Genetic Characterization and Classification of Human and Animal Sapoviruses

Abstract

Sapoviruses (SaVs) are enteric caliciviruses that have been detected in multiple mammalian species, including humans, pigs, mink, dogs, sea lions, chimpanzees, and rats. They show a high level of diversity. A SaV genome commonly encodes seven nonstructural proteins (NSs), including the RNA polymerase protein NS7, and two structural proteins (VP1 and VP2). We classified human and animal SaVs into 15 genogroups (G) based on available VP1 sequences, including three newly characterized genomes from this study. We sequenced the full length genomes of one new genogroup V (GV), one GVII and one GVIII porcine SaV using long range RT-PCR including newly designed forward primers located in the conserved motifs of the putative NS3, and also 5' RACE methods. We also determined the 5'- and 3'-ends of sea lion GV SaV and canine GXIII SaV. Although the complete genomic sequences of GIX-GXII, and GXV SaVs are unavailable, common features of SaV genomes include: 1) "GTG" at the 5'-end of the genome, and a short (9~14 nt) 5'-untranslated region; and 2) the first five amino acids (M [A/V] S [K/R] P) of the putative NS1 and the five amino acids (FEMEG) surrounding the putative cleavage site between NS7 and VP1 were conserved among the chimpanzee, two of five genogroups of pig (GV and GVIII), sea lion, canine, and human SaVs. In contrast, these two amino acid motifs were clearly different in three genogroups of porcine (GIII, GVI and GVII), and bat SaVs. Our results suggest that several animal SaVs have genetic similarities to human SaVs. However, the ability of SaVs to be transmitted between humans and animals is uncertain.

Fig 1. Sapovirus genomic organization: non-structural (NS) and structural proteins VP1 and VP2, and the conserved motifs.

Genomic organization of a common sapovirus, including two open reading frames (ORF1 and ORF2). ORF1 encodes the predicted viral NS proteins (NS1, NS2, NS3 [NTPase], NS4, NS5 [VPg], NS6-NS7 [protease-RNA-dependent RNA polymerase (RdRp)], and the major capsid protein (VP1). ORF2 encodes the minor capsid protein VP2. The following amino acid or nucleotide motifs are conserved among all available sequenced SaVs: GXPGXGKT, PL (N / D) CD, and WDE (F / Y) D of NS3, KGKXX and XDEYXX of NS5, GDCG of NS6, WKGL, KDEL, DYSXWDST, GLPSG, and YGDD of NS7, PPG and WGS of VP1, and the first three nucleotides (GTG) at the 5’–end. Also shown are the first five amino acids of NS1 (M [A / V] S [K / R] P) and around the NS7-VP1 cleavage site (FEME / G, the slash indicates the putative cleavage site by viral protease NS6) that are conserved among GI-GV, GVIII, and GXIII SaVs.

Fig 2. Phylogenetic tree of the full length genomic sequences of 34 sapovirus strains using MEGA 6.

The five strains whose complete genomes were determined in this study are boxed with dotted lines. Among these, the two SaV strains that were newly identified in this study are also indicated with arrows. The number on each branch indicates the bootstrap value. The scale represents the amino acid substitutions per site. Each sapovirus strain is indicated in the following format: Genbank accession number-strain name (species).

Fig 3. Phylogenetic tree based on the complete VP1 amino acid sequences of 74 sapovirus strains.

These strains represent all reported 14 genogroups (GI-GXIV) and the newly reported rat SaV strains using MEGA 6. The 10 animal SaV strains with additional sequences determined in this study are boxed with dotted lines. Among these, the three SaV strains that were newly identified in this study are also indicated with arrows. The number on each branch indicates the bootstrap value. The scale represents the amino acid substitutions per site. Each sapovirus strain is indicated in the following format: Genbank accession number-strain name (species).

Fig 4. Identity distribution of the VP1 amino acid sequences of 74 SaV strains.

The arrow indicates the genogrouping cut-off (≥ 57% aa identity) used in this study.The genogrouping of GV, GVII, GVIII, GIX, GXII, and GXIII SaVs sequenced in this study (as indicated as dotted box in Figs 3 and 5) based on NS7 and VP1 matched, except for the sea lion GV/CSL9775 and the porcine GVII/WGP247 strains. The Sea lion/GV/CSL9775 strain clustered together with other human and porcine GV SaV strains in the VP1 region, but it was separated from other GV strains in the NS7 region as recently discussed [1]. Similarly, GVII/ WGP247 strain clustered together with other GVII strains in the VP1 region, but it was closer to porcine GIX/WG214C in the NS7 region. The NS7 sequences of porcine GX and GXI SaVs and rat (GII and GXV) SaVs are not yet available (Table 1).

Fig 5. Phylogenetic tree of the putative NS7 amino acid sequences (approx. 500 aa) of sapovirus strains.

These 44 strains represent 12 genogroups (GI-GV, GVI, GVII, GVIII, GIX, GXII, GXIII, and GXIV) using MEGA 6. The amino acid sequences covering from “WKGL” sequence to the end of the putative NS7, “XXME”, were used. Only 44 of the 74 SaV strains in the VP1-tree (Fig 3) have sequences for this region. The 10 strains analyzed in this study are boxed with dotted lines. Among these, the eight SaV strains whose corresponding sequences were determined in this study are indicated with arrows. The NS7 sequences of porcine GIX and GX SaVs, and rat GII and GXV SaVs are not yet available. The number on each branch indicates the bootstrap value. The scale represents the amino acid substitutions per site. Each sapovirus strain is indicated in the following format: Genbank accession number-strain name (species).