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Capped RNA transcripts of full-length cDNA clones of swine hepatitis E virus are replication competent when transfected into Huh7 cells and infectious when intrahepatically inoculated into pigs.

ABSTRACT: Swine hepatitis E virus (swine HEV), the first animal strain of HEV to be isolated, is a zoonotic agent. We report here the construction and in vitro and in vivo characterizations of infectious cDNA clones of swine HEV. Eight overlapping fragments spanning the entire genome were amplified by reverse transcription-PCR and assembled into a full-length cDNA clone, clone C, which contained 14 mutations compared to the consensus sequence of swine HEV. RNA transcripts from clone C were not infectious, as determined by intrahepatic inoculation into pigs and by in vitro transfection of Huh7 cells. Multiple site-based site-directed mutagenesis was performed to generate three new cDNA clones (pSHEV-1, pSHEV-2, and pSHEV-3) which differed from each other. The transfection of capped RNA transcripts into human liver Huh7 cells resulted in the synthesis of both ORF2 capsid and ORF3 proteins, indicating that the cDNA clones were replication competent. Each of the three clones resulted in active swine HEV infections after the intrahepatic inoculation of pigs with capped RNA transcripts. The patterns of seroconversion, viremia, and fecal virus shedding for pigs inoculated with RNA transcripts from clones pSHEV-2 and pSHEV-3 were similar to each other and to those for pigs inoculated with wild-type swine HEV, suggesting that the nucleotide differences between these two cDNA clones were not critical for replication. Pigs inoculated with RNA transcripts from clone pSHEV-1, which contained three nonsilent mutations in the ORF2 capsid gene, had a delayed appearance of seroconversion and fecal virus shedding and had undetectable viremia. The availability of these infectious cDNA clones affords us an opportunity to understand the mechanisms of cross-species infection by constructing chimeric human and swine HEVs.

SUBMITTER: Huang YW

PROVIDER: S-EPMC544089 | biostudies-literature | 2005 Feb

REPOSITORIES: biostudies-literature

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Capped RNA transcripts of full-length cDNA clones of swine hepatitis E virus are replication competent when transfected into Huh7 cells and infectious when intrahepatically inoculated into pigs.

Huang Y W YW Haqshenas G G Kasorndorkbua C C Halbur P G PG Emerson S U SU Meng X J XJ

Journal of virology 20050201 3

Swine hepatitis E virus (swine HEV), the first animal strain of HEV to be isolated, is a zoonotic agent. We report here the construction and in vitro and in vivo characterizations of infectious cDNA clones of swine HEV. Eight overlapping fragments spanning the entire genome were amplified by reverse transcription-PCR and assembled into a full-length cDNA clone, clone C, which contained 14 mutations compared to the consensus sequence of swine HEV. RNA transcripts from clone C were not infectious, ...[more]

PMID: 15650181

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Project description:The understanding of the pathogenic mechanisms and the clinicopathological forms caused by currently circulating African swine fever virus (ASFV) isolates is incomplete. So far, most of the studies have been focused on isolates classified within genotypes I and II, the only genotypes that have circulated outside of Africa. However, less is known about the clinical presentations and lesions induced by isolates belonging to the other twenty-two genotypes. Therefore, the early clinicopathological identification of disease outbreaks caused by isolates belonging to, as yet, not well-characterised ASFV genotypes may be compromised, which might cause a delay in the implementation of control measures to halt the virus spread. To improve the pathological characterisation of disease caused by diverse isolates, we have refined the macroscopic and histopathological evaluation protocols to standardise the scoring of lesions. Domestic pigs were inoculated intranasally with different doses (high, medium and low) of ASFV isolate Ken05/Tk1 (genotype X). To complement previous studies, the distribution and severity of macroscopic and histopathological lesions, along with the amount and distribution of viral antigen in tissues, were characterised by applying the new scoring protocols. The intranasal inoculation of domestic pigs with high doses of the Ken05/Tk1 isolate induced acute forms of ASF in most of the animals. Inoculation with medium doses mainly induced acute forms of disease. A less severe but longer clinical course, typical of subacute forms, characterised by the presence of more widespread and severe haemorrhages and oedema, was observed in one pig inoculated with the medium dose. The severity of vascular lesions (haemorrhages and oedema) induced by high and medium doses was not associated with the amount of virus antigen detected in tissues, therefore these might be attributed to indirect mechanisms not evaluated in the present study. The absence of clinical signs, lesions and detectable levels of virus genome or antigen in blood from the animals inoculated with the lowest dose ruled out the existence of possible asymptomatic carriers or persistently infected pigs, at least for the 21 days period of the study. The results corroborate the moderate virulence of the Ken05/Tk1 isolate, as well as its capacity to induce both the acute and, occasionally, subacute forms of ASF when high and medium doses were administered intranasally.

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Capped RNA transcripts of full-length cDNA clones of swine hepatitis E virus are replication competent when transfected into Huh7 cells and infectious when intrahepatically inoculated into pigs.

Publications

Capped RNA transcripts of full-length cDNA clones of swine hepatitis E virus are replication competent when transfected into Huh7 cells and infectious when intrahepatically inoculated into pigs.

Similar Datasets

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