Characterization of St-Valerien-Like Virus Genome Detected in Japan

ABSTRACT A novel calicivirus, St-Valerien-like virus (SVV), has been identified in asymptomatic swine in Canada, Italy and the U.S.A. In this study, we characterized a new SVV strain (NUP-24/JP) detected in fecal samples of swine in Japan. The NUP-24/JP genome had 6,409 nucleotides and 2 open reading frames (ORF1 and ORF2). ORF1 and ORF2 consist of 5,940 and 453 nucleotides, respectively. Phylogenetic analysis revealed that NUP-24/JP was closely related to other SVV strains, particularly to U.S.A. strain NC-WGS93C/US. This finding suggests that SVV is prevalent in swine worldwide. Using a baculovirus expression system, we successfully produced virus-like particles, which would be useful for seroepidemiological studies of SVV.

The family Caliciviridae includes small non-enveloped viruses with single-stranded, positive-polarity RNA genomes of 7.4-8.3 kilobases [7]. The family consists of the Vesivirus (VESV), Lagovirus, Nebovirus, Norovirus (NoV) and Sapovirus (SaV) genera, as well as unassigned viruses [6,12]. Although caliciviruses are known to infect a broad range of animals, including humans, swine, cattle, rabbits and mice, each calicivirus is generally thought to have high specificity for its host species. In swine, viruses detected to date have been classified into 3 established genera in the family Caliciviridae; SaV [1, 9, 11, 13, 14, 16-19, 24, 28], NoV [15-17, 19, 21, 24, 28] and VESV [20]. Moreover, newly recognized caliciviruses, St-Valerien-like viruses (SVVs), were first identified in the feces of asymptomatic finisher swine in the province of Quebec, Canada, and were proposed as members of a potential genus of Caliciviridae [12]. SVV strains were also detected in swine in Italy and the U.S.A. [5,22]. These Italian and North American SVV strains showed a close genetic relationship. However, the prevalence and genetic diversity of SVV in countries other than Canada, Italy and the U.S.A. are unknown. In this study, we report the genome structure and phylogenetic properties of SVV detected in asymptomatic fattening swine in Japan. In addition, we describe here our attempts to produce baculovirus-expressed virus-like particles (VLPs) using the capsid protein-coding region of the Japanese SVV genome.
We collected a total of 76 fecal samples from 71 healthy and 5 sick swine at farms in Kanagawa and Tochigi prefectures. Swine were aged as follows; 16 nursing swine (1 to 3 weeks), 10 postweaning swine (4 to 10 weeks), 28 fattening swine (11 to 19 weeks), 17 finisher swine (20 to 24 weeks) and 5 unknown. All fresh fecal samples were placed in sterilized tubes and stored at −80°C until use. Samples were prepared as 10% suspensions in sterilized phosphate-buffered saline. Prepared fecal suspensions were centrifuged at 20,000 × g for 5 min at 4°C, and supernatants were collected. Total RNAs were extracted from 140 µl of supernatants using the QIAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA, U.S.A.). Yeast RNA (Applied Biosystems, Foster City, CA, U.S.A.) was used as a carrier. For screening, the specific primer set for calicivirus genome, P290 (sense; 5′-GATTACTCCAAGTGGGACTCCAC −3′) and P289 (anti-sense; 5′-TGACAATGTAATCATCACCATA −3′), was used. The primers were broadly reactive, targeting highly conserved motifs of the RNA-dependent RNA polymerase coding region of the caliciviruses [10]. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed using the PrimeScript ® One Step RT-PCR Kit Ver.2 (Dye Plus) (TAKARA, Otsu, Japan) in accordance with the manufacturer's instructions. The expected size of the amplicon (311 bp) was analyzed by 2% agarose gel electrophoresis with SYBR safe DNA gel stain (Invitrogen, Carlsbad, CA, U.S.A.) and was visualized by UV. Amplicons were purified using the QIAquick Gel Extraction Kit (QIAGEN). Nucleotide sequences of amplicons were determined using an automated sequencer ABI3130 (Applied Biosystems) and the Big Dye Terminator Cycle Sequencing Kit ver. 3.1 (Applied Biosystems) and were assembled by ATGC computer software (Genetyx Corporation, Tokyo, Japan). To find homologous sequences, we performed a BLASTn search (http://blast. ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_ PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch) of the Gene bank nucleotide database. The complete genome sequence of SVV in the sample was also determined with sequence-specific primers ( Table 1). The 5′ and 3′ terminal sequences were amplified by 5′ and 3′ RACE methods using the 5′-Full RACE Core Set (TAKARA) and the PrimeScript ® II High Fidelity RT-PCR Kit (TAKARA), respectively. In the 5′ RACE method, 5′ end phosphorylated primer, ST-V-PR, was used for the RT reaction. After degradation of RNA and circularization and/or formation of concatemers of the cDNA by T4 RNA ligase, the 5′ terminal sequence (unknown region) in the cDNA became flanked with known sequence regions. The region including the 5′ terminal sequence was amplified by 1st and 2nd PCR with external (St-V-S1F/ St-V-A1R) and internal (St-V-S2F/ St-V-A2R) primer sets, respectively (Table 1). In the 3′ RACE method, the adaptor sequence was anchored at the 3′ terminal sequence using the Anchor30+T primer in the RT reaction. The 3′ terminal sequence was amplified by PCR with P290/ 1st-Anchor25.TXR1 primer set. The SVV nucleotide sequences were determined by direct sequencing as described above. Phylogenetic analyses were performed using the Neighbor-Joining (NJ) method with Primers were published previously [9]. b) 5′ end-phosphorylated RT primer. c) Underline indicates a restriction site.
In this study, 2.6% (2/76) sample sequences (NUP-24/ JP) detected from swine aged 4 months were identified as SVV positive. Because both swine were kept in the same pen and nucleotide sequences of the 2 samples showed complete accordance, we assumed that these sequences were derived from one strain (NUP-24/JP). The NUP-24/JP genome (Genbank accession No.: AB863586) had 6,409 nucleotides (exclude poly-A) and included two open reading frames (ORF1 and ORF2) with 5,940 and 453 nucleotides, respectively. Homologies of the ORF1 and ORF2 amino acid and nucleotide sequences (ORF1: 1,979 aa and 5,940 nt, ORF2: 150 aa and 453 nt) of the NUP-24/JP with other SVV strains [5,12,22] were as follows: full-sequence, 89.0-94.5%; ORF1aa, 97.5-99.0%; ORF1nt, 88.7-94.6%; ORF2aa, 92.0-97.3% and ORF2nt, 87.6-95.8%. Amino acid motifs and putative cleavage sites of SVV [12] were also conserved (data not shown). The NJ-tree based on the complete nucleotide sequences of the NUP-24/JP and other caliciviruses indicated that the NUP-24/JP was included in the SVV cluster, particularly close to the NC-WGP93C/US strain [22] (Fig. 1). Results of phylogenetic analyses revealed that SVVs including NUP-24/JP showed high nucleotide homologies, although these strains were detected in the regions remote from one another (Canada, Italy, U.S.A. and Japan). This suggests that SVV is prevalent in swine in regions other than Europe and North America.
We attempted to propagate the NUP-24/JP strain using three cell lines; swine kidney (LLC-PK1), porcine intestinal epithelial (PIE) and Vero cells. Infection studies using LLC-PK1 cells were performed with bile acids, glycochenodeoxycholic acid and taurochenodeoxycholic acid, because it has been reported that bile acids in intestinal contents were essential for growth of swine SaVs in cultured cells [2,3]. Unfortunately, we found neither a cytopathic effect of SVV nor an increase in viral RNA in any cell line tested (data not shown). This suggests that SVV may have different infectivity against cultured cells when compared with swine SaVs, although this could be attributed to low viral concentrations in the samples used in this study.
As propagation in cultured cells was unsuccessful, we next attempted to produce VLPs using the capsid proteincoding region of NUP-24/JP genome and the Bac-to-Bac ® Baculovirus Expression System (Invitrogen), in accordance with a previously reported method [4]. Briefly, the 3′-end   approximately 55 kDa ( Fig. 2A), which corresponds to the molecular mass of VP1 of SVV [12]. Electron microscopic observation detected VLPs of two sizes (Fig. 2B). The larger type had a diameter of approximately 45 nm with a characteristic cup structure on the surface, and the smaller type had a diameter of approximately 30 nm. The assembly of VLPs of two sizes (diameters of 38 and 23 nm, respectively) has previously been reported in Norwalk virus (a prototype strain of human NoV) [27]. The authors suggested that VLPs of both sizes were composed of a 58 kDa capsid protein and that the difference in VLP size depended on the number of units of capsid protein forming the VLPs. VLPs of 2 sizes (diameters 41-48 and 26-31 nm) have been also observed in SaV (NK24 strain) [8]. In our study on SVV, as only the 55 kDa band was detected on SDS-PAGE, VLPs of 2 sizes also appear to be constructed from the intact capsid protein, VP1 of SVV. In addition, White et al. [27] indicated that the both VLPs have similar antigenic properties when analyzed with polyclonal and monoclonal antibodies. The VLPs of SVV, NUP24/JP successfully produced in this study should be useful as antigens for studying the seroprevalence rate of SVV infection in swine, such as age, species or farm-dependent differences in the prevalence of SVV-specific antibody.
In conclusion, we detected the SVV genome sequence of the NUP-24/JP strain in asymptomatic swine in Japan, thus suggesting that SVV is prevalent worldwide. Although we were unable to propagate NUP-24/JP using cell lines, we successfully produced VLPs, which would be useful for seroprevalence studies in the future.