Studies On Epidemiology, Causative Role, And Phyligenetic Analysis Of Human Bocaviruses

Objectives:Acute gastroenteritis (AGE) is one of the leading causes of death in children worldwide. Rotavirus (RV), human calicivirus (HucV), adenovirus (AdV), and a'strovirus (AstV) are the most common viral pathogens detected in infants with AGE. However, a significant proportion of AGE cases have no known etiology. Human bocaviruses (HBoVs) were recently identified in stool samples and involved in the pathogenesis of AGE. But due to the limit data about case-control studies about these bocaviruses, whether HBoV2 is an etiologic agent of gastroenteritis in children or merely a "passer-by" in the intestinal tract remains unclear. The aim of this study was to assess the role of HBoVs in gastroenteritis.Methods:We conducted a case-control study on 632 children with diarrhea and 162 healthy controls in Lanzhou, China. Viruses known or suspected to be agents of AGE, including RV, HucV, AdV, AstV, HBoVs, were detected by ELISA, PCR or RT-PCR. PCR positive samples were sequenced. Real-time PCR was used to quantify the viral load of HBoV2. All statistical analyses were performed using SPSS 11.5. Phylogenetic tree was constructed by using MEGA 4.1 package.Results:In the case group, RV was the most commonly-detected virus (45.3%), followed by HucV (10.1%), AstV (4.9%), and AdV (4.7%). HBoV, HBoV3 and HBoV4 were detected in 27 (4.3%),6 (0.9%), and 0 subjects, respectively. Whilst HBoV2, unexpectedly, was detected at a higher prevalence (129,20.4%) even than HucV. Of a total of 162 asymptomatic subjects, RV was detected in three, AstV in two, HucV and AdV in one each, and HBoV in four. HBoV3 was not detected. HBoV2 was detected at a high prevalence (20,12.3%) in this group. Multivariate logistic regression analysis indicated that both HBoV and HBoV3 was not associated with AGE, and the association between HBoV2 and AGE was weaker (OR= 1.269, CI= 0.704-2.288) than that between AGE and RV, HucV, AdV, or AstV. Mean HBoV2 viral load in the case and control groups was fewer than 55 copies/ml extract.Conclusions:This study identified HBoV2 and HBoV3 for the first time in Chinese children with AGE. HBoV2 exhibit different epidemiological features from HBoV and HBoV3.The data presented herein do not support an important causative role for HBoV2 in AGE, despite its high prevalence in stool samples.Objectives:Human bocavirus 2 (HBoV2) is a parvovirus that has been recently identified in stool samples from children. While it is frequently detected in fecal samples, HBoV2 is rarely detected in respiratory sample. To date, HBoV2 has been detected only by using nested PCR assays, which are laborious and prone to false-positive results. The aim of this study is to develop a sensitive and specific real-time PCR assay.Methods:Stool specimens were collected from 345 children with acute gastroenteritis who were hospitalized in Taiyuan Children Hospital, China, between Nov.2007 and Oct.2008. Primer and probe sets were designed for the conserved region using the Primer Express software. A 587-bp DNA subgenomic fragment of HBoV2 was amplified from a positive stool specimen using primers bracketing the real-time PCR target region in the NP-1 gene. Replicate serial 10-fold dilutions of the pGEM-TNP-1 HBoV2 plasmid were prepared. The real-time HBoV2 PCR assay was compared with a nested-primer PCR using conditions described by Kapoor et al.Results:The assay could reproducibly detect 10 copies of a recombinant DNA plasmid containing a partial region of the HBoV2 genome, with a dynamic range of 8 log units (101 to 108 copies). A clinical evaluation detected HBoV2 in 85 (24.6%) of 345 children with gastroenteritis, with viral loads ranging from 1.67×102 to 4.27×109 copies per mL of stool specimen. With conventional nested PCR,57 (16.5%, 57/345) were positive by gel electrophoresis (with sequencing,52 were confirmed as HBoV2, four as HBoV, and one as HBoV3). Nineteen conventional nested PCR positive specimens were negative for real-time PCR. Of the 19 specimens,16 were HBoV2, We also repeated the real-time PCR assay three times for the 16 HBoV2-positive specimens and found that eight of them turned positive once and the viral loads of the eight specimens were lower than ten copies per reaction, ranging from 2.01×100 to 6.99×100 copies.Conclusions:The assay was sensitive, specific, and reliable for HBoV2 DNA amplification, with a reproducible detection limit of ten target genome copies per reaction and linear amplification over a wide dynamic range, suitable for quantitative applications. In conclusion, we developed a real-time PCR assay that is more sensitive and specific than the reported conventional nested PCR.Objectives:Human bocavirus 2(HBoV2) and other human bocavirus species (HBoV, HBoV3, and HBoV4) have been discovered recently. Previous studies indicated that there is recombination phenomenon among HBoV2 genotypes, HBoV3 may be a hybrid of HBoV and HBoV2, and there are recombination signals in the genome of HBoV4. But due to the limit of sequences data and analysis methods, the precise phylogenetic relationships among these viruses are not clear yet. The aim of this study is to clarify the real phylogenetic relationships among these human bocaviruses.Methods:We collected 632 diarrhea and 162 healthy children in Lanzhou, China. Using PCR, Human bocavirus (HBoV1), HBoV2, HBoV3 and HBoV4 were screened. Complete HBoV2 sequences were amplified by using specific PCR and Genome Walking Kit. Phylogenetic analysis was conducted with Molecular Evolutionary Genetics Analysis (MEGA) version 4.1. Sequences were selected based on their similarity and then aligned and manually adjusted, the alignments were scanned by various algorithms implemented in the RDP3 package. Estimated breakpoints were verified by both the Shimodaira-Hasegawa test and manually checking phylogenetic trees for nonrecombinant segments.Result:The sequences from case and control groups had a high identity with each other. The Phylogenetic tree also indicated that there was no difference in the topological characteristics of HBoV2 between two groups. Results suggested that recombination among HBoV2 genotypes may occur. While we compared the trees of non-recombinant segments associated with the break points estimated by GARD, Shimodaira-Hasegawa test showed the topologies of these trees were not significantly different. Recombinant signals were detected in all of the HBoV, HBoV2, HBoV3 and HBoV4 genome sequences, but the signals in HBoV3 was far more significant than those in other bocaviruses. It was found that HBoV3 was a potential recombinant of HBoV and HBoV4. However, the VP1 and VP2 region of HBoV3 was as similar to HBoV2 as it was to HBoV4.Conclusions:In summary, our data suggested that HBoV2 had higher prevalence than HBoV and HBoV3 in both case and control groups. A single genetic lineagei of HBoV2 is circulating in children with and without gastroenteritis in Lanzhou, China. Recombination between HBoV2 strains doesn't have significant meanings. HBoV3 may be a hybrid virus, originating from HBoV and the common ancestor of HBoV2 and HBoV4.