Retrospective Detection,Isolation And Identification Of Swine Acute Diarrhea Syndrome Coronavirus And Establishment Of Taqman-based Real-time RT-PCR Assay

A novel coronavirus,swine acute diarrhea syndrome coronavirus(SADS-CoV),is an enveloped,positive and single-stranded sense RNA virus with a genome size of approximately 27 kb.SADS-CoV belongs to the family Coronaviridae and the genus Alphacoronavirusf.It was first discovered in Guangdong province in 2017 and caused a large scale outbreak of fatal diarrheal disease in piglets.Here,a retrospective investigation was conducted with 236 samples from 45 swine farms with a clinical history of diarrheal disease to evaluate the emergence and the distribution of SADS-CoV in Chinese pigs.The results suggested that SADS-CoV had emerged in China at least since July 2016.Eleven swine farms were tested positive for SADS-CoV.Meanwhile,a prevalence of SADS-CoV(43.5%)with detected,which was the second highest after PEDV(78.2%).The co-infection of SADS-CoV and PEDV occurred most frequently.We screened and obtained two new complete genomes,five N and five S genes of SADS-CoV.The sequences shared high sequence identities of 99.7%~100% with previous reported sequences of SADS-CoV.Phylogenetic analysis based on these sequences revealed that all SADS-CoV sequences in this study clustered with previously reported SADS-CoV strains and had a close relationship with the bat coronavirus HKU2 strains in AlphaCoVs branch.As a newly emerged swine diarrhea pathogen,there is an urgent need to establish a faster and reliable diagnostic technique for quantification and identification of SADS-CoV.A pair of specific primers and probe were designed and synthesized based on the conserved region of SADS-CoV N gene.Results showed that the method was specific for SADS-CoV,and there were no cross-reaction apperceived against other swine viruses,the lowest limit of detection was 10 times more sensitive than the conventional RT-PCR and gave higher SADS-CoV positive detection rate(70.69%,123/174)than the conventional RT-PCR(51.15%,89/174)from clinical samples.These data revealed that the TaqMan-based real-time RT-PCR assay established here was an effective method with high sensitivity,specificity and reproducibility for faster detection and more accurate quantification of SADS-CoV.In this study,four SADS-CoV strains were isolated form homogenized small intestines of infected piglets in four swine farms.With 8 ?g/mL trypsin in DMEM,the SADS-CoV strains can multiply stably and cause typical cytopathic effect(CPE)inVero cells.The four SADS-CoV strains were sequenced and alignment results showed that all SADS-CoV sequences shared 99% nucleotide identity.Under the electron microscope,coronavirus particles can be clearly observed.The identity of SADS-CoV was also verified in Vero cells by indirect immunofluorescence assay and specific fluorescence could be seen in the part of cytoplasm.These results proved that the successful isolation of four SADS-CoV strains.In order to confirm the virulence of SADS-CoV strains,pathogenicity analysis was conducted on 3-day healthy newborn piglets from a farm which had been free of diarrhea disease for a number of weeks.Two groups(6 piglets for each group)were inoculated with SADS-CoV culture supernatant with normal cell culture medium as control.Fecal swabs were collected daily from all piglets to screen known swine diarrhea viruses by RT-PCR.Meanwhile,piglets were recorded daily for signs of diseases,such as diarrhea,weight loss and death.At 48 h after infection,all piglets in infected group suffered watery diarrhea,rapid weight loss,intestinal lesions and only SADS-CoV was detected positively.At the end point of experiment,3/6 piglets died in infected group while all piglets survived in the control group.The result of quantitative real-time RT-PCR showed that the part of small intestine had the highest viral load.These results indicated that SADS-CoV could cause typical clinical signs and pathogenic for piglets.