Molecularepidemiology And Biological Characteristics Of Severe Fever With Thrombocytopenia Syndrome Virus In Taian

ObjectiveSince 2006,an outbreak of an unknown infectious disease occurred in rural areas of Henan,Anhui and Shandong provinces,China.Major clinical presentations of the patients included fever,thrombocytopenia,leukocytopenia and gastrointestinal symptoms.Such patients may present nausea,muscle aches and regional lymphadenopathy.Few severe patients would progress to multiple organ failure or even death.A novel viral pathogen was isolated from these patients and was named Severe Fever with Thrombocytopenia Syndrome virus?SFTSV?in 2010.SFTSV,also known as the new bunyavirus,belongs to the phlebovirus genus,the family of Bunyaviridae.Since then,SFTSV infection has been identified in South Kroea,Japan and at least 16 provinces from China.Shandong was the major epidemic area of SFTSV,such as Yantai and Yiyuan.SFTSV has also been detected in Taian in 2015.However,no epidemiology,genetic characteristics,evolutionary pattern and pathogenic features of SFTSV isolated from Taian have been reported.In the present study,we detected SFTSV from suspicious patients in Taian,Shandong.We then carried out an epidemiological analysis according to the confirmed SFTS patients and obtained complete genome sequences of SFTSV.The genetic variation and molecular biological features,gene recombination and reassortment of SFTSV were further analyzed.In addition,we studied the virulence of the virus and the inhibition effect of SFTSV of immune serum.Our study would provide atheoretical basis for the prevention and control of SFTSV in Taian.Methods1.Isolation,identification and epidemiological analysis of SFTSV in TaianBlood samples of the suspected SFTS patients were collected during 2015 and 2016from three sentinel hospitals in Taian..We detected the nucleic acid molecules of SFTSV to identity the SFTS positive patients and isolated the viruses from these patients.Both the Sanger and the next generation sequencing technologies were used to obtain the complete genome of the viruses.2.Bioinformatics analysis of SFTSV in TaianSFTSV reference genome sequences were obtained from Genbank.Multiple sequence alignment was performed using Muscle and sequences whose lengths were10%shorter than the gene were removed.RAxML was used to construct the phylogenetic tree using the L,M,S gene sequences,respectively.We then analyzed the potential SFTSV reassortants and recombinants.3.Virulence and antigenicity of SFTSV isolated from TaianThe virus'TCID₅₀0 was measured using Vero cells and the virulence of SFTSV was determined.The 3-4 weeks old SPF C57 mice were immunized with the epidemic strains isolated in our laboratory to obtain the immune serum.Vero cells were cultured and inoculated with the same amount of C3 and C4 strains,as well as with different concentrations of the anti-serum.The virus proliferation activity was determined to compare the cross reactivity between different subtypes of SFTSV with immune serum and to study the antigenic characteristics of different subtypes of SFTSV in Taian.Results1.29 blood samples of suspected SFTS patients were collected from three sentinel hospitals in Taian,and 15 cases?51.7%?were SFTSV nucleic acid positive.Three specific primers targeting the L,M and S fragment of SFTSV,were used.The M and S?93.3%?detection rate was higher than that of L fragment?73.3%?.Major clinical symptoms of the fifteen patients included fever?100%?and gastrointestinal symptoms?53.3%?.All cases had thrombocytopenia and most cases?86.7%?presented leukopenia,liver and kidney dysfunction?86.7%?through laboratory examinations.Moreover,all the 15 cases were identified in spring and summer,and most of them?73.3%?lived in rural areas with the ages between sixty and eigthy,Female patients accounted for 60%,and at least two patients had a clear history of tick bite.2.A total of 12 full-length genomes sequencesof SFTSV isolated from Taian were obtained in the present study.In addition,223 L,222 M and 432 S gene sequences were downloaded from GenBank,including 182 whole genome sequences of SFTSV.Our phylogenetic analyses showed that 11 SFTSV strains from Taian during the period of2015²016 belonged to the C3 subtyp,and one belonged to the C4 subtype.Apart from the previously reported C1-C5 and J1-J3 subtypes,two new subtypes,C6 and J4,were found in the phylogenetic trees of the L and S sequences.Further more,26 reassortant strains were found and they belonged to 12 different reassortment types,with 16 strains reported for the first time.In addition,there were 20 recombination events in 14 sequences,and multiple recombination events were identified to occure in a single SFTSV genome.Remarkably,recombination event was found in the S gene segnment for the first time.However,no gene reassortants and recombinants were found in the Taian strains.3.As a representative strain of the C3 subtype SFTSV,MYD,the virus virulence(1×10^7.5 TCID₅₀/0.1ml)was slightly higher than the C4 subtype strain ZCY(1×10⁵TCID₅₀/0.1ml).The antibody positive sera made by MYD,had inhibitory effect on both of the subtypes,and the cross reactivity was not significant.Conclusion1.Most of the suspected cases of SFTS were caused by SFTSV in Taian.The SFTSV peaks were found in spring and summer,and most of the patients lived in rural areas full of mountainous.2.Most SFTSV isolated in Taian belonged to the C3 subtype and only one belonged to the C4 subtype.In particular,we indentified two novel potential subtypes:C6 and J4.3.No gene reassortment and recombination events were found in the Taian strains.However,26 strains from other regions were found to be reassortants,with 16 were discovered for the first time.Multiple recombination events have also identified and some of them occurred in the S gene fragment.4.There was no significant difference in the inhibitory effect of immune serum the SFTSV between the C4 and C3 subtypes.This indicated there was no antigenic change between subtypesC3 and C4 of SFTSV isolated from Taian.