Study On The Association Between Endogenous Virus Elements In Aedes Albopictus Genome And Zika Virus Infection

Research Background:ZIKV is an important arbovirus related to public health,which can cause serious neurological diseases such as Guillain-Barré syndrome,microcephaly in infants born from infected mothers and meningitis.The major vector of ZIKV is Aedes spp.From1947,the first time of discovery,to 2007,it was little known due to the few cases of human infections and limited prevalence.From 2007 to 2013,large-scale outbreak of ZIKV occurred in Yap Island,Cambodia,Thailand,Malaysia,French Polynesia and other regions and spread rapidly to many countries and regions.Up to now,the ZIKV epidemic has spread to more than 80 countries around the world,becoming a severe threat to global human health.There is currently no effective drug or vaccine for ZIKV infection,and vector control remains the primary way to prevent the spread of the virus.More and more studies have shown that the mosquito genome contains some viral elements,which are called endogenous viral elements(EVEs),or non-retroviral integrated RNA viral sequences(NIRVS)if derived from a non-retroviral genome.In recent years,studies have shown that NIRVS in mosquitoes can participate in the antiviral response by participating in the formation of Piwi interacting RNA(pi RNA).However,the specific mechanism has not been elucidated.So,exploring the interaction between the expression of NIRVS in Aedes albopictus and ZIKV infection will help to illustrate the biology function of NIRVS,know about immunity mechanisms against viruses in moquitoes,and provide theoretical basis for the development of vector-borne disease prevention and control strategies in the future.Research content:Ae.albopictus mosquitoes from Jinghong City(JH),Yunnan Province and Guangzhou City(GZ),Guangdong Province were collected,and mosquito infection was conducted with different dose of ZIKV,the infection rate and virus load were examined,and their vector competence for ZIKV was assessed.The total RNA of the midgut and salivary gland tissues after ZIKV infection was extracted,and the small RNA and m RNA sequencing and bioinformatics analysis were conducted to study the NIRVS carried by Ae.albopictus and transcriptional analysis,and to explore the interaction relationship between NIRVS of Ae.albopictus and ZIKV infection.Research Methods:1.Jinghong and Guangzhou strain of Ae.albopictus were orally infected with ZIKV.RNA was extracted from samples taken at different days,and real-time fluorescent quantitative PCR was used to detect the virus.The infection rate and virus copyies were calculated.2.Total RNA in the midgut and salivary glands of Ae.albopictus on the 10 days post infection(dpi)after ZIKV infection was extracted.small RNA and m RNA sequencing and bioinformatics analysis were then performed.3.The differentially expressed genes(CYP304a1)of Ae.albopictus after infection with ZIKV was knocked down by RNA interference technology.Subsequently,the JH strain and GZ strains of Ae.albopictus were infected with ZIKV and the viral loads were assessed on 1 and 3 dpi.Research Results:1.Under laboratory conditions,the JH and GZ strains of Ae.albopictus were orally infected with ZIKV and the midgut and salivary gland tissues were dissected on 4,7,10,and 14 dpi,respectively.The infection rates and viral RNA copies of the JH and GZ strains increased gradually from 4 to 14 days.When it was infected with a low dose of ZIKV,the infection rate of the midgut in the JH strain was significantly lower than that of GZ,and viral RNA was detected in the salivary gland until 10 dpi.When infected with a high dose of the virus,the viral RNA copies in the midgut of the JH strain were significantly lower than that of GZ,and viral RNA was detected in the sali-vary gland until 7 dpi.2.Small RNA sequencing results showed that after ZIKV infection,si RNA of 21 nt were mainly produced in the midgut and salivary gland tissues.The s RNA of Ae.albopictus sample was mapped to 72 NIRVS,the results showed that except for the us RNA(15-17 nt)in the salivary glands of the GZ strain after ZIKV infection,the small RNA expressed in NIRVS of other groups were mainly distributed in the pi RNA interval(26-30 nt)with "1U" feature,which were typical primary pi RNA.At the same time,the small RNA of Ae.albopictus infected with ZIKV was mapped to the NIRVS,the differentially expressed NIRVS such as Alb Flavi39,Alb Flavi40,Alb Flavi38 and Alb Rha74 were screened out,which were derived from ZIKV,DENV-1 and MSPV,respectively.Sequencing data from each sample were mapped to the genomes of three viruses,which were found mainly come from ZIKV.Subsequently,s RNA was compared with the genome of ZIKV.It was found that the small RNA in samples of ZIKV infection group was mainly enriched in 21–23 nt,with the peak at 21 nt.Moreover,s RNA had a wide coverage and uniform distribution in the ZIKV genome.Finally,we analyzed the mi RNA expression profile after ZIKV infection and screened out some differentially expressed mi RNAs that may affect the vector competence.3.Analysis of the m RNA from the JH and GZ strains of Ae.albopictus after ZIKV infection revealed that only Alb Rha33 and Alb Rha58 were differentially expressed.Subsequently,m RNA differentially expressed genes(DEGs)were analyzed,and the results showed that the DEGs were very different between two tissues and two mosquito strains.When comparing DEGs between tissues,the consensus DEGs account for only1.4% of the total in the two tissues of the JH strain and 3.6% in the GZ strain.When comparing DEGs between strains,there were only 5.8% consensus DEGs shared by two strains in the midgut and 2.7% in the salivary gland.In the GO and KEGG pathway analyses,DEGs screened in four libraries were very similar in composition but differed greatly in function.Finally,a total of 59 DEGs that may affect vector competence were screened-among which,cytochrome P450 304a1(CYP304a1)was the only gene significantly downregulated in both tissues of two strains.4.The gene function of CYP304a1 was verified.The results showed that the si RNA knockdown efficiency of CYP3304 a1 in the JH and GZ strains was 65.5% and53.5%,respectively.Three days after interference,mosquitoes were infected with ZIKV by intrathoracic injection,and viral RNA were detected 1 and 3 days after infection.The results showed that there was no significant difference between the CYP304a1 interference group and GFP group in this study,indicating that CYP304a1 had no effect on virus infection and replication under the conditions set in this study.Research conclusions:1.Different geographical strains of the same mosquito species differ in their susceptibility to ZIKV.The vector competence of GZ strain was higher than that of JH strain.2.After ZIKV infection,Ae.albopictus produced 21 nt si RNA in the midgut and salivary gland tissues,indicating that the si RNA pathway played an important role in the antiviral process of mosquitoes.3.NIRVS mainly transcribe primary pi RNA.Some differentially expressed NIRVS were screened out,which provide a theoretical basis for the relationship between NIRVS and pi RNA.4.The m RNA of Ae.albopictus infected with ZIKV was mapped to NIRVS,only two differentially expressed NIRVS were screened,indicating that the main transcript of NIRVS was not m RNA.5.The m RNA sequencing data were analyzed to find the DEGs that may affect mosquito vector competence and CYP304a1 was screened and silenced.However,CYP304a1 did not influence ZIKV infection and replication in Ae.albopictus under the conditions set in this study.