Virome Analysis of Aphid Populations That Infest the Barley Field: The Discovery of Two Novel Groups of Nege/Kita-Like Viruses and Other Novel RNA Viruses

Abstract

Aphids (order Hemiptera) are important insect pests of crops and are also vectors of many plant viruses. However, little is known about aphid-infecting viruses, particularly their diversity and relationship to plant viruses. To investigate the aphid viromes, we performed deep sequencing analyses of the aphid transcriptomes from infested barley plants in a field in Japan. We discovered virus-like sequences related to nege/ kita-, flavi-, tombus-, phenui-, mononega-, narna-, chryso-, partiti-, and luteoviruses. Using RT-PCR and sequence analyses, we determined almost complete sequences of seven nege/kitavirus-like virus genomes; one of which was a variant of the Wuhan house centipede virus (WHCV-1). The other six seem to belong to four novel viruses distantly related to Wuhan insect virus 9 (WhIV-9) or Hubei nege-like virus 4 (HVLV-4). We designated the four viruses as barley aphid RNA virus 1 to 4 (BARV-1 to-4). Moreover, some nege/kitavirus-like sequences were found by searches on the transcriptome shotgun assembly (TSA) libraries of arthropods and plants. Phylogenetic analyses showed that BARV-1 forms a clade with WHCV-1 and HVLV-4, whereas BARV-2 to-4 clustered with WhIV-9 and an aphid virus, Aphis glycines virus 3. Both virus groups (tentatively designated as Centivirus and Aphiglyvirus, respectively), together with arthropod virus-like TSAs, fill the phylogenetic gaps between the negeviruses and kitaviruses lineages. We also characterized the flavi/jingmen-like and tombus-like virus sequences as well as other RNA viruses, including six putative novel viruses, designated as barley aphid RNA viruses 5 to 10. Interestingly, we also discovered that some aphid-associated viruses, including nege/kita-like viruses, were present in different aphid species, raising a speculation that these viruses might be distributed across different aphid species with plants being the reservoirs. This study provides novel information on the diversity and spread of nege/kitavirus-related viruses and other RNA viruses that are associated with aphids.

Negeviruses reduce replication of alphaviruses during co-infection

“…The use of Wolbachia-infected vectors has been widely adapted to curb mosquito-borne viral diseases, propelled by its natural ability to colonize mosquitoes [44]. Negeviruses also possess this attribute, having been discovered in numerous mosquito species on 6 continents, along with sandflies and other diverse insect species [19][20][21][22]24]. Insertion of gene-editing scFv sequences into mosquito genomes has also been used to prevent Plasmodium and DENV infection [45,46].…”

“…Members of this genus have been isolated from several species of hematophagous mosquitoes and sandflies, and negev-like viruses have also been found in other nonvector insects [17][18][19][20][21][22][23][24]. Phylogenetic studies have placed this group of viruses most closely to members of the genus Cilevirus, plant pathogens that are transmitted by mites [16,23].…”

Sequence Relationships of RNA Helicases and Other Proteins Encoded by Blunervirus RNAs Highlight Recombinant Evolutionary Origin of Kitaviral Genomes

“…The available data on the RdRp and SP24 evolutionary lineages strongly suggest the evolutionary connections between insect nege-like viruses and plant kitaviruses and occurrence of cross-kingdom transfer of positive-stranded RNA viruses between insects and plants during evolution (Kondo et al, 2019(Kondo et al, , 2020Ramos-González et al, 2020;Quito-Avila et al, in press).…”

“…However, kitavirus replicative proteins show closest similarity to arthropod-infecting negeviruses (proposed genera Nelorpivirus and Sandewavirus) and nege-like viruses (Kallies et al, 2014;Shi et al, 2016;Nunes et al, 2017;Ramos-González et al, 2020). Recent detailed phylogenetic analysis clearly showed that higrevirus RdRp are closer to cilevirus RdRp than to corresponding blunervirus protein, and these viral RdRp sequences form two separated branches in a phylogenetic dendrogram (Kondo et al, 2020;Ramos-González et al, 2020).…”

“…SP24 is the most conserved protein among non-replicative polypeptides encoded by all kitaviruses (Nunes et al, 2017;Ramos-González et al, 2020). Moreover, SP24 was found in negeviruses, many nege-like viruses (Kondo et al, 2020), and in unrelated arthropod-infecting viruses, such as chroparaviruses (Chronic Bee Paralysis Virus) (Kuchibhatla et al, 2014), which, unlike kitaviruses and negeviruses that represent order Martellivirales, show genome similarity to arthropod tombus-like viruses (Shi et al, 2016) and belong to separate virus order Tolivirales (Koonin et al, 2020). SP24-like proteins (Pfam: 16504) have several trans-membrane domains and a less conserved N-terminal region enriched with positively charged residues.…”