Genomic organization and recombination analysis of a porcine sapovirus identified from a piglet with diarrhea in China

Background Sapovirus (SaV), a member of the family Caliciviridae, is an etiologic agent of gastroenteritis in humans and pigs. To date, both intra- and inter-genogroup recombinant strains have been reported in many countries except for China. Here, we report an intra-genogroup recombination of porcine SaV identified from a piglet with diarrhea of China. Methods A fecal sample from a 15-day-old piglet with diarrhea was collected from Shanghai, China. Common agents of gastroenteritis including porcine circovirus type 2, porcine rotavirus, porcine transmissible gastroenteritis virus, porcine SaV, porcine norovirus, and porcine epidemic diarrhea virus were detected by RT-PCR or PCR method. The complete genome of porcine SaV was then determined by RT-PCR method. Phylogenetic analyses based on the structural region and nonstructural (NS) region were carried out to group this SaV strain, and it was divided into different genotypes based on these two regions. Recombination analysis based on the genomic sequence was further performed to confirm this recombinant event and locate the breakpoint. Results All of the agents showed negative results except for SaV. Analysis of the complete genome sequence showed that this strain was 7387 nt long with two ORFs and belonged to SaV GIII. Phylogenetic analyses of the structural region (complete VP1 nucleotide sequences) grouped this strain into GIII-3, whereas of the nonstructural region (RdRp nucleotide sequences) grouped this strain into GIII-2. Recombination analysis based on the genomic sequence confirmed this recombinant event and identified two parental strains that were JJ259 (KT922089, GIII-2) and CH430 (KF204570, GIII-3). The breakpoint located at position 5139 nt of the genome (RdRp-capsid junction region). Etiologic analysis showed the fecal sample was negative with the common agents of gastroenteritis, except for porcine SaV, which suggested that this recombinant strain might lead to this piglet diarrhea. Conclusions P2 strain was an intra-genogroup recombinant porcine SaV. To the best of our knowledge, this study would be the first report that intra-genogroup recombination of porcine SaV infection was identified in pig herd in China.


Background
Sapovirus (SaV) is the causative agent of gastroenteritis and has been detected in multiple mammalian species and pigs are the predominant host of SaV [1][2][3]. Based on the complete capsid protein VP1 sequences, SaV now has been divided into 15 genogroups (GI-GXV) and GIII was the predominant one infecting pigs [4]. GIII strains have been further clustered into several genotypes based on the partial VP1 or RNA-dependent RNA polymerase (RdRp) sequences reported by different researchers [5,6]. Genomic organization of a common SaV includes two open reading frames (ORF1 and ORF2), whereas in certain genogroups strains were identified an additional ORF (ORF3) [7][8][9]. ORF1 encodes the predicted viral NS proteins and the major capsid protein VP1, ORF2 encodes the minor capsid protein VP2, and ORF 3 encodes a small basic protein with unknown function [9].
SaV strains with inconsistent grouping between the nonstructural protein-encoding region (including the RdRp region) and the VP1 encoding region have been designated as "recombinant". Previous studies suggested that the recombination site was at the polymerasecapsid junction [1,2]. To date, both intra-and intergenogroup recombinant strains have been reported in humans, however, few porcine recombinant SaV strains have been reported all over the world [10][11][12][13]. In particular, no recombinant strain of SaV has been identified either in human or pig in China.
In the present study, we characterized the complete genome of a porcine SaV which might lead to a piglet diarrhea and analyzed the recombination of this strain. Phylogenetic and recombination analysis showed that this strain was an intra-genogroup recombinant, and the breakpoint for this recombination event located at the polymerase-capsid junction within ORF1. This is the first report that intra-genogroup recombination of porcine SaV related with a piglet diarrhea in China.

Specimens
A fecal sample from a 15-day-old piglet with diarrhea was collected in October, 2015 in Shanghai, China. Bacterial infection was ruled out by the diagnosis of the licensed veterinarian from the pig farm. In order to avoid sample contamination, specimen was obtained directly from the pig anus and disposable materials were used during sampling. Stool sample was freshly collected and immediately converted to 10% (w/v) suspension in phosphate-buffered saline (PBS, 0.01 M, pH 7.2-7.4) for further RNA and DNA extraction.

RNA and DNA extraction
Viral RNA/DNA was extracted from 200 μl of fecal supernatant by using the TaKaRa MinBEST Viral RNA/ DNA Extraction Kit version 5.0 (TaKaRa, Japan), according to the manual instruction. RNA/DNA was dissolved in 25 μl RNase-free water and reverse transcription was performed immediately.

Whole genome amplification
To amplify the genome of this SaV strain, the first strand cDNA was synthetized in 20 μl reaction mixture followed the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit's (Thermo, USA) introductions with 1 μl random hexamer primer supplied by the kit for the full sequences amplification or 1 μl gene-specific primer QT (Table 1) especially for the 3' end amplification (3' Q I GAGGACTCGAGCTCAAGC [17] RACE method), respectively [17]. Eleven sets of specific primers were designed based on KF204570 to amplify the remaining sequences (Table 1). All the PCR products were purified by OMEGA Gel Kit (OMEGA, USA) following the manufacturer's instructions, ligated to the pMD19-T vector (TaKaRa, Japan) and then transformed into DH5α competent Escherichia coli cells (Yeasen, China). For each product, three to five colonies were selected and sequenced (Sangon, China) in both directions with the M13+/-universal primers. The consensus sequences were assembled using the Lasergene Software package (version 8) (DNASTAR Inc., Madison, WI).

Sequence and recombination analysis
Similarity searches of the sequences were carried out in BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). After a multiple alignment with CLUSTAL W embedded in MEGA 7, the phylogenetic relationship of the strain in the present study and the reference sequences were assessed using MEGA 7. For analysis in MEGA 7, Jukes-cantor (JC) distance was utilized, employing the Neighbor joining (NJ) algorithm [18]. The reliability of different phylogenetic groupings was evaluated by using the bootstrap test (1000 bootstrap replications) available in MEGA 7. The identification of recombinants was performed by using the Recombination Detection Program 4 (RDP 4) (http:// darwin.uvigo.es/rdp/rdp.html) [19]. Prototype SaV strains used as references in the analysis with their corresponding GenBank accession numbers, source of origin and genogroups are showed in Table 2.

Result of viruses detection
RT-PCR or PCR were performed to detect the common viruses that may cause pig diarrhea. Result showed that the fecal sample was negative for porcine NoV, PCV 2, PRV, PTGV and PEDV, but was positive for porcine SaV.

Phylogenetic and recombination analysis
P2 shared the highest nucleotide homology (91%) through the entire genome and about 94% in the capsid The strains which were deposited in GenBank database by Chinese researchers and used in this study were bolded region with CH430 (GIII-3, GenBank no. KF204570) (a Chinese porcine SaV strain), respectively. However, it shared only 87% identity with strain CH430 in the RdRp region. On the contrary, p2 shared the highest 91% nucleotide identity with an American strain JJ259 (GIII-2, GenBank no. KT922089) in the RdRp region, which was opposite to the general phenomenon observed for caliciviruses that the RdRp region is more conserved than the capsid region. In the previous studies, SaV strains with inconsistent grouping between the nonstructural protein-encoding region (including the RdRp region) and the VP1 encoding region were designated as recombinant. All of these suggested that p2 strain may be a recombinant virus. Phylogenetic and recombination analysis were further performed to verify the genotype definition and recombination. Phylogenetic tree based on 42 of the complete VP1 nucleotide sequences was constructed by the NJ method, in which p2 was grouped into GIII-3 clustering with CH430 (Fig. 2). However, phylogenetic analysis based on the 3' end of RdRp nucleotide sequences gave a different grouping result, in which p2 was grouped into the GIII-2 clustering with JJ259 (Fig. 3). This finding suggested that this strain may be an intra-genogroup recombinant within GIII. To confirm the finding and detect the breakpoint where the recombination event occurred, we performed recombination analysis with p2 as the query sequence, JJ259 and CH430 as the background sequences and OH-MM280 (GIII-1, GenBank no. AY823308) as the outlier sequence using RPD software. Recombination analysis confirmed that the p2 strain was a recombinant and the major and minor parent was JJ259 and CH430, respectively. Moreover, the breakpoint for this recombination event located at position 5139 nt of the genome, which was the polymerasecapsid junction within ORF1 (Fig. 4).

Discussion
SaV causes acute gastroenteritis in humans and animals including pigs, mink, dogs, sea lions, and bats [4,9,20]. Porcine SaV and human strains were separated into a different genogroups, however, based on analyses of RdRp or capsid protein genes, porcine SaV that genetically resembled human strains rather than previously recognized porcine strains had been identified [21,22]. These findings suggested the possibility of a pig reservoir for human strains or vice versa. Meanwhile, recombination as an important survival event for all living creatures, may result in generation of new viruses with unknown pathogenic potential and altered species tropism for both animals and humans [23]. To date, both intra-and inter-genogroup recombinant strains have been reported [11][12][13][22][23][24]. So far the recombinant strain was not found in either human beings or animals of China, although the SaV infections in children and pigs were common in this area [25][26][27]. Here, we reported a complete genome of a recombinant SaV strain that identified from a piglet with diarrhea in China. Phylogenetic and recombination analysis based on the genomic sequences showed that p2 was an intragenogroup recombinant within GIII, and the breakpoint located in the RdRp-capsid junction region, which was consistent with most of other SaV recombination events. Previous reports had shown that, in the genome of SaV, recombination mostly occurred at the polymerase-capsid junction region within ORF 1 which was referred as 'hot spot' [2,13]. Chang et al. detected a 2.2 kb RNA in vitro replication assay with the replication complex of SaV Cowden strain extracted from virus-infected cells suggesting that SaV will generate subgenomic RNA during virus replication [8]. Moreover, researchers found that the RdRp-capsid junction region of SaV contained a highly conserved~20 nucleotide (nt) motif in both genomic and subgenomic RNA molecules which was considered as a transcription start signal [2]. This conserved nucleotide motif may facilitate homologous recombination during co-infection of a cell by different genogroups of virus.
SaV has been detected in a wide range of mammals with potential ability of zoonotic transmission [28][29][30][31]. Recombination was considered as a force of evolution which may produce new virus with potentially different pathogenesis and virulence [32]. Moreover, an infectious Fig. 2 Phylogenetic tree based on the complete VP1 nucleotide sequences of 42 SaV strains using MEGA 7. These strains represented all reported 8 genogroups (GIII, GV-GXI) of porcine SaV. GIII strains were grouped into 5 genotypes (GIII-1 -GIII-5). Among those strains, the p2 strain determined in this study was marked with box. Each SaV strain is presented as the following format: strain ID-GenBank accession number NoV chimera comprising the distinct biological properties from the parental viruses has been constructed and is infectious in vivo [32,33]. Therefore, detection and understanding the recombination of SaV is important. In the current study, p2 strain was identified from a pig with diarrhea, in which bacteria and the common enteric viruses that cause pig gastroenteritis were ruled out. This suggested that this recombinant virus may cause this piglet diarrhea under the nature condition. More researches such as experimental infection should be performed in the future to confirm the pathogenicity of this recombinant strain.