Recombinant porcine norovirus identified from piglet with diarrhea
© Shen et al.; licensee BioMed Central Ltd. 2012
Received: 6 January 2011
Accepted: 22 August 2012
Published: 3 September 2012
Noroviruses (NoVs) are members of the family Caliciviridae and are emerging enteric pathogens of humans and animals. Some porcine NoVs are genetically similar to human strains and are classified into GII, like most epidemic human NoVs. So far, PoNoV have been exclusively detected in fecal samples of adult pig without clinical signs.
Result showed that 2 of the 12 evaluated fecal samples were positive for PoNoVs, one of which was positive for PoNoV alone, and the other was coinfected with porcine circovirus and PoNoV. Phylogenetic and recombination analysis showed that the PoNoV positive alone strain was a recombinant new genotype strain. Experimental infection of miniature pigs with fecal suspensions confirmed that this strain can cause gastroenteritis in piglets.
This is the first report that recombinant new genotype PoNoV exised in pig herd of China, which cause diarrhea in pigs in nature condition. This find raised questions about the putative epidemiologic role of PoNoV.
KeywordsPorcine norovirus Recombinant New genotype Gastroenteritis
Noroviruses (NoVs) are members of the family Caliciviridae and are emerging enteric pathogens of humans and animals [1, 2]. They are small, non-enveloped viruses of 27–38 nm in diameter and possess a single-stranded, positive-sense genomic RNA. NoVs genome is 7.3-7.7 kb in length and contains 3 open reading frames (ORFs) . ORF1 encodes a polyprotein that is autocatalytically cleaved to produce several proteins, including RNA-dependent RNA-polymerase (RdRp) and other non-structural proteins . ORF2 encodes the major capsid protein, and ORF3 encodes for a minor structural protein . NoVs have been isolated from humans and several species of animals, including swine, canine, bovine, murine and lion [6–8]. Based on sequence analyses comprising conserved regions within the RdRp and the capsid genes, NoVs are divided into 5 genogroups (GI-GV) and further subdivided into 27 genotypes . Human NoVs are located in GI, GII and GIV. GII contains 19 genotypes, and porcine strains are found in GII-11, -18 and -19 . Some porcine NoVs are genetically related to human strains and are classified into GII, which contains most epidemic strains of NoVs in human. Some potential NoVs recombinant strains have been identified [10, 11], which raises public health concerns regarding their potential for zoonotic transmission. To date, PoNoV have been exclusively detected in fecal samples of adult pig without clinical signs.
Twelve fecal samples from piglets with diarrhea and no accurate of etiology were collected from three commercial pig farms in a Shanghai suburb, from May to August, 2009. In order to avoid sample contamination, specimens were obtained directly from the pig anus and disposable materials were used during sampling. Stool samples were freshly collected and immediately converted to 10% (w/v) suspensions in PBS (0.01 M phosphate, pH 7.2-7.4, 0.15 M NaCl,) for RNA and DNA extraction.
RNA and DNA extraction
RNA was extracted from 200ul of 10% fecal suspension by using the TRIzol reagent (Invitrogen, USA) following the manufacturer’s instructions. RNA pellets were dissolved in 25ul RNase-free water and reverse transcription was performed immediately. DNA was extracted using The QIAamp DNA Stool Mini Kit (QIAGEN, Germany) according to the manufacturer’s instructions.
RT-PCR or PCR
RT-PCR or PCR assays with different primer sets for the detection of PoNoV, and common viruses that can cause pig diarrhea including porcine circovirus type 2, porcine rotavirus, porcine transmissible gastroenteritis virus, porcine sapovirus, and porcine epidemic diarrhea virus were performed as previously described [12–14]. The amplicons were analyzed by 1% agarose gel electrophoresis in TAE buffer, followed by staining with ethidium bromide (0.5 μg/ml) and visualization under UV light.
Whole genome amplification
Primers designed to amplify the remaining sequence of pNoVs-Ch6
Nucleotide sequence (5′-3′)
3′endF1(external forward primer)
5′endF2(internal forward primer)
Sequence and recombination analysis
Eight 15-day old specific-pathogen-free (SPF) miniature pigs were purchased from Experimental Animal Center, School of Agricultural and Biology, Shanghai Jiaotong University (Shanghai, China), and maintained in a pathogen-free animal facility. This animal experiment was approved by the IACUC with a permit number of IACUC-2009-S-0202, which complies with the National Institute of Health Guide for the Care and Use of Laboratory Animals.
The pNoVs-Ch6 fecal specimen was converted to 20% (wt/vol) suspensions in phosphate-buffered saline (PBS) (0.01 M, pH 7.4) and clarified by centrifugation at 10,000 g for 10 min. The supernatant was then subjected to purification by passage through microfilters with a pore size of 0.22 μm (Millipore, Japan) to remove possible bacteria and parasites. A 1.5 ml aliquot of supernatant was used to infect five 15-day old piglets through oral inoculation. Another three piglets were only inoculated with PBS as controls. The piglets were investigated every 4 hours during the first day, and fecal samples were collected from each piglet every day. For the experimental group, one piglet was euthanized at post-inoculation day (PID) 1, 2, 4, 6, 10; meanwhile, one was euthanized at PID 1, 4, 10 for the control group. The intestinal contents were collected for RNA testing using RT-PCR, and intestinal segments were excised and immediately immersed in the different fixatives for histological examination. Tissues for histological examination were fixed in 10% neutral buffered formalin, routinely processed, sectioned at a thickness of 7 μm, and stained with hematoxylin and eosin. The tissue sections were examined and compared with negative controls.
Result of PoNoV detection
Result of detection indicated that two of the fecal samples were positive for PoNoV, one of which was coinfected with porcine circovirus. The others were negative for PoNoV, porcine circovirus type 2, porcine rotavirus, porcine transmissible gastroenteritis virus, porcine sapovirus, and porcine epidemic diarrhea virus. The fecal sample that was positive for only PoNoV was selected for further genome amplification and experimental infection.
Genome organization and recombination analysis
The entire genome of this PoNoV strain (named pNoVs-Ch6, GenBank accession number: HQ392821) consisted of 7531 nucleotides, excluding the poly (A) tail. Similar to previously reported NoVs strains, pNoVs-Ch6 has three open reading frames (ORFs), encoding 1693, 547 and 253 amino acids (aa), respectively. It shared 88% nucleotide homology with AB126320 over the entire genome sequence. The highest nucleotide homology over the RdRp sequence was shared with AB126320 (89%) and then with an American porcine strain AY823303 (88%).
Experimental infection of miniature pigs with PoNoV positive fecal suspensions
NoVs has been detected in a wide range of species, including humans, porcine, mice, cows and lions. Because PoNoV are genetically and antigenically related to human GII NoVs, there are public health concerns of potential cross-species transmission and animal reservoirs for NoVs related to human NoVs [10, 17]. So far, PoNoV have been exclusively detected in fecal samples of adult pig without clinical signs [18, 19], though in experiments with gnotobiotic piglets mild diarrhea occurred . In the current study, gastroenteritis in piglets caused by a PoNoV strain was reported. Experimental infection of miniature pigs with PoNoV positive fecal suspensions confirmed that this virus strain can cause gastroenteritis in pigs. To our knowledge, this study is the first identification of a PoNoV strain that cause piglets gastroenteritis under near natural conditions.
The genome of RNA viruses often undergoes recombination and segmentation .
Recombination is a driving force of viral evolution and has been reported for many single-stranded RNA viruses, including NoVs. It has been reported that the recombination of influenza virus leads to generation of a new strain and increases the biological fitness of the virus and its pathogenicity .
Previous reports have shown that, in the genome of NoVs, recombination mostly occurs at the junction point of ORF1 and ORF2 which is referred to as ‘hot spot’ [21–23]. Results of phylogenetic analysis and recombination identification showed that pNoVs-Ch6 was a recombinant strain, and the breakpoint located in the RdRp-capsid junction region like most other NoVs recombination events. To the best of our knowledge, this is the first report of recombinant PoNoV in China.
The detection of PoNoV that can cause gastroenteritis in piglets from commercial pig farms under natural conditions, on one hand, raises questions about the putative epidemiologic role of PoNoV, and, on the other hand, this kind of infectious strain also can be used to improve research such as virus replication, vaccine development, which has been impeded by the lack of a cell culture system for the propagation of human NoVs. Whether the recombination increased the virulence of PoNoV still needs further research.
In conclusion, the results in this study demonstrated that recombinant new genotype PoNoV exised in pig herd of China, which cause diarrhea in pigs in nature condition. This find raised questions about the putative epidemiologic role of PoNoV.
We thank Kelly Scheuer for assistance in linguistic revision of the manuscript.This work was supported by grants from the National Natural Science Foundation of China No. 31070132 and No.31270186, Key Project of Shanghai Science and Technology Committee of China under Grant No.10JC1406500, Natural Science Foundation of Jiangsu Province No.BK2012289, Research Fund for the Doctoral Program of Higher Education of China No. 20113227120008, and the Professional Research Foundation for Advanced Talents of Jiangsu University.
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