Isolation And Identification Of SFTSV And Its Vertical Transmission In C57 Mice

Fever with thrombocytopenia syndrome(SFTS)is a new viral infectious disease caused by fever with thrombocytopenia syndrome virus(SFTSV).It is the peak in summer and autumn.Most of the patients are middle-aged and old people,mainly transmitted by tick bites,and also transmitted from person to person.The main symptoms of SFTS patients are fever,nausea,asthenia,vomiting and other gastrointestinal symptoms.In laboratory tests,the levels of thrombocytopenia,leucopenia,aspartate transaminase and lactate dehydrogenase are increased.In severe cases,multiple organ failure may occur,even leading to death.Previous studies have observed that SFTSV may cause abortion,but it is not clear whether SFTSV can lead to fetal abortion through placental barrier.In this study,SFTSV was successfully isolated from the patient's serum.C57BL/6 pregnant mice were selected as experimental subjects.Different doses of SFTSV were injected intramuscularly at different stages of pregnancy,and some pregnant mice were sacrificed in the later-gestation.Various organs were collected and the remaining pregnant mice gave birth normally.By detecting SFTSV nucleic acids and antigens in the organs of the placenta and fetuses,we explored whether SFTSV can pass through the placental barrier;we investigated whether SFTSV can cause fetal damage by counting the development of fetuses.This study is helpful for people to fully understand the spreading methods and harms of SFTSV,and to provide a basis for accurate prevention and control of SFTS.Purpose:1.Isolate SFTSV from the patient's serum and investigate whether SFTSV can betransmitted vertically to the fetus through the placental barrier.2.To investigate whether SFTSV can cause fetal damage through the placental barrier and provide a basis for accurate prevention of SFTSV.Methods:1.SFTSV nucleic acid-positive patients were screened by nucleic acid extraction and nested PCR technology.Serum of SFTSV nucleic acid-positive patients was infected with monolayer Vero cells,and SFTSV virus was isolated by blind passage for 3 generations.2.SFTSV was successfully isolated using PCR,indirect immunofluorescence assay(IFA),and transmission electron microscopy.The isolated SFTSV was harvested using Vero cells and the virus titer was determined by the TCID50 method.3.C57/BL6 pregnant mice were selected as experimental subjects,and SFTSV was infected by intramuscular injection in the early-gestation and later-gestation.Pregnant mice were sacrificed on the embryonic day 17(E17)and one day after delivery,and a variety of tissue samples such as organs,placenta,and fetus were collected.4.ELISA and quantitative real-time PCR(qRT-PCR)were used to detect the serum of pregnant mice to confirm whether the pregnant mice were successfully infected with SFTSV.To observe the development of the fetus and collect the length data of intact fetuses,and to explore through statistical analysis effects of SFTSV on fetus development.5.Immunohistochemistry(IHC)method was used to explore the distribution characteristics of SFTSV in the spleen,placenta and fetus of pregnant mice;IFA method was used to investigate the presence of infectious SFTSV;Quantitative detection of SFTSV in spleen,serum,placenta and fetus viscera by qRT-PCR.Results1.Four of the six patients' sera were positive for SFTSV nucleic acid.The four sera were infected with monolayer Vero cells,and SFTSV RNA was detected in only one cell supernatant after three passages of the cells.2.IFA and transmission electron microscope observations confirmed that a new SFTSV strain was successfully isolated;the SFTSV sequence was amplified by PCR,and the whole genome sequence of the strain was successfully obtained by sequence splicing;the TCID50 of the SFTSV strain was measured by the 50%endpoint method to be 106.25/100?L.3.ELISA and qRT-PCR results showed that pregnant rats in the experimental group were infected with SFTSV successfully.Some experimental groups showed stillbirth,fetus with restricted growth,and abnormal placenta.Pregnant mice were infected with low-dose SFTSV(103TCID50)during the early-gestation(E4)fetuses showed growth restriction,and the difference was statistically significant(P<0.05).4.IHC results showed that SFTSV was mainly distributed in the red pulp of the spleen,the labyrinth of the placenta and the organs such as liver,thymus,hindbrain,and heart of the fetus;IFA results showed that infectious SFTSV was present in the fetus of the experimental group,further confirming that SFTSV can through the placental barrier was transmitted to fetus;qRT-PCR results showed that the viral load was higher in the spleen of pregnant mice,and the viral load in serum,placenta and fetus was lower.Conclusions:1.The SFTSV strain was successfully isolated.It was first confirmed that SFTSV could be transmitted vertically to offspring through placenta barrier in pregnant mice.2.The study confirmed that SFTSV could cause fetal damage through placental barrier,resulting in fetal death,fetal growth restriction and other adverse pregnancy outcomes.