| Japanese Encephalitis is an important mosquito borne infectious disease which is mainly mediated by Culex tritaeniorhynchus.The pathogen of this disease is Japanese Encephatlitis virus.Most of the affected are children and adolescents.The fatality rate of this dangerous disease can be more than 10% and among the survivors,more than half of which will suffer from neuron system defects.Otherwise,JE virus is likely to cause reproductive failure or defects in infected female pigs,which can cause huge loss to aquaculture.To illustrate the characteristics of Cx.tritaeniorhynchus and the epidemic characteristics of JE virus infection,in order to prevent and control JE scientifically and effectively,this research is conducted mainly from three aspects,the ecology,molecular biology and population genetics.1.The research of population distribution of Cx.tritaeniorhynchus.The city of Qidong and Taicang were chosen as sites of survey to investigate populations and seasonal changes of Cx.tritaeniorhynchus using chickens and pigs as bait.At the same time,the population distribution in ecological environment such as pigsty of rural areas,the residence and grassland was also investigated.By collecting mosquito monitoring data from 2009 to 2015 in vector biological monitoring network of Jiangsu province,we analyzed distribution and dynamic change of Cx.tritaeniorhynchus in different habitats within the city areas such as urban parks,residential areas and hospitals.The results showed that Cx.tritaeniorhynchus mostly feed on livestock such as pigs and can be found mainly in pigsties or bullpens,secondly in rural residential house.It starts to appear in May each year and then density increased to the peak in July and August.Density starts to decline and until October,it nearly can't be monitored.The number of Cx.tritaeniorhynchus allured by chickens is low and the population mainly appear from June to August in Jiangsu.The results also showed that Cx.tritaeniorhynchus starts to appear within the territory of urban cities in Jiangsu province at least from 2009.It mainly distributed in park and has a lesser population in residential areas and hospitals,the peak usually appears in August and maybe it's because of the living pressure caused by the change of rural ecology environment and economic model.2.Analysis of characteristics of mitochondrial DNA and micro satellites of Cx.tritaeniorhynchus.After picking up Cx.tritaeniorhynchus samples,extracting genomic DNA and mitochondrial sequencing,we downloaded mitochondria and CO? gene sequences of 5 genera and 33 species of mosquitoes from Gen Bank,to form a data with the corresponding Cx.tritaeniorhynchus sequences.All of the above were aimed to explore the possibility and advantages of using the length of mitochondrial DNA in the identification of Cx.tritaeniorhynchus classification.This study is the first time to finish mitochondrial sequencing of Cx.tritaeniorhynchus.The study of the identification and classification of Cx.tritaeniorhynchus on molecular level was carried out through the construction of evolutionary tree,and we find that the length of mitochondrial genome can be used for taxonomic identification of Cx.tritaeniorhynchus.Due to the longer sequence and more abundant biological information compared with COI gene,the length of mitochondrial genome can be more meticulous to distinguish between mosquito species.Pick up 5 Cx.tritaeniorhynchus each from 16 different geographic groups collected and extract DNA from each mosquito separately,later design primers to amplify mitochondrial ND4 and ND5 genes.The analysis result showed that there are more percentage of polymorphic sites and more number of haplotypes in ND5 than in ND4.ND5 gene is more suitable for population phylogenetic analysis as molecular marker.The ND4 and ND5 haplotypes of Cx.tritaeniorhynchus are not related to geography location.And the diversity between populations is small.Pick up 30 Cx.tritaeniorhynchus each from 20 different geographic groups collected and extract DNA from each mosquito separately,later design primers and select 10 microsatellite loci to analyze population genetics.The result showed that 30 samples in one site is enough for our study.Ho of 20 geographic groups is small than He,Fst of populations is almost above 0.1,Fst of 10 SSR loci among populations is 0.141 which is bigger than Fis(0.006).Average gene flow(Nm)of 10 SSR loci is 10.4703.64.7% of pairwise fixation index(Fst)among populations didn't show obviously diversity.These index results showed that inbreeding among populations is obvious,and the gene flow is frequent.3.Analysis of characteristics of JE viral genome in Cx.tritaeniorhynchus and the host.From 2015 to 2016,we collected Cx.tritaeniorhynchus using light trap in pigsties from 9 places in 4 cities of Jiangsu province,among which 2 collections were conducted in 2015 and 2016 at the same time and location in Xiaogaozhuang and Xintancun in Lianyungang,the rest of the sites were collected in 2016.50 mosquitos are chosen randomly as a group to extract RNA,design primer and amplify je virus nucleic acid using RT-PCR.The results showed that JE virus has a higher infection percentage in Cx.tritaeniorhynchus.In this study,the ratio of positive sample is basically more than 10%,nearly half of which is above 50%,the highest is Zhangjiayuancun in Taizhou which reached 80%.Nearly half of the minimum infection rate(IR)is above10%,the highest of which is 16.00‰.The maximum likelihood estimation(MLE)is larger than the corresponding minimum rate value,the more serious the infection status is,the bigger error between these two data will be.In 2014,we collected 13,12 and 2 serum samples of Ixobrychus minutus,little egrets and bacchius,after nucleic acid extraction we amplified these with PCR and no je virus was detected.From March to October in 2014,serum samples of young pigs and sows were collected every month to detect JE Ig G antibody with ELISA and Ig M with latex agglutination method.The results showed that Ig M and Ig G antibody of JE virus in pigs start to rise since June,the peak appeared in July and August,and start to decline from September.No positive findings of JE virus were discovered during the nucleic acid extraction from the antibody positive serum.From April to September in 2014,we collected pig brains every month from slaughterhouses to detect je virus by extracting nucleic acid,the results showed that no positive findings were detected in 228 pig brains.One or two samples were selected from JE virus positive samples from every geographic population of Cx.tritaeniorhynchus to sequence E-genes.From 2015 to 2016,a total of 17 strains of JE virus E-gene were obtained.Among which 4 strains were obtained from Cx.tritaeniorhynchus collected from 3 locations in Lianyungang in 2015,12 strains were obtained from Suqian,Nanjing,Taizhou and Lianyungang in 2015,1 strain were obtained from anopheles sinensis collected from group 14,Zhangjiayuan of Lianyungang in 2015.According to the evolutionary tree constructed on E-gene sequences,the 17 strains of JE virus belong to GI-b type.Compared with attenuated vaccine strain SA14-14-2,the key antigen sites E332?E305?E335 of the 17 strains are the same.Virulence key sites of E138 and E176 are the same between the 17 strains,but there are differences compared to SA14-14-2.The results showed that antigenicity between the 17 strains and SA14-14-2 is quite the same,but the virulence might be stronger than SA14-14-2,which indicated that the JE virus vaccine based on SA14-14-2 is still effective in Jiangsu but the monitoring of JE virus need to be further strengthened.In this study,we detected that in the same place in 2015 and 2016,the JE virus Egene and amino acid sequence are exactly the same.The reason of the same JE virus detected in Nanjing,Suqian and Taizhou might be the waterfowl migration or the Cx.tritaeniorhynchus migration. |
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