Dataset Information

Molecular characterization of a porcine enteric calicivirus genetically related to Sapporo-like human caliciviruses.

ABSTRACT: Porcine enteric calicivirus (PEC) is associated with diarrhea in pigs, and to date it is the only cultivable enteric calicivirus (tissue culture-adapted [TC] PEC/Cowden). Based on sequence analysis of cDNA clones and reverse transcription-PCR products, TC PEC/Cowden has an RNA genome of 7,320 bp, excluding its 3' poly(A)(+) tail. The genome is organized in two open reading frames (ORFs), similar to the organizations of the human Sapporo-like viruses (SLVs) and the lagoviruses. ORF1 encodes the polyprotein that is fused to and contiguous with the capsid protein. ORF2 at the 3' end encodes a small basic protein of 164 amino acids. Among caliciviruses, PEC has the highest amino acid sequence identities in the putative RNA polymerase (66%), 2C helicase (49.6%), 3C-like protease (43.7%), and capsid (39%) regions with the SLVs, indicating that PEC is genetically most closely related to the SLVs. The complete RNA genome of wild-type (WT) PEC/Cowden was also sequenced. Sequence comparisons revealed that the WT and TC PEC/Cowden have 100% nucleotide sequence identities in the 5' terminus, 2C helicase, ORF2, and the 3' nontranslated region. TC PEC/Cowden has one silent mutation in its protease, two amino acid changes and a silent mutation in its RNA polymerase, and five nucleotide substitutions in its capsid that result in one distant and three clustered amino acid changes and a silent mutation. These substitutions may be associated with adaptation of TC PEC/Cowden to cell culture. The cultivable PEC should be a useful model for studies of the pathogenesis, replication, and possible rescue of uncultivable human enteric caliciviruses.

SUBMITTER: Guo M

PROVIDER: S-EPMC113000 | biostudies-literature | 1999 Nov

REPOSITORIES: biostudies-literature

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Molecular characterization of a porcine enteric calicivirus genetically related to Sapporo-like human caliciviruses.

Guo M M Chang K O KO Hardy M E ME Zhang Q Q Parwani A V AV Saif L J LJ

Journal of virology 19991101 11

Porcine enteric calicivirus (PEC) is associated with diarrhea in pigs, and to date it is the only cultivable enteric calicivirus (tissue culture-adapted [TC] PEC/Cowden). Based on sequence analysis of cDNA clones and reverse transcription-PCR products, TC PEC/Cowden has an RNA genome of 7,320 bp, excluding its 3' poly(A)(+) tail. The genome is organized in two open reading frames (ORFs), similar to the organizations of the human Sapporo-like viruses (SLVs) and the lagoviruses. ORF1 encodes the p ...[more]

PMID: 10516074

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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:Two genetically distinct bovine enteric caliciviruses (BECs) have been identified: the norovirus (NLV) Jena and Newbury Agent-2 (NA-2) BECs, which are genetically related to human noroviruses, and the Nebraska (NB) BECs, which is related to sapoviruses and lagoviruses but may also represent a new calicivirus genus. The prevalence of these two BEC genotypes in cattle is unknown. Although reverse transcription-PCR (RT-PCR) primers for human NLV recognize NLV-BECs, the genetic relationships between NLV from humans and the NLV-BECs commonly circulating in cattle is undefined. In the present study, veal calf fecal samples were assayed for enteric caliciviruses by using six RT-PCR primer sets designed for the detection of human NLVs or BECs. Caliciviruses genetically related to the NLV-BEC Jena and NA-2 strains or to the recently characterized NB BEC strain were identified in three of four and four of four sampled veal herds, respectively. Extended 3'-terminal genome sequences of two NLV-BECs, designated CV95-OH and CV186-OH, encoding the RNA-dependent RNA polymerase (RdRp; open reading frame 1 [ORF-1]), VP1 (ORF-2), and VP2 (ORF-3) genes were determined. Phylogenetic and sequence identity analyses of each genome region demonstrated these viruses to be most closely related to the NLV-BEC Jena and NA-2 strains. In initial testing, the human P289-P290 (P289/290) primer set was found to be the most sensitive for calicivirus detection. However, its failure to identify all positive fecal pools (as determined by other assays) led us to design two new primer sets, CBECU-F/R and NBU-F/R, for the sensitive and specific detection of NLV-BEC (NLV-BEC Jena and NA-2) and BEC-NB-like viruses, respectively. The RT-PCR assays with the new primers were compared against other primer sets, including P289/290. Composite results of the tests completed by using the new assays identified 72% (54 of 75) of veal calf fecal samples as positive, with 21 of 21 sequenced reaction products specific for the target RdRp gene. The same design strategy used for the new BEC assays may also be applicable to the design of similar assays for the detection of human caliciviruses (HuCVs). Our data support the genetic relationship between NLV-BECs and NLV-HuCVs but with the NLV-BECs comprising two clusters within a third NLV genogroup.

Dataset Information

Molecular characterization of a porcine enteric calicivirus genetically related to Sapporo-like human caliciviruses.

Publications

Molecular characterization of a porcine enteric calicivirus genetically related to Sapporo-like human caliciviruses.

Similar Datasets

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