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.

FIG. 1

Genomic organization of the PEC. (A) Schematic of the genomic organization of PEC/Cowden. Two predicted ORFs include ORF1, a polyprotein fused to and contiguous with the capsid protein, forming a large polyprotein, and ORF2, a small basic protein of unknown function. The nucleotide coordinates of the predicted proteins are numbered above or below the open boxes. (B) Schematic of the conserved nucleotide sequence motifs at the 5′ termini of the genomic and predicted subgenomic RNAs. Their nucleotide sequences are aligned beneath the genomic map. Shaded boxes indicate the genomic locations of the conserved motifs in the polyprotein. The Kozak context favorable for translation initiation is underlined. The thick lines represent those overlapping clones used for assembling a full-length genome. (C) Schematic of the nucleotide and amino acid sequence differences in the predicted 3C-like proteases, RNA polymerases, and capsid proteins between TC PEC/Cowden and WT PEC/Cowden. The partial sequences are aligned, the nucleotide coordinates are indicated for each codon, and the amino acid coordinates are indicated after each amino acid.

FIG. 2

The predicted amino acid sequences of proteins encoded by the two ORFs of the PEC RNA genome. The conserved motifs including the putative 2C helicase (GPPGIGKT), 3C-like protease (GDCG), and 3D RNA polymerase (GLPSG and YGDD) in the polyprotein, as well as the first PPG motif in the predicted capsid protein, are boxed. The amino acid sequence coordinates for each of the two ORFs are on the left. The amino acids with substitutions in the RNA polymerase and capsid regions of TC PEC/Cowden are indicated by asterisks below each residue. The predicted start site (aa 1711; MEAPAP) of the capsid protein is underlined with an arrow. The three discrete regions in the predicted capsid protein are as follows: conserved region 1, aa 1711 to 1984; hypervariable region 2, aa 1985 to 2132; and region 3, aa 2133 to 2254.

FIG. 3

Phylogenetic tree generated for the sequences in the RNA polymerase region. Alignments were generated from the conserved KDEL sequence to the end of the RNA polymerase or the start of the capsid protein. Calicivirus sequences used in the alignment were retrieved from GenBank. Strain names and abbreviations (GenBank accession numbers) are as follows: for SLVs, SV (S77903), MV (X86559), Parkville virus (U73124), HuCV Houston/86 (U95643), Houston/90 (U95644), London/92 (U67857), and PEC/Cowden (AF182760); for vesiviruses, FCV (M86379), SMSV1 (U15301), and SMSV4 (U15302); for lagoviruses, RHDV (M67473) and EBHSV (Z69620); for NLVs, NV (87661), Desert Shield virus (DSV) (U04469), SHV (L07418), BEC Jena strain (JV) (AJ011099), SMA (L23831), Melksham virus (MeV) (X81879), Toronto virus (TV) (U02030), Mexico virus (MxV) (U22498), HV (U07611), LV (86557), and the Sw918-97-J swine calicivirus (AB009415) detected in Japan.