Identification and genetic analysis of a totivirus isolated from the Culex tritaeniorhynchus in northern China.

Totiviridae, a viral family of double-stranded RNA (dsRNA) viruses, contain a single dsRNA genome 4.6-7.0 kb in length. Totiviridae were initially only known to infect fungi and other eukaryotes as well as plants, but an increase in totiviruses has been detected in insects, mosquitoes, and bats. Here, we describe the isolation and characterization of a strain belonging to the family Totiviridae isolated from Culex tritaeniorhynchus in Kenli, China, in 2016. We isolated a totivirus from field-collected mosquitoes in China by cell culture in Aedes albopictus C6/36 cells, identified the virus by morphological observation and complete genome sequencing, and characterized it by phylogenetic analysis. Transmission electron microscopy identified icosahedral, non-enveloped virus particles with a mean diameter of 35-40 nm. The genome was 7612 bp in length, including two open reading frames (ORFs). ORF1 (5058 nt) encodes the capsid protein, while ORF2 (2216 nt) encodes the viral RNA-dependent RNA polymerase (RdRp). Nucleotide and amino acid homology analysis of isolate showed higher levels of sequence identity with isolate CTV_NJ2 (China, 2010) with 94.87% nucleic acid identity and 97.32% amino acid identity. The isolate was designated C. tritaeniorhynchus totivirus KL (CTV-KL). This is the first identification of a totivirus in a C. tritaeniorhynchus in northern China. Analysis of the virus's morphology, characteristic and genome organization will further enrich our understanding of the molecular and biological characteristics of dsRNA Totiviridae viruses.

Isolation and full-length sequence analysis of Armigeres subalbatus totivirus, the first totivirus isolate from mosquitoes representing a proposed novel genus (Artivirus) of the family Totiviridae.

During an investigation of arboviruses in China, a novel dsRNA virus was isolated from adult female Armigeres subalbatus. Full genome sequence analysis showed the virus to be related to members of the family Totiviridae, and was therefore named 'Armigeres subalbatus totivirus' (AsTV). Transmission electron microscopy identified icosahedral, non-enveloped virus particles with a mean diameter of 40 nm. The AsTV genome is 7510 bp in length, with two ORFs. ORF1 (4443 nt) encodes the coat-protein and a dsRNA-binding domain (which may be involved in the evasion of 'gene silencing'), while ORF2 (2286 nt) encodes the viral RNA-dependent RNA polymerase (RdRp). The AsTV coat protein shows a higher level of amino acid identity with Drosophila totivirus (DTV, 52 %) than with infectious myonecrosis virus (IMNV, 29 %). Similarly, the RdRp shows higher identity levels with DTV (51 %) than with IMNV (44 %). Identity levels to other members of the family Totiviridae, in either the coat protein or the RdRp, ranged from 6 to 11 %. Based on a recent reassessment of the coding strategy used by IMNV, we suggest that an AsTV coat-RdRp fusion protein could be synthesized via a -1 frameshift. Elements favouring -1 frameshift such as 'slippery heptamers' and pseudonkots, were identified in the AsTV, DTV and IMNV genomes. AsTV was shown to grow in both mosquito and mammalian cells, suggesting that it is an arbovirus that can infect mammals.

Ded1p, a conserved DExD/H-box translation factor, can promote yeast L-A virus negative-strand RNA synthesis in vitro.

Viruses are intracellular parasites that must use the host machinery to multiply. Identification of the host factors that perform essential functions in viral replication is thus of crucial importance to the understanding of virus-host interactions. Here we describe Ded1p, a highly conserved DExD/H-box translation factor, as a possible host factor recruited by the yeast L-A double-stranded RNA (dsRNA) virus. We found that Ded1p interacts specifically and strongly with Gag, the L-A virus coat protein. Further analysis revealed that Ded1p interacts with the L-A virus in an RNA-independent manner and, as a result, L-A particles can be affinity purified via this interaction. The affinity-purified L-A particles are functional, as they are capable of synthesizing RNA in vitro. Critically, using purified L-A particles, we demonstrated that Ded1p specifically promotes L-A dsRNA replication by accelerating the rate of negative-strand RNA synthesis in vitro. In light of these data, we suggest that Ded1p may be a part of the long sought after activity shown to promote yeast viral dsRNA replication. This and the fact that Ded1p is also required for translating brome mosaic virus RNA2 in yeast thus raise the intriguing possibility that Ded1p is one of the key host factors favored by several evolutionarily related RNA viruses, including the human hepatitis C virus.