Isolation,Identification And Gene Sequence Analysis Of Japanese Encephalitis Virus From Bats

Background and objectiveJapanese encephalitis is an acute infectious disease caused by Japanese Encephalitis Virus(JEV)through mosquito,which destroy human and animals'central nervous system.Encephalitis virus is one of the arthropod vectors virus epidemic in tropical and subtropical regions.It belongs to the genus Flavivirus,family Flaviviridae.The viral genome is a positive single-strand RNA of about 11kb that contains a single open reading frame(ORF)encoding three structural proteins(capsid protein(C),precursor membrane protein(prM),envelope protein(E))andseven nonstructural(NS)proteins(NS1,NS2a,NS2b,NS3,NS4a,NS4b,and NS5).Only a single serotype of JEV has been identified while five genotypes(GI-V)of the virus have been described on the basis of the nucleotide sequence of the envelope(E)gene.This disease is one of the most dangerous viral encephalitis with an estimated 35,000 to 50,000 cases and 10,000 to 15,000 deaths worldwide annually,mainly in South-East Asia and other Pacific coast areas.Japanese encephalitis is a severe zoonotic disease with high fatality ranging from 25%to 50%and high incidence of persistent neurological sequelae in nearly 50%of survivors,especially in young children.JE is epidemic in many countries of the world.Except for Qinghai,JE cases have been reported in almost all regions of China.The diagnosis of JE depend clinical diagnosis,however,JE clinical symptoms are similar to many diseases.So it can not only be diagnosed based on clinical manifestations.In addition,bats infected with Japanese encephalitis virus have no observable clinical manifestations.Laboratory diagnosis must be carried out.The laboratory diagnosis includes virus isolation and identification,etiology and serological tests.The virus isolation is reliable,which can be used as the gold standard.In this study,BHK-21 cells and suckling mice were used to isolate suspicious bat JEV strains.According to manual for the Laboratory Diagnosis of Japanese Encephalitis Virus Infection from the World Health Organization(2010),our study use indirect immunofluorescence(IFA),RT-PCR assay and nucleotide sequencing to conform and identify the virus.The animal reservoir hosts of JEV are mainly pigs and birds.Bats have been known to be an important animal reservoir host of infectious virus.Some studies have shown that Japanese encephalitis virus or Japanese encephalitis virus serum antibodies may exit in bats in China and Japan.Chinese scholars have reported that JEV were isolated from bats.However,the molecular biology information of the bats JEV strains is very limited.In recent years,scholars of Yunnan province in China have been isolated four JEV from bats(B58,GB30,HB49 and HB97)and the genome sequence has been identified.All four bat JEV isolates belonged to the G?.In this study,we isolated nine suspected positive JEV strains from four species named Myotis Ricketti,Scotophilus kuhli,Miniopterus schreibersi and Rhinolophus affinis in Guangdong,Hainan and Hunan China in our laboratory.Virus identification and analysis of the genome sequence were also finished.We compared and analyzed the gene sequence of JEV strains isolated by our laboratory and 23 JEV genome sequences,12 JEV sequences of E gene,6 JEV gene sequences of M gene and 1 JEV gene sequences of NS5 from GenBank selected representing various regions,host and time.Methods1.Isolation of bat JEV strainsThe filtrated bat brain tissues supernatants of the nine suspected positive JEV strains(GD1,HN1,HN2,HN3,YY1,YY2,YY3,SY87,YY158)were inoculated into BHK-21 monolayer.The observation lasted for seven days.The Nakayama strain was treated as a positive control and the normal cells as blank control.Supernatants with virus solution were obtained and frozen at-80 ?.Each group of five 2-3 days old NIH suckling mice was inoculated with nine filtrated bat brain tissues supernatants.The Nakayama strain was treated as positive control and negative control group were inoculated DEPC water.Weight-reducing,refused milk,slow movement,neck rigidity,convulsions,hemiparesis and lethargy were observed.Within 24h after inoculation,observation was carried out every 2h.After 24h,the observation was carried out three times a day lasted for 14 days.The suckling mice brain tissues were obtained under aseptic operation and preserved in RNA later at-80?.2.Detection of bat JEV strains2.1 Detection of specific nucleic acid with bat JEVRNA extraction,reverse transcription and PCR were done in BHK-21 cell suspension and suckling mice brain tissues,then suspicious positive amplicons by PCR were sequenced by the company.2.2 Determination of bat JEV titerThe third generation of viruses with stable virulence was used.Virus was diluted for continuous 10-fold dilution from 10-1 to 10-8.BHK-21 cells were inoculated with each dilution for 8 holes.Positive control and blank control were used.Recorded the numbers of lesions holes under the microscope.TCID50 was calculated using the Reed-Muench method.2.3 Indirect immunofluorescence assayAccording to the reference and the antibody test kit instructions,the antibody concentrations were detected.2.4 Determination of median lethal dose(LD50)with bat JEVThe nests of suckling mice as the unit were divided into 12 groups.Each group contained 5 to 6 suckling mice.Mice were inoculated with 0.02ml virus by transcranial.Inoculated with Nakayama strain was as a positive control group.Negative control group inoculated with 5%MEM culture medium.The other 10 groups were inoculated with the 10-0-10-4 GD1 and HN2 strains.Observed and recorded the mortality.Improved Cole's method was used to calculate the median lethal dose.The brain samples from dead mice in the 10-2 dilution group were decteced for the E gene of JEV.2.5 BiopsyThe 10-0 GD1 and 10-0 HN2 group,positive group and negative group of suckling mice's brains were preserved in 10%formalin immediately after they died.Biopsy were made and observed.3.Analysis of bat JEV sequence23 JEV genome sequences,12 JEV sequences of E gene,6 JEV gene sequences of M gene and 1 JEV gene sequences of NS5 were selected from GenBank representing various regions,host and time.Mutiple sequence alignments were performed by MEGA 4.0 and percentage identities between aligned nucleotide were carried out using MegAlign.DNAMAN were used to predict RNA secondary structure.Geneious 5.5.6 was used to show nucleotide and amino acid differences.MEGA 4.0 was used for phylogenetic analysis.Phylogenetic trees were constructed based on E gene,M gene and NS5 gene using the neighbor-joining model.The robustness of phylograms was evaluated by 1,000 bootstrap replicates.Nucleotide distances were estimated from bootstrapped datasets with the method of Maximum composite likelihood.The West Nile virus was used as an outgroup.4.Quality control1)All supplies used in experiment,such as pipette tip,EP tube,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 PCR reactions should be particularly careful to prevent cross-contamination.Results1 Isolation of bat JEV strains BHK-21 cells in control group grown in spindle-shape,however cells inoculated with Nakayama strain turned round gradually and appeared CPE at the third day.Some cells inoculated with suspicious JEV turned round,but the lesions did not as obvious as the positive control group.After 3 generations blind passages,all groups appeared CPE,but only GD1 and HN2 groups appeared the same CPE as the Nakayama group.Weight-reducing,refused milk,slow movement,neck rigidity,convulsions,hemiparesis and lethargy appeared in suckling mice of the positive control group within 2-5 days after inoculation.The suckling mice of experimental group appeared similar symptoms 1-7 days after inoculation,with the mortality rate of 0-60%.No symptoms were observed in negative control group.They were still healthy 14 days after inoculation.2 Detection of bat JEV strains2.1 Detection of specific nucleic acid with bat JEV Only GD1 the HN2 strain had amplicons by PCR in BHK-21 cells.No amplicons were detected in suckling mice inoculation with bat suspected positive JEV strains.2.2 Determination of bat JEV titerReed-Muench was used to calculate the virus titer.The virus titer of GD1 was 5.48 TCID50/ml and the virus titer of HN2 was 5.98TCID50/ml.2.3 Indirect immunofluorescence assayGD1,HN2 had green fluorescence as positive control.No fluorescence but very weak background light was seen in negative control.2.4 Determination of median lethal dose(LD50)with bat JEVImproved Cole's method was used to calculate the median lethal dose.LD50 of GD1 was 4.28TCID50(95%CI,4.03,4.53)/0.02ml.LD50 of HN2 was 4.51TCID50(95%CI,4.34,4.68)/0.02ml.Dectection of the E gene of JEV in the brain samples from dead mice in the 10-2 dilution group showed that all samples were positive by RT-PCR and confirmed by nucleotide sequencing.2.5 BiopsyThe biopsy showed hyperemia,hemorrhage and thrombosis in cerebral vasodilation,punctate necrosis in brain cells and inflammatory cells infiltration in GD1 and HN2 gruops.Lesions characteristic were also observed in the positive control group.The negative group did not observe any pathological phenomena.3 Analysis of bat JEV sequenceThe GD1 strain and the HN2 strain had a 99.9%identity for the complete nucleotide sequences.They had 99.4%to 99.6%nucleotide sequence identities to the other bat JEV strains(GB30,B58,HB49 and HB97)and 79.7%to 99.2%nucleotide sequence identities to other JEV genomes.They had a 99.9%identity of E gene sequences.Compared E gene sequences of the two strains to other four strains of bat JEV,they shared 99.2%to 99.3%nucleotide sequence identities.They had 78.0%to 99.3%E sequence identities to others.The GD1 strain and the HN2 strain had a 99.9%amino acid identity.They had 99.4%to 99.6%amino acid identities to the other bat JEV strains and 91.3%to 99.4%amino acid identities compared to other JEV full-length amino acid sequences.The GD1 strain and the HN2 strain had a 99.9%E protein amino acid identity.They had 98.6%to 99.0%amino acid identities of the E protein compared to other four bat JEV strains and shared 91.0%to 99.2%amino acid identities of E protein compared to other JEV strains.The comparison of four bat JEV strains(B58,GB30,HB49 and HB97),the SA 14-14-2 strain,Chinese mosquito P3 strain,Chinese pig strain WHE and the human Beijing-1 strain with GD1 and HN2 strain nucleotide and amino acid sequence showed that there were mutations and deletion,but most are silent mutations.Based on the E gene analysis revealed that the GD1,HN2,SY87 and YY158 strains were close and clustered in a phylogenetic branch,belonged to G?.Besides,they were phylogenetically closed to the mosquito strain(BN 19)isolated in 1982 and the human strain(Liyujie)isolated in 1979 in China.They had small distance to four bat JEV strains(B58,GB30,HB49 and HB97).Based on the M and NS1 gene analysis also revealed that GD1 and HN2 strains were closed to B58,GB30,HB49 and HB97 strains and clustered in a phylogenetic branch,belonged to G?.Conclusions1.The GD1 and HN2 strains were virulent for BHK-21 cells and suckling mice.2.The GD1,HN2,SY87 and YY158 strains and four other isolates of bat JEV(B58,GB30,HB49 and HB97)were clustered together in a phylogenetic tree branch,belonged to G?.3.The GD1,HN2,SY87 and YY158 strains were closed to the mosquito isolated strain BN 19 and Chinese human strain Liyujie.Although there were some different amino acids among them.4.The bats probably are reservoir host of Japanese encephalitis virus.They may play a potential role in the spread of Japanese encephalitis virus.