Serological Of Filovirus Infection In Bats

The Filoviridae family in the Mononegavirales has three genera,consisting of Ebolavirus(EBOV),Marburgvirus(MARV),and Cuevavirus.EBOV and MARV are mainly distributed in Africa,Cuevavirus is distributed in Europe.Filovirus can cause severe hemorrhagic fever diseases that are highly contagious and lethal to humans,so some members of filovirus were as potential biological weapon agents in the word,such as Zaire ebolavirus(ZEBOV)and MARV.Except for Reston ebolavirus(REBOV)that its natural host is considered to be pig,the rest members of filoviruses are bats.Our laboratory found a novel filovirus in Asia first time in 2015,indicating that the bat in Asia also infect the filovirus,but its species,infection rate and the significance of public health is not clear,and there is a lot of bat species in China,so it is of great scientific significance to establish a rapid,efficient and accurate method for serological surveillance of filovirus infection in bats.In this study,we established an indirect ELISA method for REBOV,ZEBOV,MARV,Lloviu cuevavirus(LLOV)and the novel bat-borne filovirus DH04 based on expression of their C-terminal sequences of nucleoproteins,respectively.Firstly,five recombinant plasmids were constructed,then transformed into E.coli BL21 and induced.The recombinant proteins were soluble which was further confirmed by Western blot using Anti-His monoclonal antibody,indicating that the protein was expressed correctly.The indirect ELISA methods were established by using purified protein as coating antigen and an optimized coating concentration of antigen(2 ?g/ml),blocking solution(5% skim milk powder),secondary antibody dilution(1: 20000).The specific tests showed that the new ELISA method cannot cross-react with the bat sera against group A bat rotavirus,three bat and human Bunia viruses,Laibin virus,Seoul virus,Xuan son virus,indicating a high performance of specificity.This method can detect a positive serum with a dilution of 1:1600,and the coefficient of variation of intra/inter-batch were all less than 10%,meaning an excellent sensitivity and repeatability.We applied ELISA methods to screen bat sera collected from Yunnan,Guangxi,and Zhejiang,and 45.2%(141/312)of them were positive,the positive rate of Yunnan was significantly higher than that of the other two provinces(P <0.05),and no significant difference between Zhejiang and Guangxi.65.7%(94/143)of bat sera were positive,46.9% were reacted with DH04,36.4% with REBOV,29.4% with ZEBOV and 21.0% with MARV,in Yunnan.26.2%(32/122)of bat sera were positive,16.4% were reacted with REBOV,10.7% with DH04,9.8% with ZEBOV and 4.1% with MARV,in Guangxi.31.9%(15/47)of bat sera were positive,27.7% were reacted with DH04,6.4% with ZEBOV,4.3% with MARV and 2.1% with REBOV,in Zhejiang.Seventy five bat sera were selected for Western blot according to the ELISA result,fifty nine were positive,nineteen were negative,the results showed that the WB total consistancy with ELISA of positive bat sera was between 80% and 100%,and negative sera was between 78% and 94%.Western bloting using monoclonal antibodies showed that DH04 cannot cross-react with the other three viruses,while MARV can cross-react weakly with REBOV,ZEBOV,and a strong cross reaction observed between REBOV and ZEBOV.In this study four indirect ELISA methods based recombinant proteins were successfully established to detect nucleoprotein antibody,and applied to detect bat sera in south of China,the results showed that there is a wide range of filovirus infection in bats,China,and there were several situations of filovirus mixed infections in bats,while different viruses have different characteristics in different bat species and regions: DH04 is the the most popular virus in Yunnan and Zhejiang,instead of REBOV in Guangxi;the positive rate of Rousettus leschenaultii was significantly higher than that of the other four species,DH04 is the most of in the active sera in Rhinolophus ferrumequinum,instead of REBOV in Scotophilus kuhlii,but did not detect positive sera in Miniopterus australis.A potential risk of filovirus infection is posed to these local livestock,and a systematic surveillance in these areas should be conducted in the future.