| Porcine teschovirus?PTV?and Porcine sapelovirus?PSV?belongs to the genuses of Teschovirus and Sapelovirus,respectively,within the family Picornaviridae,and are widely distributed in pig populations.Infected swine with PTV or PSV can lead to a variety of clinical symptoms,including diarrhea,respiratory disease,polioencephalomyelitis, and even death.For investigate the prevalence and molecular characterization of PTV and PSV in pig populations of Hunan province.Firstly,two RT-PCR methods specially for PTV and PSV detection were established,and there were not specially bands occurred when amplify the DNA or cDNA of other porcine viral pathogens with this two methods.The sensitivity of this two RT-PCR methods was evaluated by using plasmids containing known amounts of viral cDNA.Detection limits with PTV-plasmid and PSV-plasmid were 36.4 copies/?L and 58.9 copies/?L,respectively,which indicated a high sensitivities for PTV and PSV detection.To investigate the prevalence of PTV and PSV circulating in Hunan,China,460 fecal and 118 intestinal content samples were collected from 2014 to 2017 in 42 farms.An overall PTV-positivity rate of 14.88%was detected with the RT-PCR method of PTV detection.Specifically,PTV was identified in 9.18%of the suckling pigs,in 16.44%of the nursery pigs,and in 20.55%of the fattening pigs.Thus,the high PTV prevalence was found in fattening and nursery pigs,which was significantly higher than that in suckling pigs.Moreover,the pigs without diarrhea had a significantly higher infection rate?19.06%?than those with diarrhea?7.87%?,and the PTV genotypes identified in pigs without diarrhea?15 genotypes?were much more than in those with diarrhea?8 genotypes?.PTV comprises at least 13 genotypes?PTV 1–13?.Here,the genotypes of field strains prevalent among pig populations in Hunan Province,China,were identified.Multiple PTV genotypes,including all genotypes except PTV 7 and 8,were found co-circulating in the pig populations,reflecting a high genetic diversity.Moreover,widespread co-infection with different PTV isolates?genotypes?,which make up 26.74%of the PTV-positive samples was reported in this study.In addition,we identified four novel PTV genotypes,provisionally designated as PTV 14–17.To our knowledge,this is the first such report.An overall PSV positivity rate of 46.39%was detected with the RT-PCR method of PSV detection.PSV was identified in 17.39%of the suckling pigs,in 60.44%of the nursery pigs,and in 49.32%of the fattening pigs.The highest PSV prevalence was found in nursery and fattening pigs,which was significantly higher than that in suckling pigs.In addition,the pigs without signs of diarrhea had a significantly higher infection rate?48.34%?than those with diarrhea?31.94%?.Moreover,co-infection with PTV and PSV was identified in 9.86%of the pig populations.In general,PTV and PSV were widely spread and co-infected in the pig populations.PTV and PSV have a similar infectious pattern with peak period in the nursery and fattening pigs,moreover,the healthy pigs had the high infection rates of PTV and PSV indicated this two viruses infected swine are most often asymptomatic.The samples identified as PTV-positive and PTV 13-17 were selected and incubated with PK-15 and ST cells,respectively.After three passages in PK-15 and ST cells and RT-PCR identification,66 PTV strains were successfully isolated.The nearly complete genomes of PTV-HuN1–42 and complete VP1 genes of PTV-HuN43–45were sequenced and submitted to GenBank.Alignment of coding sequences of the 45PTV-HuNs with those of other known PTVs showed that the polyprotein genomic sequence contained 6,609–6,651 nucleotides,encoding a 2,202–2,217-amino acid sequence.Phylogenetic analyses based on the VP1 gene and capsid protein gene exhibited 17 main lineages corresponding to PTV genotypes 1–17,and eight PTV genotypes?PTV 2–6,9,11,and 17?were identified in the strains obtained in our study;this is the first report of isolation of PTV 3–6,9,and 11 in China,and the isolation of two PTV 17 strains is the first report in the world.Recombination analysis of the polyprotein gene of these 40 PTV-HuNs and other known PTVs suggested that9 recombination events?15 PTV-HuNs suggested as recombinants?occurred within the PTV-HuNs.Eight inter-genotype and an intra-genotype recombination events were identified.Two hotspots of breakpoints were also found adjacent to positions1410 and 3500 of the polyprotein gene,respectively.To estimate the rate of evolution and the time to the most recent common ancestor?TMRCA?of PTV,70 complete polyprotein gene sequences,including the 25 non-recombinant PTV-HuNs,were analyzed.The mean substitution rate for the complete polyprotein gene dataset was5.00×10-4 nucleotide substitutions per site per year.The TMRCA for PTV was estimated to be 511 years before present.Selection pressure analysis suggest that purifying selection is the predominant driving force contributing to PTV nucleotide mutations,and only a few positive selection sites are found.The samples identified as PSV-positive were selected and incubated with PK-15 cells.After three passages in PK-15 cells and RT-PCR identification,43 PSV strains were successfully isolated.The nearly complete genomes of PSV-HuN1–33 were sequenced and submitted to GenBank.Alignment of coding sequences of the 33 PSV-HuNs with those of other known PSVs showed that the polyprotein genomic sequence contained 6,972–7,005 nucleotides,encoding a 2,323–2,334-amino acid sequence,with most PSV-HuN strains containing 6996 nucleotides and encoding a 2332-AA sequence.Differences in the encoded sequences were significantly related to the insertions or deletions of AAs occurring in the C-terminal region of VP1.Phylogenetic and genetic divergence analyses of capsid-protein gene sequences indicated substantial genetic diversity among the PSVs in Hunan,China.However,phylogenetic analysis of all available 3CD gene sequences of PSVs showed a distinct phylogenetic topology with those of capsid-protein gene sequences.Therefore,we speculated that potential recombination events also potentially exist in the PSV-HuNs.Recombination analysis of the polyprotein gene of these 33 PSV-HuNs and other known PSVs suggested that 6 recombination events?8 PSV-HuNs suggested as recombinants?occurred within the PSV-HuNs.A putative recombinant hotspot near the 3?end of the P1 region was identified.Similarly,selection pressure analysis suggest that purifying selection is the predominant driving force contributing to PSV nucleotide mutations,and only a few positive selection sites are found.In conclusion,the simplex PTV/PSV-RT-PCR with the characterization of strong specificity,high sensitivity,and stable repeatability were established.The epidemic feature and rule of PTV and PSV in pig populations of Hunan province were clarified with the investigation of molecular prevalence using the methods.Moreover,genetic analysis of all available PTV and PSV sequences indicated high genetic diversities exists within PTVs and PSVs in Hunan,China,and strong purifying selection and recombination play important roles in the evolution of this two viruses.Thereby,this study providing a solid foundation for the research of epidemiology and molecular characterization of PTV and PSV and early warning and surveillance with the aim to prevent and control this potential pathogen,and avoiding economic losses in the pork industry. |
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