A Survey Of Japanese Encephalitis Virus In Bats In Several Areas Of Hunan, China

Background and objective:Epidemic encephalitis B which is also called Japanese encephalitis is an important arbovirus communicable disease caused by Japanese encephalitis virus （JEV）.Japanese encephalitis was reported firstly in Japan in 1871,and JEV was firstly isolated in1924.China diagnosed the first case of Japanese encephalitis by the serological and virus isoIated methods in 1940.Japanese encephalitis will be epidemic every 2 or 15 years in most countries.WHO estimated up to 50,000 Japanese encephalitis patients occurred annually with a mortality rate of about 25%,and 20 to 40 percents of the survivors had the sequela of neural paralysis and changes of psychology,so once it had been given the name of "oriental plague" because it caused a hard economic and psychological burden to the society and the family.In nature,Japanese encephalitis virus exists in the circulation transmited by the mosquitoes and the wild water bird.Swine have been documented as important amplifying hosts in epidemic areas.Japanese encephalitis is a mosquito-borne disease.30 species of mosquitoes were found as transmiting vectors of Japanese encephalitis virus all over the world,and 20 in China.Among them Culex tritaeniorhynchus Giles is the most sensitive,and JEV is transmissed through the Culex tritaeniorhynchus Giles among the wild birds,human beings and the domestic animals.Most arboviruses have more than one host,so it is the case with the Japanese encephalitis.Sheep,house swallows,monkeys,fair shrewmouse also the small white mouse can be infected.Besides.it has been isolated from the bats as reported.Chinese scholars have been reported from Yunnan,Guangxi,Guangdong, Hainan,Anhui,Guizhou bats to detect Japanese encephalitis virus antibody in serum.However,there is no separation of the Japanese encephalitis virus or Japanese encephalitis virus nucleic acid detected in the reports.To understand the situation of bats carrying human viral pathogens in different regions has the theoretical and practical significance in delving into the evolutionary relations of relevant virus in the human beings and animals and in scienctifically preventing and responding to the possible occurrence of diseases caught by both human beings and animals.So,we intend to investigate whether bats can carry Japanese encephalitis virus in part region of Hunan Province.Methods:1.Samples and handling.From 2007 to 2008,bats were collected from several areas of Hunan province. Brain tissues were obtained with aseptic technique after the bats were sorted and then grinded with normal sodium and Hanks buffer solution in high pressure-handled grinders.After being centrifuged,the brain tissue supernatants were conserved in -80℃for subsequent detection.2.RT-PCR.Three pairs of consensus primers were designed to anneal to JEV RNA was extracted from brain tissue by QIAamp Viral RNA Mini Kit.Secondly,RNA was converted to a DNA copy（cDNA） prior to enzymatic DNA amplification by use of SUPERSCRIPTⅢ1ST STRAND（RT） and the random primer.Subsequent Taq polymerase amplification was performed on the resulting cDNA with the upstream and downstream consensus primers.3.SYBR GreenⅠreal-time RT-PCR.①We detected the bats’ tissue by SYBR GreenⅠreal-time RT-PCR,our primers have been confirmed and recognized as specificity for the JEV which cited from reference.②We inoculated supernatant of positive or Suspicious positive samples into VERO cells to Separate viruses,then daily observation and three blind-generation.③We inoculated culture solution of cell or supernatant of bat brain into the encephalocoele of BALB/c suckling mice and observe the symptom,if there are Suspicious positive symptom we will continuet to blind passage three generation,if symptom emerging we will identity it with SYBR GreenⅠreal-time PCR.4.Cell culture.The brain tissue supernatants were 1:10 diluted in Hanks buffer solution containing 100U penicillin and 100U streptomycin sulfate per milliliter solution.Five hundred micro liters of each fresh brain tissue was inoculated into confluent Vero monolayer and gently rocked every 10min for 2h in cell culture box with 37℃、5%CO₂ settings. After being washed with medium without FCS,the Vero cell monolayer were added RPMI-1640 with 2%FCS for subsequent culture.Positive samples display pathological appearances,and will be detected in further cell culture and RT-PCR. Negative samples will be detected in RT-PCR,meanwhile be put in blind passage for three passages and be affirmed in RT-PCR detection method.5.Animal experimentsThe centrifugal supernatants from12 cases of positive samples were inoculated to neonatal rats brains each inoculation 0.03ml.Daily observation of 2 or 3 times, continuous observation of 1 week.Neonatal rats died within 48h after inoculations were judged to as non-specific death.Symptoms of neonatal rats:refusal to milk, convulsions,lateral,camponotus until death.After 1 week if no symptoms appear, biography for blind.If three generations of blind inoculation as yet been no symptoms, and non-amplified gene fragments by RT-PCR detection were judged as negative.6.Sequence and genetic analysis①Sequence RT-PCR products;②Compare sequences of the PCR products with known sequences of the JEV genes of coronaviruses in GeneBank using on line server BLAST;③Align viral sequences by using Clustal W;④Employ MEGA 4 program package to construct the phylogenetic trees by using the neighbor-joining（NJ） method with 1000 bootstrap replicates.Results:1.We collected bats 4 times in Hunan locations from 2007 to 2008.2 families,8 species,a total of 851 bats were sampled,in which 2 families were Rhinolophidae, and Vespertilionidae,and 8 species were 496 Rhinolophus affinses,7 Rhinolophus macrotises,12 Myotis rickettis,2 Rhinolophus blythis,293 Miniopterus schreibersis, 17 Rhinolophus ferrumequinums,20 Rhinolophus sinicus,2 Rhinolophus pearsoni.2.We found 2 cases carrying Jev from 344 bats brain tissue samples by use of RT-PCR.The carring rate of encephalitis B virus is 0.694%in YY Hunan and 0.5%in SY Hunan.3.10 cases carrying Jev were detected from 507 bats brain tissue samples by use of real-time PCR.The carring rate of JEV was 2.67%in YY Hunan and 1.68%in SY Hunan.4.To identify Vero cells after virus isolation and culture results,2 cases of J ev-positive samples collected in 2007 transfected Vero cells,Obviously happened CPE under the microscope.10 cases of jev-positive samples collected in 2008 transfected Vero cells,obviously happened CPE under the microscope too.5.12 positive specimens were identified,of which two cases of the positive samples from 2007 were inoculated suckling mice.and the symptoms of seizure,side, Camponotus are apparent in the fifth day;10 cases of the positive samples from 2008 were inoculated suckling mice,3 cases only slight symptoms and the other 7 cases no apparent symptoms.6.Phylogenetic trees constructed showed that the bat JEV origin from a branch of aggregation with NAKAYAMA,Nakayama-NIH and YNDL04-45.the bat JEV genotypes are typeⅢ.Conclusions:The study showed that bats in the Hunan regions carry JEV,but the carrying rate is not high.2 positive samples of JEV were detected from bats brain tissues of 2007 with the method of RT-PCR.The carrying rate was 0.69%in YY Hunan.The carrying rate was 0.5%in SY Hunan.These 2 samples could cause regular attack and death to baby rats once vaccinated.10 positive samples of JEV were detected from bats brain tissues of 2008 by use of Real-time PCR.The carrying rate was 2.67%in YY Hunan. The carrying rate was 1.68%in SY Hunan.The result display that the homology compared with known sequences of the genes of Japanese encephalitis virus ranges between 96%～100%after depuration and sequencing.