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Reverse genetics system for porcine enteric calicivirus, a prototype sapovirus in the Caliciviridae.

ABSTRACT: A porcine enteric calicivirus (PEC), strain Cowden in the genus Sapovirus of the Caliciviridae family, can be propagated in a porcine kidney continuous cell line (LLC-PK) in the presence of bile acids in the cell culture medium. A full-length cDNA copy of the Cowden PEC genome was cloned into a plasmid vector directly downstream from the T7 RNA polymerase promoter, and capped RNA transcripts derived from this clone were infectious when transfected into LLC-PK cells. The recovery of PEC after transfection of RNA transcripts was dependent on the presence of bile acids, consistent with our recent identification of a bile acid-mediated signaling pathway required for PEC replication (Chang et al., Proc. Natl. Acad. Sci. USA 101:8733-8788, 2004). Recovery of virus was verified by detection of PEC antigen in transfected cells by immunofluorescence and enzyme-linked immunosorbent assays, direct observation of recovered viral particles by electron microscopy, and partial sequence analysis of their genomes (first 1,070 nucleotides) to differentiate them from tissue culture-adapted parental virus. The recovered virus retained its ability to infect piglets when administered by the oral route and showed an attenuated phenotype similar to that of the tissue culture-adapted parental virus. This reverse genetics system for PEC provides a new tool to study the molecular basis of replication and pathogenesis for caliciviruses associated with diarrheal disease.

SUBMITTER: Chang KO

PROVIDER: S-EPMC544127 | biostudies-literature | 2005 Feb

REPOSITORIES: biostudies-literature

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Reverse genetics system for porcine enteric calicivirus, a prototype sapovirus in the Caliciviridae.

Chang Kyeong-Ok KO Sosnovtsev Stanislav V SV Belliot Gaël G Wang Qiuhong Q Saif Linda J LJ Green Kim Y KY

Journal of virology 20050201 3

A porcine enteric calicivirus (PEC), strain Cowden in the genus Sapovirus of the Caliciviridae family, can be propagated in a porcine kidney continuous cell line (LLC-PK) in the presence of bile acids in the cell culture medium. A full-length cDNA copy of the Cowden PEC genome was cloned into a plasmid vector directly downstream from the T7 RNA polymerase promoter, and capped RNA transcripts derived from this clone were infectious when transfected into LLC-PK cells. The recovery of PEC after tra ...[more]

PMID: 15650167

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Project description:UnlabelledThe porcine sapovirus (SaV) (PoSaV) Cowden strain is one of only a few culturable enteric caliciviruses. Compared to the wild-type (WT) PoSaV Cowden strain, tissue culture-adapted (TC) PoSaV has two conserved amino acid substitutions in the RNA-dependent RNA polymerase (RdRp) and six in the capsid protein (VP1). By using the reverse-genetics system, we identified that 4 amino acid substitutions in VP1 (residues 178, 289, 324, and 328), but not the substitutions in the RdRp region, were critical for the cell culture adaptation of the PoSaV Cowden strain. The other two substitutions in VP1 (residues 291 and 295) reduced virus replication in vitro. Three-dimensional (3D) structural analysis of VP1 showed that residue 178 was located near the dimer-dimer interface, which may affect VP1 assembly and oligomerization; residues 289, 291, 324, and 328 were located at protruding subdomain 2 (P2) of VP1, which may influence virus binding to cellular receptors; and residue 295 was located at the interface of two monomeric VP1 proteins, which may influence VP1 dimerization. Although reversion of the mutation at residue 291 or 295 from that of the TC strain to that of the WT reduced virus replication in vitro, it enhanced virus replication in vivo, and the revertants induced higher-level serum and mucosal antibody responses than those induced by the TC PoSaV Cowden strain. Our findings reveal the molecular basis for PoSaV adaptation to cell culture. These findings may provide new, critical information for the cell culture adaptation of other PoSaV strains and human SaVs or noroviruses.ImportanceThe tissue culture-adapted porcine sapovirus Cowden strain is one of only a few culturable enteric caliciviruses. We discovered that 4 amino acid substitutions in VP1 (residues 178, 289, 324, and 328) were critical for its adaptation to LLC-PK cells. Two substitutions in VP1 (residues 291 and 295) reduced virus replication in vitro but enhanced virus replication and induced higher-level serum and mucosal antibody responses in gnotobiotic pigs than those induced by the tissue culture-adapted strain. Structural modeling analysis of VP1 suggested that residue 178 may affect VP1 assembly and oligomerization; residues 289, 291, 324, and 328 may influence virus binding to cellular receptors; and residue 295 may influence VP1 dimerization. Our findings will provide new information for the cell culture adaptation of other sapoviruses and possibly noroviruses.

Project description:The New World mammarenavirus Tacaribe virus (TCRV) has been isolated from fruit bats, mosquitoes, and ticks, whereas all other known New World mammarenaviruses are maintained in rodents. TCRV has not been linked to human disease, but it has been shown to protect against Argentine hemorrhagic fever-like disease in marmosets infected with the New World mammarenavirus Junín virus (JUNV), indicating the potential of TCRV as a live-attenuated vaccine for the treatment of Argentine hemorrhagic fever. Implementation of TCRV as a live-attenuated vaccine or a vaccine vector would be facilitated by the establishment of reverse genetics systems for the genetic manipulation of the TCRV genome. In this study, we developed, for the first time, reverse genetics approaches for the generation of recombinant TCRV (rTCRV). We successfully rescued a wild-type (WT) rTCRV (a trisegmented form of TCRV expressing two reporter genes [r3TCRV]) and a bisegmented TCRV expressing a single reporter gene from a bicistronic viral mRNA (rTCRV/GFP). These reverse genetics approaches represent an excellent tool to investigate the biology of TCRV and to explore its potential use as a live-attenuated vaccine or a vaccine vector for the treatment of other viral infections. Notably, we identified a 39-nucleotide (nt) deletion (?39) in the noncoding intergenic region (IGR) of the viral large (L) segment that is required for optimal virus multiplication. Accordingly, an rTCRV containing this 39-nt deletion in the L-IGR (rTCRV/?39) exhibited decreased viral fitness in cultured cells, suggesting the feasibility of using this deletion in the L-IGR as an approach to attenuate TCRV, and potentially other mammarenaviruses, for their implementation as live-attenuated vaccines or vaccine vectors.IMPORTANCE To date, no Food and Drug Administration (FDA)-approved vaccines are available to combat hemorrhagic fever caused by mammarenavirus infections in humans. Treatment of mammarenavirus infections is limited to the off-label use of ribavirin, which is partially effective and associated with significant side effects. Tacaribe virus (TCRV), the prototype member of the New World mammarenaviruses, is nonpathogenic in humans but able to provide protection against Junín virus (JUNV), the causative agent of Argentine hemorrhagic fever, demonstrating the feasibility of using TCRV as a live-attenuated vaccine vector for the treatment of JUNV and potentially other viral infections. Here, we describe for the first time the feasibility of generating recombinant TCRV (rTCRV) using reverse genetics approaches, which paves the way to study the biology of TCRV and also its potential use as a live-attenuated vaccine or a vaccine vector for the treatment of mammarenavirus and/or other viral infections in humans.

Dataset Information

Reverse genetics system for porcine enteric calicivirus, a prototype sapovirus in the Caliciviridae.

Publications

Reverse genetics system for porcine enteric calicivirus, a prototype sapovirus in the Caliciviridae.

Similar Datasets

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