Epidemiological Survey Of Japanese Encephalitis Virus And Serum Antibody Prevalences In Bats In Guangdong And Hainan Province

Background and objectiveJapanese encephalitis(JE)is a severe zoonotic disease caused by Japanese Encephalitis Virus(JEV)through mosquito,which destroys the central nervous system of human and animals.The fatality rate ranges from 25%to 50%in humans.Nearly 50%of survivors suffer from persistent neurological sequelae.Thus,JE has become an important public health problem which has received global attention.JEV belongs to the genus Flavivirus of the family Flaviviridae.JEV is an enveloped virus with a positive single-strand RNA genome approximately 11 kb in length.It contains a single open reading frame(ORF)flanked by 5’and 3’nontranslated regions(NTRs),encoding three structural proteins[capsid(C)protein,precursor membrane(prM)protein,envelope(E)protein]and seven nonstructural(NS)proteins(NS1,NS2a,NS2b,NS3,NS4a,NS4b,and NS5).Five genotypes(GI-V)of JEV have been identified on the basis of the nucleotide sequence of the E gene.About 50%of the survivors have severe neurological sequelae in the form of behavioral abnormality,cognitive impairment,seizures,a variety of movement disorders and so on.JE mainly occurs in China,Indian and Southeast Asia.An estimated 67900 cases occur globally each year(overall incidence:1.8/100000 people).Approximately half(33900)of these cases occur in China(excluding Taiwan)and approximately three-quarter(51000)occur in children aged 0～14 years with the incidence of 5.4/100000.Except for Qinghai,JE cases have been reported in almost all regions of China(excluding Qinghai).The average incidence in the highly endemic areas is considered to be approximately>0.5/100000.The highly endemic areas include Sichuan Province,Henan Province,Guizhou Province,Chongqing City and Yunnan Province.Annually,the number of morbidities in these five areas accounts for 50%of the total cases nationwide.The combined population of these areas,however,represents only 26%of the national population.The average incidence in moderately endemic areas is considered to be between 0.2/100000 and 0.5/100000.The areas include Shanxi Province,Anhui Province,Hubei Province,Hunan Province,Jiangxi Province and Guangxi Province.The other areas(except Qinghai Province)are slightly endemic areas.The number of JE cases rapidly increases after June.The highest morbidity occurs in July or August.The high morbidity is maintained in September and then significantly decreases in October.The morbidity in August accounts for 41.14%of the total annual morbidity.The number of JE cases in southern and northern China differs significantly.Normally,the morbidity in northern China increases after August and significantly decreases in the middle of September,whereas in southern China the morbidity increases after July and significantly decreases at the end of August.JEV is transmitted through a zoonotic cycle among mosquitoes,pigs and birds.Bat has been known as an important animal reservoir host of a large number of infectious viruses,more than 80 kinds of viruses have been detected or isolated from bats,such as SARS-coronavirus-like viruses of bats,Hendra virus,Ebola virus and JEV.Some studies have shown that JEV or JEV serum antibodies may exit in bats in China and Japan.In recent years,the nucleotide sequences of four JEV isolates(B58,GB30,HB49 and HN97)derived from bats in Yunnan Province were determined.We isolated four JEV isolates(GDI,HN2,YY87 and YY158)from bats captured in Guangdong,Hainan and Hunan Provinces.Our study showed that eight bat JEV isolates belonged to GⅢ of JEV and shared a high genetic identity.But the role bats play in the cycle between human and animals is still unclear.To further investigate the role bats play in the JEV transmission,we investigated the prevalence of JEV from bats in Guangdong and Hainan Provinces of southern China.Methods1.Collection of bat samplesBats were captured and sampled from habitats in Guangdong and Hainan Province,during June to November 2013.Bat species were identified by Professor Wu Yi(bat expert,academy for life science,Guangzhou University)and Professor Chen Zhong(academy for life science,Hainan Normal University).After ether anesthesia,bat blood was collected by hemospasia from heart.By sterile dissection,brain tissue samples of bats were preserved in the tubes with the presence of RNAlater.Then the blood and bat brain tissues were transported back to the laboratory by foam boxes with ice packs in 12 hours.The samples were stored at-80℃.2.Serum antibody of batsWe collected serum specimens from bats.ELISA was conducted to detect bat serum JEV IgG.3.Detection of JEV in bat brain tissues by using TaqMan Real-time PCRRNA extraction,reverse transcription and TaqMan Real-time PCR were done in bats brain tissues.4.Cell cultureThe supernatants of suspicious brain tissue and postitive control were inoculated into BHK-21 cell.The inoculated BHK-21 cells were slightly shaked every 15 min for 1 hour in cell culture box with 37℃,5%CO2 settings.Morphological changes were observed.We determined the results according to the change.5.Suckling mice inoculated with virusWe inoculated supernatant of suspicious positive samples into 3 days of encephalocoele of NIH sucking mice,and then observed every day.If there were no suspicious positive symptoms,then we would continue to blind passage 3 generations.If there were still no symptoms observed,it was diagnosed as negative.6.Detection of specific nucleic acid with bat JEV RNA extraction,reverse transcription and TaqMan Real-time PCR were done in BHK-21 cell suspension and suckling mice brain tissues,then the company sequenced suspicious positive amplicons by PCR.7.Quality control(1)All supplies used in experiment,such as EP tubes,pipette tips,frozen pipes and gloves were disposable.(2)During RNA extraction and reverse transcription,pipette tip,EP tube and grinding devices were treated by DEPC in order to prevent contamination of RNase.(3)All the experiments were processed on the biosafety cabinet in a biological clean room to prevent from pollution.(4)Positive,negative and blank controls were included in the process of experiments.(5)Cell culture was carried out in the Biosafety Class Ⅱ laboratory to prevent pollution.(6)Preparation for PCR reactions should be particularly careful to prevent cross-contamination.Results1.General information of batsIn this study,343 bats from 6 species were collected from natural habitats in some regions of Guangdong and Hainan Province.These bats belong to 3 families including Vespertilionidae,Rhinolophidae and Pteropodidae.2.Detection of JEV serum antibody using ELISA93 out of 201 bat serum samples were ELISA-positive.The total positive rate was 30.61%(45/147)for bats from Guangdong Province,the positive rate was 31.06%(41/132),30.77%(4/13),0%and 0%for Pipistrellus abramus,Rousettus leschenaultia,Rhinolophus blythi and Rhinolophus macrotis,respectively.For bats in Hainan Province,the total positive rate was 88.89%(48/54),the positive rate was 95.56%(43/45)for Miniopterus schreibers,and 4 out of 8 Rousettus leschenaultias were diagnosed as ELISA-positive.3.Detection of JEV in bats using TaqMan Real-time PCRFive bat brain tissue specimens were positive for JEV by TaqMan Real-time PCR.Four from Guangdong Province(positive rate:1.62%(4/247))and one of five was collected from Hainan Province(positive rate:1.06%(1/96)).JEV was detected from two species of bats,namely Pipistrellus abramus and Miniopterus schreibersi.4.The result of cell culture and suckling mice inoculatedCells inoculated by the supernatant of JEV positive(TaqMan Real-time PCR)bat brain tissue showed slight cytopathic effect(CPE).However,no viral suspension of the cells inoculated by JEV positive bat brain tissue was posotive by TaqMan Real-time PCR.5.Three out of five bats whose brain tissue specimens were positive for JEV(TaqMan Real-time PCR)were found serum antibody positive for JEV.Conclusions1.We used TaqMan Real-time PCR to investigate the JEV infections in bats in Guangdong and Hainan Province.JEV was detected from two species of bats,namely Pipistrellus abramus and Miniopterus schreibersi,which indicates that under some circumstance,JEV exists in some spicies of bats,but the viral load is low.Further studies are still needed to investigate the potential role of bats in the spread of JEV.2.The high JEV serum antibody positive-rates in the Pipistrellus abramus,Miniopterus schreibersi and Rousettus leschenaultia indicate that with high sensitivity of JEV,these species of bats are involved in the JEV transmission cycle in the nature.