Phylogenetic Analysis Of Porcine Torovirus And Development Of Multiplex Detection Methods For Swine Diarrhea-related Viruses

Diarrhea is a common disease in pig breeding.It can infect pigs of all ages and cause growth inhibition or even death,bringing huge economic losses to pig industry.Viral infection is one of the main factors causing diarrhea in pigs.Members of family Coronaviridae,such as porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus(TGEV),swine acute diarrhea syndrome coronavirus(SADS-CoV),and porcine deltacoronavirus(PDCoV),are common causes of swine diarrhea.Besides,the latent possibility of some viruses to cause swine diarrhea are often been ignored.Porcine torovirus(PToV)is an example.PToV belongs to family Tobaniviridae,order Nidovirales,sharing similar genomic structure and component with coronaviruses.In this study,we conducted an epidemiological survey for PToV,and developed multiplex RT-PCR and RT-qPCR clinical detection methods for the prevention and control of swine diarrhea diseases.1.Epidemiological survey and phylogenetic analysis of PToVIn this study,307 swine diarrhea samples collected from Henan,Guangdong,Anhui and Jiangsu provinces in China during 2017 to 2019 were detected with PCR,in which 18 were positive for PToV(positive rate 5.9%).Co-infection analysis showed that the co-infection rate of PToV with PEDV,PDCoV,porcine teschenvirus(PTV),and porcine sapelo virus(PSV)are 38.9%?5.6%?27.8%and 16.7%,separately.5 whole genome sequences,9 Spike(S)gene sequences and 17 Hemagglutinin-esterases(HE)gene sequences of PToV were obtained through genomic amplification of positive samples.Phylogenetic analysis was performed using those newly acquired sequences and other available PToV strain sequences in public databases.Results showed that PToV strains can be divided into 2 clades according to phylogenetic trees based on S gene,HE gene and the whole genome.The newly obtained PToV full-length sequences have a nucleic acid similarity of 95.0%-99.7%,all belonging to the same clade.Based on the topological differences among different phylogenetic trees,we performed recombination analysis on the whole genome of PToV,which revealed large scales of recombination events in HE and S genes of PToV.All results above exhibited the epidemic circumstances and genetic diversity of PToV in China,enriched the genetic data of PToV,and provided scientific evidence for further research and prevention of this virus.2.Development of a multiplex RT-PCR detection method for the simultaneous detection of 5 swine diarrhea viruses including PToVIn this study,5 pairs of PCR primer specific for PEDV,PDCoV,PToV,SADS-CoV and TGEV were designed for the simultaneous detection.The annealing temperature and concentration of the primers were then optimized to assure good sensitivity,specificity and repeatability of the detection method.Our method can detect single or mixed plasmid standards as low as 1×103 copies/?L.All 29 simulations of co-infection events were detected precisely,and there were no non-specific amplification caused by other swine viruses observed.A total of 87 clinical samples were tested using this multiplex RT-PCR detection method,the result of which was 100%consistent with the result of singleplex RT-PCR detection method,indicating that our new method has good specificity,sensitivity,and repeatability.3.Development of a multiplex RT-qPCR detection method for the simultaneous detection of 4 swine diarrhea viruses including PToVBesides,a multiplex TaqMan-probe-based RT-qPCR detection method was also developed in this study for the simultaneous detection of PEDV,PDCoV,PToV and SADSCoV.We designed 4 sets of specific primers and TaqMan probes based on available sequence data.The final concentration of primers and probes was optimized to achieve best fluorescent signals.Our multiplex RT-qPCR detection method can detect single or mixed plasmid standards as low as 1×102 copies/?L stably,and can adapt to various simulations of coinfection events,showing good repeatability.A total of 101 clinical samples were detected using this method,the result of with showed 100%consistency with the result of singleplex RT-PCR detection method.In conclusion,this study investigated the epidemic circumstances and phylogenetic features of PToV in center and south-eastern China,as well as developed 2 multiplex detection methods for swine diarrhea viruses,offering reliable and powerful tools for the epidemiological investigation,clinical detection,prevention and surveillance of swine viral diarrhea diseases.