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Establishment of a reverse genetics system for studying human bocavirus in human airway epithelia.

ABSTRACT: Human bocavirus 1 (HBoV1) has been identified as one of the etiological agents of wheezing in young children with acute respiratory-tract infections. In this study, we have obtained the sequence of a full-length HBoV1 genome (including both termini) using viral DNA extracted from a nasopharyngeal aspirate of an infected patient, cloned the full-length HBoV1 genome, and demonstrated DNA replication, encapsidation of the ssDNA genome, and release of the HBoV1 virions from human embryonic kidney 293 cells. The HBoV1 virions generated from this cell line-based production system exhibits a typical icosahedral structure of approximately 26 nm in diameter, and is capable of productively infecting polarized primary human airway epithelia (HAE) from the apical surface. Infected HAE showed hallmarks of lung airway-tract injury, including disruption of the tight junction barrier, loss of cilia and epithelial cell hypertrophy. Notably, polarized HAE cultured from an immortalized airway epithelial cell line, CuFi-8 (originally derived from a cystic fibrosis patient), also supported productive infection of HBoV1. Thus, we have established a reverse genetics system and generated the first cell line-based culture system for the study of HBoV1 infection, which will significantly advance the study of HBoV1 replication and pathogenesis.

SUBMITTER: Huang Q

PROVIDER: S-EPMC3431310 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Establishment of a reverse genetics system for studying human bocavirus in human airway epithelia.

Huang Qinfeng Q Deng Xuefeng X Yan Ziying Z Cheng Fang F Luo Yong Y Shen Weiran W Lei-Butters Diana C M DC Chen Aaron Yun AY Li Yi Y Tang Liang L Söderlund-Venermo Maria M Engelhardt John F JF Qiu Jianming J

PLoS pathogens 20120830 8

Human bocavirus 1 (HBoV1) has been identified as one of the etiological agents of wheezing in young children with acute respiratory-tract infections. In this study, we have obtained the sequence of a full-length HBoV1 genome (including both termini) using viral DNA extracted from a nasopharyngeal aspirate of an infected patient, cloned the full-length HBoV1 genome, and demonstrated DNA replication, encapsidation of the ssDNA genome, and release of the HBoV1 virions from human embryonic kidney 29 ...[more]

PMID: 22956907

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Project description:Influenza D virus (IDV) was initially isolated in the United States in 2011. IDV is distributed worldwide and is one of the causative agents of the bovine respiratory disease complex (BRDC), which causes high morbidity and mortality in feedlot cattle. The molecular mechanisms of IDV pathogenicity are still unknown. Reverse genetics systems are vital tools not only for studying the biology of viruses, but also for use in applications such as recombinant vaccine viruses. Here, we report the establishment of a plasmid-based reverse genetics system for IDV. We first verified that the 3'-terminal nucleotide of each 7-segmented genomic RNA contained uracil (U), contrary to previous reports, and we were then able to successfully generate recombinant IDV by cotransfecting 7 plasmids containing these genomic RNAs along with 4 plasmids expressing polymerase proteins and nucleoprotein into human rectal tumor 18G (HRT-18G) cells. The recombinant virus had a growth deficit compared to the wild-type virus, and we determined the reason for this growth difference by examining the genomic RNA content of the viral particles. We found that the recombinant virus incorporated an unbalanced ratio of viral RNA segments into particles compared to that of the wild-type virus, and thus we adjusted the amount of each plasmid used in transfection to obtain a recombinant virus with the same replicative capacity as the wild-type virus. Our work here in establishing a reverse genetics system for IDV will have a broad range of applications, including uses in studies focused on better understanding IDV replication and pathogenicity, as well as in those contributing to the development of BRDC countermeasures.IMPORTANCE The bovine respiratory disease complex (BRDC) causes high mortality and morbidity in cattle, causing economic losses worldwide. Influenza D virus (IDV) is considered to be a causative agent of the BRDC. Here, we developed a reverse genetics system that allows for the generation of IDV from cloned cDNAs and the introduction of mutations into the IDV genome. This reverse genetics system will become a powerful tool for use in studies related to understanding the molecular mechanisms of viral replication and pathogenicity and will also lead to the development of new countermeasures against the BRDC.

Project description:The genome of recombinant adeno-associated virus 2 (rAAV2) remains a promising candidate for gene therapy for cystic fibrosis (CF) lung disease, but due to limitations in the packaging capacity and the tropism of this virus with respect to the airways, strategies have evolved for packaging an rAAV2 genome (up to 5.8 kb) into the capsid of human bocavirus 1 (HBoV1) to produce a chimeric rAAV2/HBoV1 vector. Although a replication-incompetent HBoV1 genome has been established as a trans helper for capsid complementation, this system remains suboptimal with respect to virion yield. Here, a streamlined production system is described based on knowledge of the involvement of HBoV1 nonstructural (NS) proteins NS1, NS2, NS3, NS4, and NP1 in the process of virion production. The analyses reveal that NS1 and NS2 negatively impact virion production, NP1 is required to prevent premature termination of transcription of the cap mRNA from the native genome, and silent mutations within the polyadenylation sites of the cap coding sequence can eliminate this requirement for NP1. It is further shown that preventing the expression of all NS proteins significantly increases virion yield. Whereas the expression of capsid proteins VP1, VP2, and VP3 from a codon-optimized cap mRNA was highly efficient, optimal virion assembly, and thus potency, required enhanced VP1 expression, entailing a separate VP1 expression cassette. The final NS protein-free production system uses three-plasmid co-transfection of HEK293 cells, with one trans helper plasmid encoding VP1 and the AAV2 Rep proteins, and another encoding VP2-3 and components from adenovirus. This system yielded >16-fold more virions than the prototypic system, without reducing transduction potency. This increase in virion production is expected to facilitate greatly both research on the biology of rAAV2/HBoV1 and preclinical studies testing the effectiveness of this vector for gene therapy of CF lung disease in large animal models.

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Establishment of a reverse genetics system for studying human bocavirus in human airway epithelia.

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Establishment of a reverse genetics system for studying human bocavirus in human airway epithelia.

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