 genes. Phylogenetic analysis of these 3 ORFs in 15 HBoV strains did not reveal any genotypic differences between the strains from Taiwan and other countries (similarity 99-100%). Analysis of the nucleotide sequences encoding the complete genome (positions 1-5262) of Taiwanese strains showed that most of strains were genetically close to ST1 or ST2 (GenBank accession number DQ0000495 and DQ0000496, identified by Allander et al. 1 , with the phylogenetic tree clearly divided into two major groups (I and II) (Fig. 1A ) . Group I could be further divided into three subgroups, the first comprised of ST1 and TW674 07viruses (Ia), the second comprised of ST2 viruses (Ib), and the third comprised of 7 Taiwanese strains (Ic). Group II was comprised of 3 strains (TW2715 06, TW2717 06, and CU74). These data demonstrate that at least 3 clusters of HBoV co-circulated in Taiwan during the study period. Amino acid sequences encoded by the NS1, NP1, VP1, and VP2 genes are shown in Table 3 . All 7 Taiwanese strains in group Ic had amino acid changes at position S590T of VP1 numbering in comparison with sequence of ST2 (Table 3) . Similarity plots of the HBoV TW2717 06 sequence with CU74 and CU74W consensus sequences suggested the presence of one point of crossover (Fig. 2B) with the most likely breakpoints located at 1500-1600 bp. This report is the first to evaluate HBoV infection in Taiwan. The percentage of HBoV-positive specimens among children with LRTIs was similar to that found in other studies of HBoV infection. In our study, HBoV was the only virus detected in nearly 10% of children less than 2 years old with LRTIs, a greater percentage than reported in other studies from Asia. 22, [31] [32] [33] It is possible that there were additional coinfections not detected in our study since we did not test our respiratory samples for other potential pathogens such as rhinoviruses and enteroviruses. Our findings support the hypothesis that HBoV might play a role in the pathogenesis of LRTIs. Further studies are required to determine whether HBoV plays a causative role in LRTI or acts as an exacerbating factor that simple increases the severity of infections caused by other pathogens. Phylogenetic analysis demonstrates that at least 2 groups and 3 clusters of one group of HBoV co-circulated during the study period. There was no temporal link between any of the groups as they were equally distributed throughout the study period. Each individual gene (Fig. 1) of HBoV was constructed and our results show that the greatest sequence variations occurred in the VP1/VP2 gene. Furthermore, our findings indicate that the two different groups of HBoV identified in Taiwan are genetically distinct. One is remarkably similar to the initial strains of HBoV identified in Sweden and the other one is similar to the strain (CU74) detected in Thailand. Likelihood-mapping analysis suggests that each ORF may have some "unresolved" phylogenetic signal, and that NS1 may not be the best gene with which to construct a phylogenetic tree. Phylogenetic trees constructed by NJ and ML methods showed a similar topology. A monophyletic group with longer branch length consisted only of Taiwanese HBoV isolates suggests that this virus might have circulated in Taiwan for a certain period (Fig. 1A) . Moreover, computing the evolutionary networks from sequence data by applied splitstree software, 29 there appeared to be at least 2 networks among Asian strains ( Fig. 2A) . This finding suggests that the virus might have circulated simultaneously in several locations and that simultaneous infections with different strains might have occurred, resulting in an opportunity for interbreeding of virus and genetic recombination among the viral genes. It should be noted that recombinant HBoV has been reported; however, we are unaware of other HBoV mosaic genomes with a breakpoint pattern similar to the one presented here. This leads to speculation that if the recombinant variant continues to circulate, further genetic evolution may allow more efficient transmission in an exposed population. Our study evaluated the phylogenetic relationships of the complete coding sequences of HBoV as well as each gene, and clearly demonstrates the value of analyzing the complete genetic composition in order to understand fully its molecular epidemiology. In conclusion, our data demonstrate that HBoV is circulating in Taiwan and suggest that HBoV might be involved in the pathogenesis of lower respiratory tract disease in children. Furthermore, our results indicate that genetically distinct HBoVs co-circulated in Taiwan during 2006-2007 and that recombinance among these viruses occurred contributing to the genetic diversity of the circulating strains. Importantly, data presented here provide evidence suggesting that HBoV may undergo gene recombination to evade host immunological response. Continued clinical and molecular surveillance in the future will be necessary to monitor the spread and epidemiological impact of HBoV. None of the authors had conflicts of interest. Cloning of a human parvovirus by molecular screening of respiratory tract samples Human bocavirus: prevalence and clinical spectrum at a children's hospital Detection of human bocavirus in Canadian children in a 1-year study Clinical characteristics of human bocavirus infections compared with other respiratory viruses in Spanish children Clinical and epidemiologic characteristics of human bocavirus in Danish infants: results from a prospective birth cohort study Human Bocavirus infection Human bocavirus (HBoV) in Thailand: clinical manifestations in a hospitalized pediatric patient and molecular virus characterization Bocavirus infection in hospitalized children Human bocavirus in French children Isolation of human bocavirus from Swiss infants with respiratory infections Evidence of human coronavirus HKU1 and human bocavirus in Australian children Human bocavirus Human bocavirus infection in young children in the United States: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus Clinical and molecular epidemiology of human bocavirus in respiratory and fecal samples from children in Hong Kong Human bocavirus in infants Role of Human Bocavirus infections in outbreaks of gastroenteritis Human bocavirus in children hospitalized for acute gastroenteritis: a case-control study Human bocavirus, a respiratory and enteric virus Human bocavirus infection in children hospitalized with acute gastroenteritis in China Realtime PCR assays for detection of bocavirus in human specimens Realtime PCR for diagnosis of human bocavirus infections and phylogenetic analysis Human bocavirus infection, People's Republic of China Complete coding sequences and phylogenetic analysis of Human Bocavirus (HBoV) Using the quantitative genetic threshold model for inferences between and within species MRBAYES: Bayesian inference of phylogenetic trees MrBayes 3: Bayesian phylogenetic inference under mixed models TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing Bayesian phylogenetic analysis of combined data Application of phylogenetic networks in evolutionary studies Full-length human immunodeficiency virus type 1 genomes from subtype Cinfected seroconverters in India, with evidence of intersubtype recombination The association of newly identified respiratory viruses with lower respiratory tract infections in Korean children Human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in Thailand Detection of human bocavirus in Japanese children with lower respiratory tract infections This study was funded by Centers for Diseases Control, Taiwan (grant DOH96-DC2402), and was funded in part by the National Science Council (grant NSC 96-2314-B-195-010) and Mackay Memorial Hospital (grant MMH 9724).The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the funding agency