The Comparative Transcriptome Analysis Of RDV-infected And Uninfected Salivary Glands Of Green Rice Leafhopper And Characterization Of Two Novel RNA Viral Genomes

Viruses ubiquitously exist in organisms and exhibit the most ecological and genomic diversity.As the most speciose group of animals,insects harbor various RNA viruses and act as critical vectors in arthropod-borne diseases.While insects are a major reservoir of virus biodiversity,viruses are remained unexamined in many insect species.Traditional virus researches are mainly driven by and focus on the causative agents infecting humans or the animals and plants of economic importance,so the characterized viruses are restricted.Nowadays,new RNA viruses are emerging owing to the utilization of RNA sequencing technologies,therefore,the knowledge of RNA virus is rapidly expanding.The green rice leafhopper,Nephotettix cincticeps,is one of the important pests on rice in Asia,transmitting rice dwarf virus(RDV)that causes rice dwarf disease.In order to investigate the interactions between N.cincticeps and RDV,RDV-uninfected and infected transcriptome analyses were performed.We found that RDV could affect other viruses in N.cincticeps.Among the differentially expressed viruses,ML gene,a positive-and a negative-stranded RNA virus were selected to be further characterized.The mean results are summarized as follows:1.The comparative transcriptome analysis of RDV-infected and uninfected salivary glands of N.cincticepsBy analyzing the transcriptomes of RDV-infected and uninfected salivary glands of N.cincticeps,622 differential expressed genes(DEGs)were identified,with 342 genes up-regulated and 280 genes down-regulated.The DEGs were used for gene set enrichment analysis.82 DEGs were selected for q PCR verification.52 insect and 10viral-like DEGs were confirmed.The viral-like sequences c12476_g1,c13775_g1,c31720_g1 and c84382_g1 were significantly up-regulated,while c31757_g1,c44831_g1,c53292_g1,c57866_g1,c57866_g2 and c18405_g1 were down-regulated when N.cincticeps was infected with RDV.1.Identification of N.cincticeps ML genes and interaction verification between ML proteins and RDV viral proteins.Six ML genes were identified in N.cincticeps,and NcML1,NcML2 and NcML6 genes were selected for multiple sequence alignment,phylogenetic analysis and tertiary structure modelling.The expression model of NcML1,NcML2 and NcML6 were tested and these three genes were most abundant in brain,especially NcML6.Yeast two-hybrid and Pull-Down were used to detection the interaction between NcML proteins and RDV.The results demonstrated that NcML6 could interact with Pns6,P8 and Pns12 of RDV.In order to investigate the potential function of NcML genes,ds RNAs of NcML genes were injected into N.cincticeps.However,no symptom was observed so far.3.Identification and characterization of a novel iflavirus in green rice leafhopperNephotettix cincticeps positive-stranded RNA virus-1(NcPSRV-1),found in N.cincticeps,consists of 10,524 nucleotides excluding the poly(A)tail and contains one predicted open reading frame encoding a polyprotein of 3,192 amino acids,flanked by5' and 3' untranslated regions.NcPSRV-1 has a typical iflavirus genome arrangement and is clustered with the members of the family Iflaviridae in the phylogenetic analysis.NcPSRV-1 could be transmitted horizontally and vertically.Moreover,NcPSRV-1 had high prevalence in the laboratory populations.NcPSRV-1 could also infect the twostriped leafhopper,N.apicalis,and the parasite of N.cincticeps,Pipunculus multillatus,but was absent in the zigzag leafhopper,Recilia dorsalis,and rice Oryza sativa variety TN1.The infection of RDV altered the viral load and infection rate of NcPSRV-1 in N.cincticeps,for which it seems that RDV has an antagonistic effect on NcPSRV-1infection in the host.4.Identification and characterization of a novel rhabdovirus in green rice leafhopperThe genome sequence of Nephotettix cincticeps negative-stranded RNA virus-1(NcNSRV-1)was verified with 12,361 nucleotides in length,flanked by untranslated 3'leader and 5' trailer.The anti-sense viral genome consists of five major structural protein genes(N,P,M,G and L),which shares a typical architecture with the family Rhabdoviridae.An additional gene,P6,is interposed between G and L genes.NcNSRV-1 is phylogenetically clustered with the unclassified rhabdoviruses isolated from insects and exhibits low sequence identities with other viruses.The transcription regulatory sequences in NcNSRV-1 gene junctions were determined,revealing a unique transcription initiation sequence.In view of the phylogeny,sequence identity and genome organization,NcNSRV-1 is likely to be an insect rhabdovirus.NcNSRV-1could not long exist in the laboratory populations,may due to the missing of host or transmission route.