Identification Of Feline Infectious Peritonitis Virus HF1902 Strain And Eatabishment Of A SYBR Green Assay For FIPV

Feline coronavirus(FCoV)is an enveloped,positive-stranded RNA virus of subfamily Coronavirinae of the family Coronaviridae of the order Nidovirales.FCoV is classified into two biotypes:feline enteric coronavirus(FECV),considered as a mild infection of the enteric tract,and feline infectious peritonitis virus(FIPV),that can cause a highly lethal disease,known as feline infectious peritonitis(FIP)in cats.Currently,FCoV infection is widespread among domestic feline populations,and become one of major pathogenic virus in cats.However,it is critical to investigate FIPV in laboratory cell culture to understand the basic virology of FIPV infection,characterize FIPV isolates,test novel therapeutics,and develop effective vaccines with broader coverage.At present,there are few epidemiological data and effective detection methods about FIPV in China.In this study,we try to isolate wild FIPV and establish a method for detecting FIPV.The research contents of this paper include the following three aspects:Firstly,isolation and identification of FIPV HF1902 strain.In this study,a case of suspected FIP sample was collected.Histopathological examination showed that macrophages clustered around blood vessels accompanied by dense lymphocytes(mainly B cells)and plasma cells in the liver,kidney and intestine.And then,the total RNA was extracted from the liver.Subsequently,the N gene was amplified by RT-PCR.After the filter-sterilized material inoculated into fcwf-4 cells for third generation,the cells showed the characteristic cytopathic effect(CPE),including cell rounding,fall of and other stable cytopathic effect.The cultured viruses were collected and delivered to centrifuge under a high speed for purifying.We could observe a 100 nm crown-like particle under the electron microscope.At the same time,the fifth generation of inoculated filter-sterilized material fcwf-4 cells was confirmed the expression of FIPV N protein by indirect immunofluorescence(IFA)and Western blot.These results proved that we succes sfully isolated a FIPV strain and named it as HF1902 strain.Secondly,FIPV HF1902 strain genome was sequenced and analysis.According to the sequences of the referenced FIPV strains from Gen Bank,we designed 17 pairs of specific primers for determining the full-length genome of FIPV HF1902 strain.It is showed that the 29244 nt HF1902 strain genome,contain 38.11%G+C,has 11reading code box(ORFs)which encoded four structural protein(S,E,M,N),5accessory protein(7a and 7b,3a,3b,3c),and two non-structural protein(1a and 1b).At the ends of coding regions exist non-coding regions(Untranslated region,UTR),the 5?UTR comprises 303 nt and the 3?UTR consists of 255 nt.Using DNASTAR and MEGA6.0 software,FIPV and the reference stains'N and S gene sequences were analyzed.The phylogenetic analysis shows that FIPV HF1902 as other isolates in China which belongs to the FIPV gene type I.The molecular bioinformatics analysis showed that the nucleotide homology between FIPV HF1902 strain and the reported FIPV reference strain were 90.6%-92.4%(N gene)and 53.1%-90.0%(S gene),respectively.In addition,by comparing the S1/S2 site of S gene it is showed that FCoV II S1/S2 site is obviously missing 6 amino acids(KRSRRP),and compared with standard FCoV?type strains HF1902 isolates S1/S2 site has three amino acid substitution(R mutated as S,S mutated as A,P mutated as S).The prediction of the secondary structure of the N protein shows that the protein was a hydrophilic stable protein with 27.57%?-helix(h),15.12%extended chain(e),3.71%?turn(t)and 67.11%random spiral(c).There were no signal peptide region,transmembrane domain.It maybe has 48 potential phosphorylation sites.Moreover,there may exist six B cell epitopes,two CTL epitopes and two Th epitopes.Finally,to establish a real-time PCR method for detecting FIPV,a pair of specific primers was designed based on FIPV N gene.After optimized PCR conditions,a SYBR Green?real-time PCR method,that can quantitative detect FIPV was set up.This method not only has good specificity,but also has strong sensitivity.The minimum amount of detection was 10~2 copies/L,that is 1000 times higher than the sensitivity of conventional RT-PCR.Meanwhile,the repeatability coefficient of the method is less than 5%.Using this method,we quantitatively analyzed the viral load in the tissues of cats infected with FIPV,and the results showed that the viral load in the spleen was highest,and the viral load in other organs varied widely,up to 100 times.In summary,this study successfully isolated and identified a FIPV strain,and its full-length genome was determined and analyzed.We also successfully established a specificity,sensitivity of SYBR Green?real-time PCR detection method.The results of this study laid a foundation for further developing antiviral drugs and vaccines against FCoV infection.