Study On Genetic Differentiation And Infection Of Zika Virus Among Aedes Aegypti And Aedes Albopictus Populations

Aedes aegypti and Aedes albopictus are major vectors for massive mosquito-borne viruses,including ZIKV,and threaten human health world-widely.The diversified climatic regions of our country facilitate the genetic differentiation and transmission capacity of these two vector mosquitoes;thus,it is essential to investigate the genetic variation and differentiation of Ae.aegypti and Ae.albopictus populations and their transmission capacity of Zika virus in China,which will provide scientific basis for monitoring and early warning of Zika virus epidemic.In present study,combined with mitochondrial COI,ND4 and ND5 genes,the newly isolated microsatellite loci were employed to evaluate the genetic variation and differentiation of 17 Ae.albopictus populations sampled from three different climatic regions across China and 32 Ae.aegypti populations sampled during 2017?2018 from Yunnan province from the perspectives of population genetic diversity,structure and diffusion.The endogenous virus fragments related to Zika virus infection were screened out from the genomes of Ae.albopictus and Ae.aegypti via small RNA sequencing technology.To determine the distribution and diversity of these endogenous virus fragments and whether the distribution of the fragments is related to the genetic differentiation of mosquito populations,multiple PCR amplifications were done in natural Ae.albopictus and Ae.aegypti populations.To explore the influences of geographical and species differences for mosquito transmission,the ZIKV transmission differences were investigated among different geographical strains of Ae.albopictus and Ae.aegypti population.To assess the influence of temperature changes on the transmission of Zika virus by mosquitoes,the Ae.albopictus and Ae.aegypti populations were infected and tested under different temperature zones in China.The main results of this research are as follows:1.The results of the population diversity and genetic differentiation of Ae.aegypti populations sampled from Yunnan during 2017?2018,showed that the average number of alleles of the Ae.aegypti populations from Xishuangbanna prefecture was higher than that of Dehong prefecture in both 2017 and 2018 populations.Based on COI and ND4genes,31 haplotypes were detected among 2017 Ae.aegypti populations and the Hd value of Xishuangbanna prefecture(0.694)was higher than that of Dehong prefecture(0.377)and Lincang city(0.046),meanwhile,nearly all Ho values were lower that of He values except population RLWD and MDXL,significantly deviating from the Harvin equilibrium and showing a significant population expansion trend(P<0.05).while only ten haplotypes were detected among 2018 Ae.aegypti populations and the Hd value of Xishuangbanna prefecture(0.404)was higher than that of Dehong prefecture(0.225)and Lincang city(0.150),meanwhile,nearly all Ho values were high that of He values except population JGYH and RLHP.The STRUCTURE test showed that the 2017populations were divided into three branches,and AMOVA test showed that the factors that caused population differentiation were mainly among populations within regions and within individuals among the same region,and the variation rates were 19.36%and74.18%,respectively;while the 2018 populations were divided into two branches,and AMOVA test showed that he factors that caused population differentiation mainly came from the populations of different regions and the individuals in the same region,and the variance rates were 57.42%and 36.19%,respectively.All the geographical distance was significantly correlated with genetic distance(P=0.001).Diffusion analysis showed that Ae.aegypti population from Mengla County in Xishuangbanna Prefecture and RLYB in Dehong Prefecture spread to Dehong and Xishuangbanna Prefectures respectively,which led to the genetic similarity of the populations in the two places in2017;while,among 2018 populations,the population JGTA in Dehong Prefecture and MDAJ in Lincang City spread to Xishuangbanna and Dehong Prefectures and other Ae.aegypti populations mainly spread within the regions,resulting in genetic differences between the two populations.2.Population diversity and genetic differentiation of Ae.albopictus populations among different climatic regions showed that the number of alleles in populations of different temperature regions was very high,and there was an upward trend from temperate zone(3.876)to tropical zone(4.144).Based on the COI and ND5 genes,a total of 25 haplotypes were detected and the haplotypes in the tropics were significantly higher than those in the temperate and subtropical regions;the Ho value of all populations(0.557)was significantly lower than the He value(0.684)with all populations deviated significantly from Harvin equilibrium and showed a significant population expansion trend(P<0.05).Based on the structure and haplotype cluster analysis,the populations were divided into two groups and 3 haplotype branches,and the tropical populations were completely separated from the other two regions.AMOVA test showed that the molecular variation mainly came from individuals within the populations and between individuals,accounting for 31.4%and 63.04%,respectively,and only the geographic distance and genetic distance for the tropical populations was significantly correlated(R~2=0.6614,P=0.048).Dispersal analysis showed that there were four main dispersal routes among Ae.albopictus populations of different climatic regions.3.The study on the endogenous virus fragments distribution and diversity analysis showed that the endogenous inserts can be used for the analysis of the population structure of Ae.aegypti and Ae.albopictus at population structure level and only endogenous Rhabdovirus fragments can be used for population differentiation studies of Ae.aegypti.The Aa Rha033 fragment of Ae.aegypti can be used to monitor the invasion and spread of this population,and the Ab Rha58 fragment of Ae.albopictus may be a potential molecular marker for detecting population structure of Ae.albopictus.4.The cell kinetical analysis of ZIKV showed that the propagation of ZIKV was similar in BHK-21,Vero,C6/36 and Aag2 cells.Compared with Aag2 and Vero cells,C6/36 and BHK-21 cells were more suitable for the propagation of ZIKV with a significant difference(P<0.001);meanwhile,compared with BHK-21,Vero and Aag2cells,the growth and CPE of C6/36 cell were accelerated with the raising of temperature,while it didn't influence the ZIKV propagation rate.Transcriptome analysis results showed that:on the second day of infection,expression level of 1358 genes were significantly changed(P<0.05),with 707 genes up-regulated and 651 genes down-regulated on the second day post infection;while 3640 genes were significantly changed(P<0.05)with 2218 genes up-regulated and 1422 genes down-regulated on the fourth day post infection and These genes mainly participated in cell catalysis,binding,cell metabolism and biological regulation functions.5.the life cycle of Ae.albopictus was significantly influenced by temperature(P<0.0001).The survival time of Ae.albopictus was 34.82±8.65 days under low temperature conditions(21?),29.13±7.57 days under standard temperature conditions(28?),and only 8.23±4.13 days under high temperature conditions(35°C).The egg-laying behavior of Ae.albopictus in different climatic regions was significantly influenced by temperature and ZIKV.Egg numbers laid by different geographic strains of Ae.albopictus changed with temperature,and the ZIKV-infection would promote the egg production in latter subtropical strain NJDX and tropical strain HKWN under low temperature conditions(21?).6.The ZIKV infection analysis of different Ae.albopictus strains showed that the infection rate of ZIKV in each tissue of the subtropical strain of Ae.albopictus NJDX was significantly higher than that of the tropical strain HKWN and the temperate strain BHBG,with the positive infection rate of each tissue greater than 60%on the second day post infection.Among subtropical strain NJDX and the tropical strain HKWN,the infection scenario lasted from day 2 to 14,while it lasted from day 3 to 10 for the temperate strain BHBG.The positive infection rate for the salivary glands of the tropical strain HKWN and the temperate strain BHBG was significantly enhanced via raising the temperature and the positive infection rate for the salivary glands of the temperate strain BHBG was zero under low temperature conditions(21?).Similarly,the positive infection rate for the midgut of the tropical strain HKWN increased with increasing temperature,while the positive infection rate for the midgut of the temperate strain BHBG decreased with the increase of temperature.In subtropical strain NJDX,the positive infection rate of the midgut increased with the increasing temperature.The ovarian positive infection rate of the subtropical strain NJDX and tropical strain HKWN showed a decreasing trend with increasing temperature,while the positive infection rate for the ovary of the temperate strain BHBG increased with increasing temperature.7.The affection of temperature on the transmission of ZIKV by Ae.aegypti showed that the replication and spread of ZIKV in various tissues of Ae.aegypti was promoted along with temperature rise during the early days post ZIKV infection(Day 3),while the ZIKV infection rate of Ae.aegypti was promoted via low temperature on Day14th post infection.the ZIKV transmission capacity of Ae.aegypti was higher than Ae.albopictus under all conditions.