Establishment Of A Reverse Genetics System For Studying Human Bocavirus And In Vitro Modeling Infection In Human Airway Epithelia

In2005, a previously unknown DNA virus was identified in nasopharyngeal specimens from children with respiratory tract infection by Allander. Researchers used random polymerase chain reaction (PCR) amplification and highthroughput sequencing methods specifically developed for detection of unknown viral sequences. Analysis of the recovered gene sequences showed resemblance to bovine and canine minute parvoviruses, and the virus was named human bocavirus (HBoV). Later,3other HBoV were identified in stool and named HBoV2,3and4. Disease associations of HBoV are not entirely clear, but recent studies provide evidence that HBoVl causes pneumonia and other respiratory tract diseases, in particular during primary infections. HBoV is a small DNA virus with a nonenveloped icosahedral capsid similar to other Parvoviridae. The5kb linear and single-stranded genome is organized in3open reading frames that encode2forms of the nonstructural protein NS1, a nuclear phosphoprotein NP1, and2structural capsid proteins, VP1and VP2. HBoV types2-4have similar genomic organizations as HBoVl and10%-30%sequence dissimilarities. Replication mechanisms of HBoV and the pathogenesis of HBoV infections are poorly characterized. This is largely because no animal model is available and tissue culture of HBoV is difficult. A head-to-tail junction of an HBoV1episome identified in an HBoV1-infected HAE was found to possess two sequences identical to parts of the BPV1left-end hairpin (LEH). This finding suggested that the head sequence is part of the HBoV1LEH.In the current study, we have successfully sequenced the full-length HBoVl genome and cloned it in a plasmid referred to as pIHBoVl. Furthermore, we have demonstrated that transfection of human embryonic kidney293(HEK293) cells with pIHBoV1results in efficient production of HBoVl virions at a high titer, and that these virions are able to productively infect both primary and conditionally transformed polarized HAE. The main results of this study are summarized as follows:A head-to-tail junction of an HBoVl episome identified in an HBoVl-infected HAE was found to possess two sequences identical to parts of the BPV1left-end hairpin (LEH). This finding suggested that the head sequence is part of the HBoV1LEH. We therefore used the head sequence as the3’end of a reverse primer together with a forward primer, which anchors the3’end of the HBoVl genome predicted from the BPV1LEH, we amplified the hairpin of the LEH from a viral DNA extract prepared from a nasopharyngeal aspirate taken from an HBoVl-infected patient. Only one specific DNA band was detected. Sequencing of this DNA revealed a novel sequence of the HBoVl LEH. We thus speculated that the entire HBoV1REH is similar in structure to its MVC counterpart. We were able to amplify a specific～300-bp-long DNA fragment. Sequencing confirmed the presence of the palindromic hairpin of the predicted REH, and revealed two novel nucleotides at the end of the hairpin. These results indicate that we have identified, for the first time, both the LEH and REH of the HBoV1genome from a clinical specimen, and confirm that the HBoVl genome structure is typical of the genus Bocavirus. We also cloned and sequenced the non-structural (NS) and capsid (VP) protein-coding (NSVP) genes of the HBoV1Salvadorl isolate from the patient-extracted viral DNA. We then ligated the LEH, NSVP genes and REH into pBBSmal vector, and refer to this full-length clone as pIHBoV1. We have deposited the sequence of the full-length genome of the isolate in GenBank (JQ923422).We first investigated whether pIHBoV1replicates in HEK293cells. Specifically, we transfected pIHBoVl into HEK293cells (untreated or infected with adenovirus), alone or with pHelper. Interestingly, we found that pIHBoVl replicated well in the absence of helper virus. Indeed, all the three representative forms of replicated bocavirus DNA (DpnI digestion-resistant dRF DNA, mRF DNA and ssDNA, all these DNA forms were observed). DpnI digestion-resistant DNA bands are newly replicated DNA in cells. To confirm the specificity of DNA replication and the identity of the DpnI-resistant DNA bands, we disrupted the ORFs encoding viral proteins NS1and NP1in pIHBoV1; When the NS1ORF was disrupted, no DpnI digestion-resistant DNA was detected, confirming that replication of this DNA requires NS1. Notably, when the NP1ORF was disrupted, an RF DNA band was detected but it was very weak, suggesting that NP1is also involved. The presence of the ssDNA band in pIHBoVl-transfected cells suggested that progeny virions were produced. To prove this, we carried out large-scale pIHBoV1transfection and CsCl equilibrium centrifugation to purify the virus that was produced. Electron microscopy analysis revealed that purified virus displayed a typical icosahedral structure, with a diameter of～26nm.In2008, Dijkman demonstrated that HBoV1infects and replicates in HAE, but limited information of virus infection was obtained in this study. To prove whether the purified HBoVl is infectious, three sets of primary HAE were generated, and these were infected with HBoV1from the apical side of ALI (air-liquid interface). The results demonstrate that the HBoV1virion produced by pIHBoVl transfection is capable of infecting polarized primary HAE cultures from cells derived from various donors. More importantly, we found that productive HBoVl infection was persistent. We also found that productive HBoV1infection disrupted the tight junction barrier, lead to the loss of cilia and airway epithelial cell hypertrophy. These are hallmarks of respiratory tract injury when a loss of epithelial cell polarity occurs.Although HBoV1persistently infects polarized primary HAE, in our previous study, we used a high MOI for the infection because of extensive virus inactivation likely occurred during the CsCl equilibrium ultracentrifugation process. In the current study, to ensure the accuracy of virus infection, we used apical washes collected from the apically infected HAE cultures as a source of virus for infection, in this way, most of the virions were active in the washes, and then we have a relatively accurate MOI for HBoVl infection. We analyzed infection apically at an MOI from100to0.001vgc/cell. Notably, HBoV1virions were released from all these infected HAE cultures. We infected primary HAE basolaterally with apically harvested HBoVl virions as used above at an MOI of1vgc/cell. We found HBoVl can infect primary HAE both apically and basolaterally, even at a low MOI. And that both of the apical infection and basolateral infection are persistently productive, cause loss of the cilia, and ultimately disruption of the tight junction barrier of the epithelium. Thus, our results strongly suggest the HBoV1is a highly infectious respiratory virus.Taken together, in this study, we have identified the terminal hairpins of the HBoV1genome and cloned the full-length HBoV1genome. Virions produced from transfection of this clone into HEK293cells are capable of infecting polarized HAE cultures. Thus, we have established a reverse genetics system that overcomes the critical barriers to studying the molecular biology and pathogenesis of HBoV1, using an in vitro culture model system of HAE. More importantly, we found that productive HBoV1infection was persistent.