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Rescue of a genotype 4 human hepatitis E virus from cloned cDNA and characterization of intergenotypic chimeric viruses in cultured human liver cells and in pigs.

ABSTRACT: Hepatitis E virus (HEV) is an important but extremely understudied human pathogen. Genotypes 1 and 2 are restricted to humans, whereas genotypes 3 and 4 are zoonotic, infecting both humans and pigs. This report describes, for the first time, the successful rescue of infectious HEV in vitro and in vivo from cloned cDNA of a genotype 4 human HEV (strain TW6196E). The complete genomic sequence of the TW6196E virus was determined and a full-length cDNA clone (pHEV-4TW) was assembled. Capped RNA transcripts from the pHEV-4TW clone were replication competent in Huh7 cells and infectious in HepG2/C3A cells. Pigs inoculated intrahepatically with capped RNA transcripts from pHEV-4TW developed an active infection, as evidenced by faecal virus shedding and seroconversion, indicating the successful rescue of infectious genotype 4 HEV and cross-species infection of pigs by a genotype 4 human HEV. To demonstrate the utility of the genotype 4 HEV infectious clone and to evaluate the potential viral determinant(s) for species tropism, four intergenotypic chimeric clones were constructed by swapping various genomic regions between genotypes 1 and 4, and genotypes 1 and 3. All four chimeric clones were replication competent in Huh7 cells, but only the two chimeras with sequences swapped between genotypes 1 and 4 human HEVs produced viruses capable of infecting HepG2/C3A cells. None of the four chimeras was able to establish a robust infection in pigs. The availability of a genotype 4 HEV infectious clone affords an opportunity to delineate the molecular mechanisms of HEV cross-species infection in the future.

SUBMITTER: Cordoba L

PROVIDER: S-EPMC3541786 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Rescue of a genotype 4 human hepatitis E virus from cloned cDNA and characterization of intergenotypic chimeric viruses in cultured human liver cells and in pigs.

Córdoba Laura L Feagins Alicia R AR Opriessnig Tanja T Cossaboom Caitlin M CM Dryman Barbara A BA Huang Yao-Wei YW Meng Xiang-Jin XJ

The Journal of general virology 20120725 Pt 10

Hepatitis E virus (HEV) is an important but extremely understudied human pathogen. Genotypes 1 and 2 are restricted to humans, whereas genotypes 3 and 4 are zoonotic, infecting both humans and pigs. This report describes, for the first time, the successful rescue of infectious HEV in vitro and in vivo from cloned cDNA of a genotype 4 human HEV (strain TW6196E). The complete genomic sequence of the TW6196E virus was determined and a full-length cDNA clone (pHEV-4TW) was assembled. Capped RNA tran ...[more]

PMID: 22837416

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Project description:We developed a reverse genetics system for infectious pancreatic necrosis virus (IPNV), a prototype virus of the Birnaviridae family, with the use of plus-stranded RNA transcripts derived from cloned cDNA. Full-length cDNA clones of the IPNV genome that contained the entire coding and noncoding regions of RNA segments A and B were constructed. Segment A encodes a 106-kDa precursor protein which is cleaved to yield mature VP2, nonstructural protease, and VP3 proteins, whereas segment B encodes the RNA-dependent RNA polymerase VP1. Plus-sense RNA transcripts of both segments were prepared by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of chinook salmon embryo (CHSE) cells with combined transcripts of segments A and B generated infectious IPNV particles 10 days posttransfection. Furthermore, a transfectant virus containing a genetically tagged sequence was generated to confirm the feasibility of this system. The presence and specificity of the recovered virus were ascertained by immunofluorescence staining of infected CHSE cells with rabbit anti-IPNV serum and by nucleotide sequence analysis. In addition, 3'-terminal sequence analysis of RNA from the recovered virus showed that extraneous nucleotides synthesized at the 3' end during in vitro transcription were precisely trimmed or excluded during replication, and hence these were not incorporated into the genome. An attempt was made to determine if RNA-dependent RNA polymerase of IPNV and infectious bursal disease virus (IBDV), another birnavirus, can support virus rescue in heterologous combinations. Thus, CHSE cells were transfected with transcripts derived from IPNV segment A and IBDV segment B and Vero cells were transfected with transcripts derived from IBDV segment A and IPNV segment B. In either case, no infectious IPNV or IBDV particles were generated even after a third passage in cell culture, suggesting that viral RNA-dependent RNA polymerase is species specific. However, the reverse genetics system for IPNV that we developed will greatly facilitate studies of viral replication and pathogenesis and the design of a new generation of live attenuated vaccines.

Dataset Information

Rescue of a genotype 4 human hepatitis E virus from cloned cDNA and characterization of intergenotypic chimeric viruses in cultured human liver cells and in pigs.

Publications

Rescue of a genotype 4 human hepatitis E virus from cloned cDNA and characterization of intergenotypic chimeric viruses in cultured human liver cells and in pigs.

Similar Datasets

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