Molecular characterization and phylogenetic analysis of the complete genome of a porcine sapovirus from Chinese swine
- Shixing Yang†1,
- Wen Zhang†2,
- Quan Shen1,
- Fen Huang1,
- Yan Wang1,
- Jianguo Zhu1,
- Li Cui1,
- Zhibiao Yang1 and
- Xiuguo Hua1Email author
© Yang et al; licensee BioMed Central Ltd. 2009
Received: 18 August 2009
Accepted: 6 December 2009
Published: 6 December 2009
Porcine sapovirus was first identified in the United States in 1980, hitherto, several Asian countries have detected this virus. In 2008, the first outbreak of gastroenteritis in piglets caused by porcine sapovirus in China was reported. The complete genome of the identified SaV strain Ch-sw-sav1 was sequenced and analyzed to provide gene profile for this outbreak.
The whole genome of Ch-sw-sav1 was amplified by RT-PCR and was sequenced. Sequence alignment of the complete genome or RNA dependent RNA polymerase (RdRp) gene was done. 3' end of ORF2 with 21-nt nucleotide insertion was further analyzed using software.
Sequence analysis indicated that the genome of Ch-sw-sav1 was 7541 nucleotide long with two ORFs, excluding the 17 nucleotides ploy (A) at the 3' end. Phylogenetic analysis based on part of RdRp gene of this strain showed that it was classified into subgroup GIII. Sequence alignment indicated that there was an inserted 21-nt long nucleotide sequence at the 3' end of ORF2. The insertion showed high antigenicity index comparing to other regions in ORF2.
Ch-sw-sav1 shared similar genetic profile with an American PEC strain except the 21-nt nucleotide at the 3' end of ORF2. The insert sequence shared high identity with part gene of Sus scrofa clone RP44-484M10.
Caliciviridae is a family of positive sense single-stranded RNA viruses comprised of both human and animal pathogens . Caliciviridae family contains four genera, Lagovirus, Vesivirus, Norovirus and Sapovirus . Various caliciviruses possess common features. For example, they are small, non-enveloped virus, 27-38 nm in diameter. They possess a single-stranded, 7.3-8.3 kb plus-sense RNA genome, a single 56-71 kD capsid protein , and a polyprotein containing confering motifs of a putative 2C helicase, 3C-like protease, and 3D RdRp. SaV are recognized as emerging enteric pathogens in humans, swine and mink . SaV infection may cause diarrhea especially in the younger . It is currently divided into eight distinct genetic groups (GI-GVIII) based on the RdRp gene. Among these genetic groups, GIII can't infect humans but can be cultured in vitro in the presence of bile acid . The genome of SaV consists of 7.1-7.5 kb nucleotide and encodes two or three open reading frames (ORFs). ORF1 encodes one polyprotein that contains coding sequences for the nonstructural proteins and the major capsid protein (VP1), ORF2 encodes the minor structural protein (VP2), while ORF3 is only present in strains from genotypes GI, GIV and GV, and encodes a small basic protein . SaV is considered as a significant global enteropathogen of acute gastroenteritis . Recently, it was shown that the host tropism of some calicivirus is less specific. Some calicivirus may have zoonotic potential, and animals such as domestic pig may be a reservoir for caliciviruses [9–11]. Porcine sapovirus was first identified in the United States by electron microscopy in 1980  and genetically characterized as a sapovirus in 1999 . Recently, SaV infections have been identified in Japan, South Korea, Venezuela, Hungary and Belgium [14–18]. In the United States, porcine sapovirus was also detected from Oyster . Although porcine SaV was mainly detected in pigs, some studies indicated that some porcine SaV might be potential pathogencity transmitting to humans. For example, the porcine SaV strain (Sapovirus pig/43/06-18p3/06/ITA) isolated from Italy was most closely related to human SaV through the alignment of RdRp sequences, suggesting the possibility of a pig reservoir for human strains or vice versa . We previously reported an outbreak of gastroenteritis in piglets in China caused by the first Chinese porcine SaV strain . In this study, gene profile of this strain was investigated, the entire viral genome and 3' end of Ch-sw-sav1 were cloned and sequenced.
Porcine SaV positive fecal samples were collected from commercial pig farms in Shanghai as introduced in our previous study. Samples were converted to 20% (wt/vol) suspensions in phosphate-buffered saline (PBS) (0.01 M, pH 7.2 to 7.4) and clarified by centrifugation at 10,000 g for 10 min.
Nucleotide sequences of the oligonucleotides used for PCR amplification and sequencing
RNA extraction and cDNA synthesis
Viral RNA was extracted with TRIzol Reagent from supernatants of fecal suspensions, according to the manufacture's instructions. The cDNA synthesis was primed by Oligo dT16 or the reverse one of each set of primers using TaKaRa RNA PCR kit (TaKaRa, Japan) in a 10 μL reaction volume. The reaction condition was 40 min at 42°C, then 15 sec at 86°C.
PCR and RACE amplifications of the full-length SaV genome
PCR was carried out in 50 μL reaction volume, containing 8 μL dNTP Mixture (25 mM), 5 μL 10×Ex-taq buffer, 0.2 μL Ex Taq, 1 μL (25 mM) of each primer, 10 μL of template and adding sterilize H2O to 50 μL. The reaction was done with the following profile: Activation of DNA polymerase at 95°C for 5 min, followed by 35 cycles of denaturation of DNA at 95°C for 40 sec, annealing at the 50°C for 1 min, extension at 72°C for 1 min and then followed by a final extension step at 72°C for 10 min. Purfied PCR products were ligated to pMD-18T vector (TaKaRa, Japan) and 3 to 5 positive colonies were sequenced.
The 3' RACE was carried out with TaKaRa RNA PCR Kit (TaKaRa, Japan) following the manufacture's instructions. Briefly, ten microliters of RNA were used as template to synthesize cDNA with AMV Reverse transcriptase for 1 h at 42°C. The external reverse primer which has a poly (T) tract was used to prime the cDNA synthesis. The cDNA was then amplified with the external forward primer (5'-TCAATTGGCTGGG TCACGTGAAG-3', nucleotide position numbers 7027-7049) and internal forward primer (5'- CAAACACCTTTGGTCCACCAAGG-3', nucleotide position numbers 7070-7092) with Ex Taq DNA polymerase (TaKaRa, Japan). The PCR reaction mixture was incubated for 2 min at 94°C, followed by 35 amplification cycles comprising denaturation at 94°C for 30 s, annealing at 65°C for 30 s, and extension at 72°C for 30 s. The product was extended for another 7 min at 72°C to ensure a full extension.
The PCR products were purified from 1% agarose gel using the QIAquick Gel Extraction kit (Qiagen, Gemany). Purified PCR products were ligated into pMD18-T Vector. For each product, three to five positive colonies were selected and sequenced.
Summary of sapovirus strains and representative strains for Lagovirus, Vesivirus, and Norovirus genera and NB-like viruses used in sequence analysis
GenBank accession no.
Porcine enteric sapovirus/USA
Human calicivirus strain Mc37/Japan
San Miguel sea lion virus serotype 1
European brown hare syndrome virus
European brown hare syndrome virus
3' end of ORF2 partial sequences analysis
Six available Porcine SaVs partial sequences of 3' end of ORF2 were retrieved from GenBank, according to sequence alignment. As follows: OH-MM-280-03-US (AY823308), PEC-USA (AF182760), strain LL14 (AY425671), OH-JJ-259-00-US (AY826423), NC-QW-270-03-US (AY826426). Nucleotide sequence and protein were aligned by CLUSTAL W method using DNAstar software, antigen index was analysed by protean using DNAstar software.
Genomic organization of Ch-sw-sav1 virus
Percentages of nucleotide sequence identity of Ch-sw-sav1 with other caliciviruses in regions aligned for phylogeny
GenBank accession no.
Porcine enteric sapovirus/Japan
Porcine enteric sapovirus/K8/JP
Porcine enteric sapovirus/K7/JP
Sapporo virus was identified in 1982 from an outbreak of diarrhea in an orphanage in Sapporo, Japan . Schuffenecker  classified them into three major genetic groups. Furthermore, it has been divided into eight genogroups based on the genetic diversity of the viral polymerase . PEC, the first of pig origin, was discovered in 1980s in the United States and belongs to SaV GIII . Hitherto, SaV has been identified in many countries [14–18]. Traditionally, we thought only SaV GIII infected pig. However, strains detected in USA and Italy that belonged to new genotype showed high homology with human SaVs respectively. It indicated that animals might act as reservoirs for human caliciviruses. So it is necessary to analyze the genetic profile of porcine SaV for the first step of controlling the pathogen. In February 2008, we reported the first outbreak of gastroenteritis caused by porcine SaV in piglets in China mainland. It may be caused by simultaneous contact with virus polluted water or food and the virus gene profile was further investegated. Ch-sw-sav1 was chosen to be sequenced and compared with other SaV published. Results showed that it shared high homology with PEC for the similar gene structure and similar sequence motif at 5' terminus that was favorable for translation initiation of eukaryotic sequence . However, there was 21-nt nucleotide insertion at the 3' end of ORF2 of Ch-sw-sav1. The inserted sequence had a high antigenicity index analyzed with DNAstar software. It's predicted that ORF2 encodes capsid protein that is correlative with the assembly, antigenicity and receptor interations of SaV. So the inserted sequence may affect antigenicity profile or other profiles of capsid protein which need to be further identified . Accordingly, in phylogenetic analysis, we classified Ch-sw-sav1 into Genogroup III of SaV basing on the partial RdRp gene sequence, and it shared highest nucleotide identity with the Hungary SaV (91.2%) which was isolated from a diarrheaed pig .
The porcine SaV strain in the present study came from an outbreak of gastroenteritis in piglets group, which had inserted sequence at the 3' end of ORF2. The role of the inserted sequence was unknown, but it is highly divergent in sequence and differs in size in caliciviruse s. Since the ORF2 protein is functionally conserved and may be involved in protein-protein interactions or protein-nucleic acid interactions during replication based on its strong positive charge. The inserted sequence likely has special biological function. So establishing full-length infectious clones containing or not containing this inserted fragment would now be the next step towards the identification of this fragment involved in symptomatology and pathogenicity.
Complete sequence of the first Chinese porcine SaV was determined and analyzed providing a gene profile of porcine SaV presented in swine population in China today. Sequence analysis showed that it was classified into genogroup III with two ORFs. A 21-nt insertion in ORF2 changed antigenicity index of capsid protein.
This work was supported by Key Project of Shanghai Science and Techology Committee of China under Grant No.063919121.
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