Epidemiology Of Lymphocytic Choriomeningitis Virus In Cattle And Sheep In Hulun Buir

Lymphocytic choriomeningitis virus(LCMV)is a single negative stranded RNA virus of genus Mammarenavirus(MARV),the family of Arenaviridae,Bunyavirales.LCMV infection in humans can lead to influenza-like illness or aseptic meningitis,and lead to serious systemic infection and death in immunocompromised patients.Prenatal infection of LCMV may cause abortion,stillbirth,malformations,congenital microencephaly and other central nervous system diseases.A neglected zoonotic disease caused by LCMV often poses a serious threat to mankind's physical health and has become an urgent public health problem.Isolation of LCMV from ticks was newly reported in northeastern China in 2018,and the infection rates of ticks ranged from 2.5%to 12.2%.Ticks are the second largest pathogen vector,transmitting various zoonotic diseases by biting and sucking blood.Vertical or horizontal transmission of LCMV may occur between ticks and mammals,but there is no evidence to support it up to now.Tick-borne diseases were prevalent in Hulun Buir region of Inner Mongolia.Epidemiological investigation of LCMV on cattle and sheep from this region was helpful to understand that the infection situation of LCMV in local mammals and the circulating transmission mode of LCMV among different hosts,providing scientific basis for the effective prevention and control of LCMV.In this study,prokaryotic expression of LCMV nucleocapsid(NP)protein was conducted on the basis of previous studies,indirect ELISA and fluorescence quantitative PCR methods were established for the detection of serological and molecular epidemiological of LCMV.Prokaryotic expression of LCMV NP protein.Specific primers were designed according to the published LCMV NP sequences from 4 ticks(MG554174,MG554175,MG554176,MG554177).The target gene was amplified from total RNA by RT-PCR,and the prokaryotic expression vector pET-30a-LCMV-NP was constructed and transformed into E.coli BL21(DE3)competent cells.The expression was induced by IPTG,and the recombinant proteins were purified by affinity chromatography and detected by SDS-PAGE and Western blotting.The results showed that the 1785 bp target gene was amplified by RT-PCR,and sequence comparison showed 100%identity with tick LCMV;The molecular weight of the recombinant protein was 56.4kDa,and the concentration was 0.626 mg/mL,showing a good immunogenicity.Seroepidemiological of LCMV in cattle and sheep.From May 2017,234 cattle serum samples and 221 sheep serum samples were collected in Hulun Buir.The recombinant protein LCMV-NP(0.2?g/mL)was used as the encapsulated antigen to detect LCMV-specific antibodies in cattle and sheep serum by indirect ELISA.The results showed that the positive rates of specific antibodies in cattle and sheep were 2.56%(6/234)and 4.52%(10/221),respectively.Molecular epidemiology of LCMV in cattle and sheep.In order to establish more sensitive LCMV nucleic acid detection methods,qPCR primers were designed according to the conserved region of LCMV NP gene in GenBank,and then the primers were screened and the reaction conditions were optimized.The results showed that the R~2 and amplification efficiency of the established RT-qPCR method were 0.997 and 96.498%,which has good sensitivity and specificity.The positive rates of LCMV RNA in the serum of the cattle and sheep were21%(49/234)and 19.9%(44/221),respectively,by RT-qPCR method established in this experiment.The positive rate was significantly higher than that of the specific antibody(P<0.001).Past infection was noted in serological results and ongoing infection was noted in molecular biological results of LCMV in cattle and sheep,suggests that LCMV may lead to persistent infection in cattle and sheep.It was found that the highest similarity of LCMV NP gene sequence between cattle and ticks is 98%.Phylogenetic analysis showed that LCMV of cattle belongs to severe disease related LCMV(type?),and LCMV of cattle and ticks were in the same evolutionary branch,suggesting that LCMV in cattle may be derived from ticks.In this study,LCMV NP recombinant protein was successfully expressed,the prevalence of LCMV in cattle and sheep in Hulun Buir of Inner Mongolia was investigated by established indirect ELISA and RT-qPCR.Provided an evidence of LCMV infection in cattle and sheep,and it provided theoretical basis for transmission of different hosts and study of genetic evolution LCMV.It also laid a foundation for the effective prevention and control of LCMV.