Severe Fever With Thrombocytopenia Syndrome Virus Aerosol Infection In C57/BL6 Mice

Fever with Thrombocytopenia Syndrome(SFTS)is a new tick-borne infectious disease that has recently spread in East Asia.The disease is caused by Bunyavirus’s purpose syndrome virus(SFTSV).The main clinical manifestations are fever,thrombocytopenia,leukopenia,abdominal pain and diarrhea and other gastrointestinal symptoms.Some patients may have neurological symptoms.In severe cases,they may experience multiple organ failure or even death.At present,the disease is mainly transmitted through tick bites,but many patients have no history of tick bites before the onset of the disease.Some clinical studies and epidemiological investigations have shown that SFTS patients without a history of tick bites may be infected with the disease through contact with non-biological samples such as blood and secretions of positive patients and animals,but some cases still lack relevant exposure history and the unexplained cluster cases persisted.In this study,serum samples of suspected SFTS patients from a hospital in Jinan were collected from June to December 2019 according to the Diagnosis and Treatment Plan for Fever with Thrombocytopenia Syndrome.Among them,66 cases were positive by nucleic acid test.Analysis of demographic information,epidemiological history,and clinical symptoms revealed that among 66 confirmed patients,59%had no known suspicious exposure history(tick bites,recent activities in the grass,or farming).This suggests that in addition to the above transmission routes,SFTSV may also have one or more other transmission routes,such as aerosol transmission.In this study,SFTSV was used to infect human bronchial epithelial cells BEAS-2B,and the possibility of SFTSV transmission through aerosols was initially explored.The results showed that SFTSV can infect BEAS-2B cells,which provides a biological basis for SFTSV aerosol transmission;C57/BL6 mice were infected with SFTSV through aerosol route.According to the virus infection titer,three experimental groups were set up:high-dose group,middle-dose group and low-dose group,and a negative control group was also set.Different doses of virus aerosol exposure were carried out in the aerosol exposure device,and the negative control group replaced the virus suspension with normal saline.On the 3rd,5th,7th,10th,and 14th days after exposure,2 mice in each dose group were sacrificed.The antibody in the serum and the viral load and antigen distribution in the lung and spleen of the mice were detected,the possibility of aerosol transmission and the main target organs after infection were analyzed.This study will futher expand people’s understanding of the transmission routes of SFTSV,and will help to provide scientific basis protection and update treatment guidelines for the further improvement of SFTSV prevention and control measures.Purpose:To study the ability of SFTSV to infect C57/BL6 mice through aerosol route,suggesting the possibility of aerosol transmission of SFTSV in the population;analyze the target organs of virus replication and antibody production after infection,and provide scientific basis for the further improvement of SFTSV prevention and control measures.Methods:1.Clinical data and serum samples of suspected patients with SFTSV infection were collected according to the Diagnosis and Treatment Plan for Fever with Thrombocytopenia Syndrome.Nucleic acid extraction,nested PCR,agarose gel electrophoresis and gene sequencing were used to screen the SFTS from suspected cases in Jinan Infectious Diseases Hospital to confirm the confirmed cases.2.Determining the whole genome sequence of the existing virus strain JS2011-013-1 in our group after adaptive mutation。3.Inoculate SFTSV in a single layer of human bronchial epithelial cells(BEAS-2B),perform nucleic acid detection and use indirect immunofluorescence methods to test whether SFTSV can infect BEAS-2B cells4.Choose C57/BL6 mice as the research object,divide them into high-dose group,middle-dose group,low-dose group,negative group,12 mice in each group.The mice in each experimental group were exposed to different doses of aerosol,and the negative control group replaced the virus suspension with normal saline.On the 3rd,5th,7th,10th,14th,and 20th days after the exposure,2 mice in each group were sacrificed,and the eyeball blood,lung and spleen of the mice were taken.5.Detect the total antibody of SFTSV in mouse serum by ELISA.6.Use real-time fluorescent quantitative PCR to detect the viral load in the lungs and spleen of mice.7.Use immunohistochemistry to detect the distribution of SFTSV in mouse lung and spleen.Results:1.From June to December 2019,98 serum samples of suspected SFTS patients were collected at the Jinan Infectious Disease Hospital.After verification,there were 66 confirmed SFTS cases2.Indirect immunofluorescence shows that SFTSV can infect human bronchial epithelial cells(BEAS-2B).3.The total antibody to SFTSV in the serum of mice in different dose groups was detected by ELISA.The results showed that serum antibodies were positive on day 7 after aerosol exposure in the high-dose and medium-dose groups,and remained positive on day 10,14 and 20 after aerosol exposure.Two mice in the low-dose group were positive for antibodies at 10 and 20 days after infection,while the other mice were negative for antibodies.The antibody test results show that SFTSV can infect C57/BL6 mice through aerosol route,and the mice can produce antibodies after infection,and the existence of antibodies is related to the time and dose of infection4.The results of qRT-PCR showed that SFTSV RNA was present in the spleen and lung of mice.The viral load in the spleen and lung of mice in the high-dose group was higher than that in the medium-dose group,and the viral load in the medium-dose group was higher than that in the low-dose group.The maximum viral load in the lungs was higher than that in the spleen,and viral nucleic acid was detected earlier in the lungs than in the spleen.The results showed that SFTSV could infect C57/BL6 mice through the aerosol route,and the infection dose was positively correlated with the viral load in mice.Compared with other infection routes,the lungs of mice replaced the spleen as the main target organ of viral infection after SFTSV infection through the aerosol route.5.Use immunohistochemistry to detect the distribution of antigen in lung tissue and spleen tissue of experimental group and negative group.The results showed that there was SFTSV antigen distribution in lung tissues and spleen tissues of some mice in the high-dose group and the middle-dose group.Conclusion:Under experimental conditions,exposure to higher concentrations of SFTSV aerosol can infect C57/BL6 mice.The virus first infects and replicates in lung tissue,where viral nucleic acid is detected earlier than in the spleen and viral load is higher.C57/BL6 mice can produce antibodies after infection,and the production of antibodies in mice is related to the time and dose of infection.