Porcine NoVs were classified into 3 genotypes within GII based on the complete capsid sequences: 1 genotype with prototype Japanese strains Sw43 and Sw918 and 2 new genotypes. A total of 19 of 275 samples showed a potential positive band after agarose gel electrophoresis of the RT-PCR products of primer pair p290/110. Fourteen samples representative of each potentially positive farm or the slaughterhouse were sequenced. After performing BLAST search, we identified 6 NoVs (QW48, Michigan farm A; QW101, QW125, and QW126, Ohio farm B; and QW170 and QW218, Ohio slaughterhouse), 3 sapoviruses, and 5 sequences that had no significant hit in the database. Because the QW126 shared 99% nucleotide (nt) identity with the QW101 and QW125 strains in the 274-nt RdRp region, it was not sequenced further.
We sequenced the 3-kb 3´ end of the genome containing the partial RdRp, VP1 and VP2 genes, and the 3´ untranslated region of the 5 strains. The porcine NoVs represented 3 distinct clusters: 1) Sw43, Sw918, and QW48; 2) QW101 and QW125; and 3) QW170 and QW218, on the basis of the size of each gene and the ORF1-ORF2 overlap region (). Across the 3 kb, the QW101 and QW125 strains and the QW170 and QW218 strains shared 99% nt identity.
Sizes of the putative capsid protein VP1 and the minor capsid protein VP2, the overlap regions, and the 3´ UTR of GII NoV*
The amino acid identity of the predicted complete and S and P domains of the capsid protein of the 5 porcine NoVs, the previously reported porcine NoVs (Sw43 and Sw918), and representative human, bovine, and murine NoV strains is summarized in . In the complete capsid, the QW48 strain was most closely related to the porcine NoV prototype Sw43 strain (98% amino acid identity); the QW170 and QW218 strains shared the highest amino acid identities (81%) to porcine Sw43 and Sw918 strains; the QW101 and QW125 strains showed the highest amino acid identity to human GII-3/Mexico (71.4%), then to human GII-6/Baltimore (71.0%), porcine QW218 (71.0%), and porcine Sw43 (70.6%) strains. The S and P domains of these NoVs showed similar relationships. A neighbor-joining phylogenetic tree based on the amino acid sequences of the complete capsids () showed that QW48 grouped with Sw43 and Sw918 strains into GII-11 and that QW170 and QW218 formed a new genotype (GII-19), which was closer to porcine than to human strains. However, QW101 and 125 formed a new genotype (GII-18) between human and porcine GII NoVs.
Percentage amino acid identities of noroviruses within the capsid region
Figure 1 Neighbor-joining phylogenetic tree of genogroup II noroviruses (NoVs) based on the complete capsid region. The 5 newly identified porcine NoV strains are in boldface. Genogroups (G) and genotypes (numbers after G) are indicated. The human NoV GI-1/Norwalk (more ...)
Further analysis of the predicted C-terminal ≈260 amino acids of the RdRp region () showed similar grouping results for QW48, QW101, and QW125 strains but different for QW170 and QW218 strains, which were in the same cluster (GII-11) as Sw43, Sw918, and QW48 in the RdRp region. This finding suggested that a recombination event occurred between QW170/218-like and Sw43-like NoVs. The complete VP2 sequences of representative strains were also analyzed (data not shown). Results were similar to those of the capsid sequence classification.
Figure 2 Neighbor-joining phylogenetic tree of genogroup II noroviruses (NoVs) based on the partial RNA-dependent RNA polymerase region (C-terminal 260–266 amino acids). The 5 newly identified porcine NoV strains are in boldface. Genogroups (G) and genotypes (more ...)
A potential recombination event occurred between QW170/218-like and Sw43-like strains. To examine where the recombination occurred, we performed RIP analysis by placing the 3´-end RdRp and the capsid sequence of QW170 or QW218 as a query sequence and the corresponding sequences of Sw43 and QW101 as background sequences. The resulting diagram () showed that QW170 had high similarity to Sw43 in the RdRp but not in the capsid region. This abrupt change happened in the RdRp-capsid junction region. Therefore, we performed sequence alignments of the RdRp-capsid junction of NoVs, including the calicivirus genomic-subgenomic conserved 18-nt motif (20
) (). Between Sw43, QW170, and QW218, all 18 nt were identical, but identities decreased downstream of this motif. QW170 and QW218 grouped with Sw43 with a high bootstrap value of 95 in the RdRp tree (), whereas they segregated from Sw43 with the highest bootstrap value of 100 in the capsid tree (). We could not clarify which was the parent or progeny strain.
Figure 3 Identification of a potential recombination event between QW170 and Sw43 strains. A) Recombination Identification Program analysis of QW170 strain. At each position of the window, the query sequence (QW170) was compared to each of the background genotype (more ...)
The porcine NoVs replicated in gnotobiotic pigs. Two pigs were inoculated with QW101-like GII-18 porcine NoVs (QW126 and QW144 strains) to verify their replication in pigs as confirmed by quantitative RT-PCR and IEM and to produce convalescent-phase serum to examine antigenic reactivity with human NoVs. These 2 strains were confirmed as QW101-like porcine NoVs in both the RdRp (169-nt) and the capsid S domain (363-nt) regions by sequence analysis of the RT-PCR products (Q.H. Wang and L.J. Saif, unpub. data). They shared 99% and 100% amino acid identities to the QW101 strain in the 2 regions, respectively. Porcine NoV shedding, assessed by quantitative RT-PCR with primer pair PNV7/8, was detected at PID 3–5 (euthanized) after QW144 exposure, coincident with mild diarrhea. The RT-PCR–detectable units of the rectal swab RNA increased from negative at PID <2, 103 at PID 3–4, and 104 at PID 5 (large intestinal contents). Norovirus shedding was detected only at PID 5 without diarrhea after QW126 exposure. Examination of the intestinal contents of the pig inoculated with QW144 by IEM with pig convalescent-phase antiserum LL616 showed clumps of ≈32-nm NoV particles (). The 2 control pigs had no virus shedding or diarrhea. Detailed studies of the pathogenesis of porcine NoVs in gnotobiotic pigs are in progress (S. Cheetham and L.J. Saif, unpub. data).
Figure 4 Immune electron micrograph of porcine noroviruses (NoVs). The diluted intestinal contents of a gnotobiotic pig euthanized on postinoculation day 5 to QW101-like porcine NoVs (QW144) were incubated with convalescent-phase serum LL616 from another gnotobiotic (more ...)
Antisera to QW101-like (QW126) porcine NoVs cross-reacted with VLPs of human GII NoVs in ELISA and Western blot. In ELISA (), the pig convalescent-phase antiserum (LL616) to QW101-like porcine NoV QW126 strain showed higher titers (1:400–1:800) to GII-3/Toronto, GII-4/MD145, GII-4/HS66, and GII-6/Florida strains; a lower titer (1:100) to GII-1/Hawaii strain; and lowest titer (1:10) to GI-3/Desert Shield strain. In Western blot (), the capsid proteins (59–60 kDa) of Toronto, MD145, HS66, and Florida strains, but not the Hawaii and Desert Shield strains, were detected by pig antiserum LL616 but not the negative control serum (data not shown). Thus, 1-way antigenic cross-reactivity exists between human NoV antigens and porcine NoV (GII-18) antiserum, with moderate cross-reactivity to human NoVs GII-3, 4, and 6; low cross-reactivity to GII-1; and very low cross-reactivity to GI-3.
Antigenic cross-reactivity between human GII NoV antigens (VLPs) and a pig convalescent-phase antiserum against porcine GII NoVs, as determined by ELISA*
Figure 5 Antigenic cross-reactivity between human genogroup (G) II norovirus (NoV) capsid proteins and a pig convalescent-phase antiserum (LL616) against porcine QW101-like (GII-18) NoV was determined by Western blot. The CsCl-gradient purified viruslike particles (more ...)