Genome Characterization Of Bocavirus And The Exploratory Researches On The Construction Of Bocavirus Infectious Clones

Objective:The genus Bocavirus belongs to the family Parvoviridae that cause a variety of diseases in animals. As a novel virus, the pathogenicity of bocavirus has not been recognized clearly, with limited evidence. Given the lack of an effective cell culture or animal model, studies on the replication and pathogenesis of bocavirus remain limited. Construction of infectious molecular clones of bocaviruses has also been hampered by the sequencing barrier represented by the viral genome termini. Porcine bocavirus was first discovered in our lab in2009. Compared to HBoV, PBoV showed high genomic similarity and close biological characteristics. And meanwhile, compared with HBoV, specimens, especially specimens with the high viral load of PBoV were easier to obtain. Therefore, PBoV is an ideal alternative to HBoV for further research. In this study, a novel bocavirus was indentified and its genome characteristics were analyzed. A full-length genomic clone was also constructed based on identification of a porcine bocavirus episome (PBoVG2e) and partial ITR sequence from linear PBoV genome. These exploratory researches will lead to greater understanding of the biological characteristics, pathogenesis and even the strategy for constructing infectious clones of bocavirus in a future study.Methods:1. Genome characterization of a novel porcine bocavirusWe used a high-throughput DNA sequencing method to screen novel species of PBoV in healthy piglet fecal samples. A novel species of PBoV (PBoV3C) with a nearly full-length genome sequence was identified using genome walking method. Phylogenetics, recombination and further secondary structure of VP2of PBoV3C were analyzed and predicted using software and net servers.2. Exploratory researches on the construction of bocavirus infectious clones.â‘  Detected episomes of PBoV in piglet fecal samples using a semi-nested PCR method. Prediction of secondary structure and putative promoters was performed in the noncoding region (NCR).â‘¡Partial ITR sequence of linear PBoV group2genome were obtained using adaptor-PCR assay.â‘¢A full-length genomic clone based on PBoVG2e genomic sequence was constructed using restriction enzyme digestion.â‘£We present real-time PCR assays based on NP1gene for further detection and quantification of PBoVG2e.Result:1. Using a high-throughput DNA sequencing method, one DNA sequence (contig01006), suspected to belong to a novel porcine bocavirus (PBoV), was found with a high rate of detection (19.6%) in healthy piglet fecal samples. Moreover, a novel species of PBoV (tentatively named PBoV3C) with a nearly complete genome sequence (5235bp) was identified. PBoV3C exhibits typical genome characteristics of bocaviruses, and shows the highest genomic identities (78%to81%) with PBoV3A/B (PBoV3/4-UK) and PBoV3D/E (PBoV3/4-HK), respectively. Phylogenetic and recombination analyses indicated a high diversity, prevalence and complexity within PBoVs. Additionally, we propose a uniform method of PBoV nomenclature based on the VP1gene.2.â‘ The episomal forms of two porcine bocaviruses (PBoVG2e and PBoVG3e) were identified in fecal samples of healthy piglets. The relevant terminal sequences of the noncoding region (405and511nt, respectively) were also obtained. Sequence analyses and second structure prediction indicated that the PBoVG2e was more similar to that of HBoV3â‘¡Partial ITR sequence of linear PBoV group2genome were obtained using adaptor-PCR assay, therefore, the head-tail junction of these circular episomes was located.â‘¢A full-length genomic clone based on PBoVG2e sequence was constructed and verified correctly by sequencing.â‘£A sensitive and stable real-time PCR assay for further detection and quantification of PBoVG2e was also constructed in this study.Conclusion:In this study, a novel species of PBoV (PBoV3C) with a nearly full-length genome sequence was identified. Its biological characterization, phylogenetics and secondary structure of VP2protein were analyzed and predicted. We also propose a uniform PBoV nomenclature based on the VP1gene. Furthermore, a full-length genomic clone based on PBoVG2e sequence was constructed. The corresponding real-time PCR assay for PBoVG2e was also carried out. Our study contributes to a possible alternative strategy for constructing infectious clones of bocavirus in a future study.