Identification Of Aphid Bunyavirus ABV-1 And Exploration Of Its Interaction With Aphid Hosts

Viruses are a class of small organism with simple structure,which rely on the host for self-proliferation.RNA viruses take RNA as their genetic material.They are extremely diverse and rapidly evolving.In addition to spreading plant viruses,key agricultural pest aphids are often infected by a variety of insect specific RNA viruses.Investigation of the RNA virus can enrich the aphid viral database and provide resources for development of viral based-aphid control approach which also which also provide new perspective for understanding of the interaction between viruses and aphids.In this thesis,we discovered18 novel RNA viruses from 10 aphid species by high-throughput sequencing technology,and analyzed the widespread presence bunyavirus（tentatively named Aphid bunyavirus1—ABV-1）in both the infection and transmission patterns as well as the influence to pea aphid,Acyrthosiphon pisum.The main results are as follows:1.Discovery and characterization of RNA viruses in aphidsHigh-throughput sequencing technology was used to explore the RNA viruses in Ac.pisum,Aphis aurantia,Aphis citricidus,Aphis gossypii,Melanaphis sacchari,Myzus persicae,Rhodobium porosum,Rhopalosiphum padi and Sitobion avenae,which discovered a total of 18 novel RNA viruses,including 14 positive-stranded RNA viruses and 4 negative-stranded RNA viruses.Rd Rp were found in these 18 RNA viruses by conserved domain analysis of viral sequences.With phylogenetic tree based on the Rd Rp aphids were abundant in Bunyavirus,Narnavirus and Dicistrovirus.The expression profiles of the viral-derived small RNA（vs RNA）in aphids were analyzed by using small RNA sequencing technology,the results showed that most viruses exhibited vs RNA distribution patterns with 22 nt peaks and uniformly distributed in the viral genome,indicating that these discovered viruses replicated in aphids and triggered the immune response of host’s RNAi.Importantly,a novel bunyavirus（ABV-1）,was found in a variety of aphids.This virus contained three structural proteins,Rd Rp,Nucleoprotein and Glycoprotein（Gc and Gn）,and two non-structural proteins,NSm and NSs.ABV-1 assigned with Phasmaviridae family of Bunyavirales,and this imply ABV-1 as an insect-specific virus.2.Infection and transmission patterns of ABV-1 in pea aphidsThe infection pattern of ABV-1 in different stages（1-4 instars,1,3,7,14-day-old adults）and tissues（gut,embryo,fat body,integument,central nerve system,stylet,muscle,hemolymph）of Ac.pisum were analyzed by RT-q PCR.ABV-1 was presented in all developmental stages and tissues of Ac.pisum.The titer of ABV-1 in different developmental stages of Ac.pisum was relatively stable,the titer of ABV-1 in hemolymph and integument was significantly higher than other tissues.In addition,ABV-1 was localized in the gut,salivary gland and integument of Ac.pisum by immunofluorescence.The transmission pattern of ABV-1 in pea aphid population was analyzed by PCR.The results showed that vertical transmission rate of ABV-1 in Ac.pisum was 100%.The presence of ABV-1 on aphid-fed plants and honeydew could be detected,suggesting the possible presence of horizontal transmission.Based on the transmission patterns of ABV-1 in Ac.pisum,the individual rearing and PCR-based screening strategy were adopted to obtain low titer of ABV-1(ABV-1^low strain)from the pea aphid of high titer of ABV-1(ABV-1^high strain)through generations of selection.3.The influence of ABV-1 on the biological characteristics of pea aphidsBased on ABV-1^low and ABV-1^high strains,we compared the key biological characteristics of Ac.pisum.The results showed that ABV-1 could reduce the total nymphal duration and induce the reproduction.Through the electrical penetration graph（EPG）,we found that ABV-1 significantly reduced the time for aphids to probe plants and prolong the feeding time.In summary,this thesis discovered 18 novel RNA viruses in 10 aphid species,including a new bunyavirus—ABV-1 that widely detected in aphids.Taking the model organism Ac.pisum as the main research object,the infection and transmission patterns of ABV-1 were analyzed.It was preliminarily found that ABV-1 could promote the rapid reproduction and plant feeding of aphids.Taken all together,we proposed that ABV-1may have a symbiotic-like relationship with aphids.The research results may establish a model for virus-aphid interactions.