Establishment And Application Of Real-Time Fluorescent Quantitative RT-PCR For The Detection Of SFTSV

Severe fever with thrombocytopenia syndrome virus(SFTSV)belongs to the Bunyaviridae,a member of the Phlebovirus genus.The virus was first discovered in China,and subsequently reported in Japan,South Korea and the United States.SFTSV mainly infects humans through tick bites and causes severe fever with thrombocytopenia syndrome(SFTS),with a mortality rate of between 6%and 30%.Since SFTSV was discovered in 2009,cases of SFTSV infection have been reported in 23 provinces of China,with an increasing trend every year.SFTSV has a wide host range and can infect a variety of animals in epidemic areas.Researchers have detected viral nucleic acids in a variety of domestic and wild animals in epidemic areas.Epidemiological investigation found that ticks collected from animals with positive SFTSV nucleic acid test could also be detected as positive nucleic acid,proving that ticks are its transmission medium.At present,there is neither effective vaccine to prevent SFTSV infection,nor special drugs to treat SFTSV infection,and only symptomatic treatment can be carried out clinically Therefore,it is particularly important to establish an easy-to-operate and highly sensitive detection method for the prevention and control of SFTSV.In this study,primers and probes were designed according to the Rd Rp gene of SFTSV,and a standard plasmid containing the target fragment was constructed.By optimizing the reaction system and conditions,a real-time fluorescence quantitative RT-PCR method for detecting SFTSV was established.The results showed that the linear relationship between Ct values of the standard curve and the template was good when the standard plasmid concentration at a range of 4.22×10~2 copies/μL to 4.22×10~9copies/μL,and the correlation coefficient(R~2)was 0.9998.The minimum detection limit for SFTSV was 4.22×10~1 copies/μL,which was 100 times more sensitive than the conventional RT-PCR.The results showed that the detection of SFTSV was specific,while other viruses in the same family were detected negative by this method.And the variable coefficient of intra-and inter-reproducibility assay was below 2%.The 200 ticks collected were tested using the established real-time RT-PCR and the positive rate was 3.5%,while the positive rate was only 2%when these samples were detected by the conventional RT-PCR.The real-time RT-PCR detection method has high specificity and sensitivity,good repeatability.The result provided a useful tool for clinical sample detection of SFTSV.The established real-time quantitative RT-PCR method was used to detect SFTSV in 200 bovine serum samples collected from Heilongjiang province,and 17 samples were found to be SFTSV positive,the positive rate was 8.5%.In order to evaluate the pathogenicity of SFTSV to ferrets,six ferrets were inoculated with 200μL of SFTSV by subcutaneous injection and spot eye injection respectively.The results showed that ferrets infected with SFTSV showed typical symptoms such as fever,weight loss,decreased platelet count and white blood cell count.Histopathological observation showed that in the infected group,juvenile erythrocytosis and hemosiderin deposition occurred in the red pulp part of the spleen,degeneration of some neurons and proliferation of glial cells occurred in the brain tissue,perivascular infiltration of glial cells was observed around small blood vessels,degeneration and necrosis of some acinar cells were observed in the stomach.Subsequently,the real-time fluorescence quantitative RT-PCR method established in this study was used to detect the viral load in the tissues,organs and serum of SFTSV infected ferrets,and it was found that viral nucleic acid was detected in all tissues,organs and serum of SFTSV infected ferrets.In the infected ferret group,the spleen had the highest viral load,followed by the stomach and brain,suggesting that the spleen may be a susceptible organ to SFTSV.The viral load in all tissues peaked at day 6 and gradually decreased thereafter.Serum viral load also reached its peak at day 6 and then began to decrease.This study successfully established an animal model of SFTSV infection in ferrets.In this study,a convenient and sensitive real-time quantitative RT-PCR detection method was successfully established,which can be used for the detection of clinical SFTS,and provides a technical means for the epidemiological monitoring of SFTS.The pathogenicity of SFTSV to ferrets was studied,which laid a foundation for further research on the pathogenesis of SFTSV and screening of antiviral drugs and vaccines.