Characterization and Tissue Tropism of Newly Identified Iflavirus and Negeviruses in Glossina morsitans morsitans Tsetse Flies.

Tsetse flies cause major health and economic problems as they transmit trypanosomes causing sleeping sickness in humans (Human African Trypanosomosis, HAT) and nagana in animals (African Animal Trypanosomosis, AAT). A solution to control the spread of these flies and their associated diseases is the implementation of the Sterile Insect Technique (SIT). For successful application of SIT, it is important to establish and maintain healthy insect colonies and produce flies with competitive fitness. However, mass production of tsetse is threatened by covert virus infections, such as the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV). This virus infection can switch from a covert asymptomatic to an overt symptomatic state and cause the collapse of an entire fly colony. Although the effects of GpSGHV infections can be mitigated, the presence of other covert viruses threaten tsetse mass production. Here we demonstrated the presence of two single-stranded RNA viruses isolated from Glossina morsitans morsitans originating from a colony at the Seibersdorf rearing facility. The genome organization and the phylogenetic analysis based on the RNA-dependent RNA polymerase (RdRp) revealed that the two viruses belong to the genera Iflavirus and Negevirus, respectively. The names proposed for the two viruses are Glossina morsitans morsitans iflavirus (GmmIV) and Glossina morsitans morsitans negevirus (GmmNegeV). The GmmIV genome is 9685 nucleotides long with a poly(A) tail and encodes a single polyprotein processed into structural and non-structural viral proteins. The GmmNegeV genome consists of 8140 nucleotides and contains two major overlapping open reading frames (ORF1 and ORF2). ORF1 encodes the largest protein which includes a methyltransferase domain, a ribosomal RNA methyltransferase domain, a helicase domain and a RdRp domain. In this study, a selective RT-qPCR assay to detect the presence of the negative RNA strand for both GmmIV and GmmNegeV viruses proved that both viruses replicate in G. m. morsitans. We analyzed the tissue tropism of these viruses in G. m. morsitans by RNA-FISH to decipher their mode of transmission. Our results demonstrate that both viruses can be found not only in the host's brain and fat bodies but also in their reproductive organs, and in milk and salivary glands. These findings suggest a potential horizontal viral transmission during feeding and/or a vertically viral transmission from parent to offspring. Although the impact of GmmIV and GmmNegeV in tsetse rearing facilities is still unknown, none of the currently infected tsetse species show any signs of disease from these viruses.

A second capsidless hadakavirus strain with 10 positive-sense single-stranded RNA genomic segments from Fusarium nygamai.

A unique capsidless virus with a positive-sense, single-stranded RNA genome (hadakavirus 1, HadV1), a member of the extended picorna-like supergroup, was isolated previously from the phytopathogenic fungus Fusarium oxysporum. Here, we describe the molecular and biological characterisation of a second hadakavirus strain from Fusarium nygamai, which has not been investigated in detail previously as a virus host. This virus, hadakavirus 1 strain 1NL (HadV1-1NL), has features similar to the first hadakavirus, HadV1-7n, despite having a different number of segments (10 for HadV1-1NL vs. 11 for HadV1-7n). The 10 genomic RNA segments of HadV1-1NL range in size from 0.9 kb to 2.5 kb. All HadV1-1NL segments show 67% to 86% local nucleotide sequence identity to their HadV1-7n counterparts, whereas HadV1-1NL has no homolog of HadV1-7n RNA8, which encodes a zinc-finger motif. Another interesting feature is the possible coding incapability of HadV1-1NL RNA10. HadV1-1NL was predicted to be capsidless based on the RNase A susceptibility of its replicative form dsRNA. Phenotypic comparison of multiple virus-infected and virus-free single-spore isolates indicated asymptomatic infection by HadV1-1NL. Less-efficient vertical transmission via spores was observed as the infected fungal colonies from which the spores were derived became older, as was observed for HadV1-7n. This study shows a second example of a hadakavirus that appears to have unusual features.

Identification and characterization of a novel virus associated with an eriophyid mite in extracts of fruit trees leaves.

Eriophyid mites are commonly found on the leaf surface of different plant species. In the present study, a novel virus associated with an eriophyid mite species was detected using high-throughput sequencing (HTS) of total RNA from fruit tree leaves, primarily growing under greenhouse conditions. The complete genome sequence was characterized using rapid amplification of cDNA ends followed by Sanger sequencing, revealing a genome of 8885 nucleotides in length. The single positive-stranded RNA genome was predicted to encode typical conserved domains of members of the genus Iflavirus in the family Iflaviridae. Phylogenetic analysis showed this virus to be closely related to the unclassified iflavirus tomato matilda associated virus (TMaV), with a maximum amino acid sequence identity of 59% in the RNA-dependent RNA polymerase domain. This low identity value justifies the recognition of the novel virus as a potential novel iflavirus. In addition to a lack of graft-transmissibility evidence, RT-PCR and HTS detection of this virus in the putative host plants were not consistent through different years and growing seasons, raising the possibility that rather than a plant virus, this was a virus infecting an organism associated with fruit tree leaves. Identification of Tetra pinnatifidae HTS-derived contigs in all fruit tree samples carrying the novel virus suggested this mite as the most likely host of the new virus (p-value < 1e-11), which is tentatively named "eriophyid mite-associated virus" (EMaV). This study highlights the importance of a careful biological study before assigning a new virus to a particular plant host when using metagenomics data.

An expanded class of histidine-accepting viral tRNA-like structures.

Structured RNA elements are common in the genomes of RNA viruses, often playing critical roles during viral infection. Some viral RNA elements use forms of tRNA mimicry, but the diverse ways this mimicry can be achieved are poorly understood. Histidine-accepting tRNA-like structures (TLSHis) are examples found at the 3' termini of some positive-sense single-stranded RNA (+ssRNA) viruses where they interact with several host proteins, induce histidylation of the RNA genome, and facilitate processes important for infection, to include genome replication. As only five TLSHis examples had been reported, we explored the possible larger phylogenetic distribution and diversity of this TLS class using bioinformatic approaches. We identified many new examples of TLSHis, yielding a rigorous consensus sequence and secondary structure model that we validated by chemical probing of representative TLSHis RNAs. We confirmed new examples as authentic TLSHis by demonstrating their ability to be histidylated in vitro, then used mutational analyses to imply a tertiary interaction that is likely analogous to the D- and T-loop interaction found in canonical tRNAs. These results expand our understanding of how diverse RNA sequences achieve tRNA-like structure and function in the context of viral RNA genomes and lay the groundwork for high-resolution structural studies of tRNA mimicry by histidine-accepting TLSs.

Multiple clades of Husavirus in South America revealed by next generation sequencing.

Husavirus (HuV) is an unclassified virus of the order Picornavirales that has already been identified worldwide in various locations. The genetic, epidemiological, and pathogenic characteristics are, however, little understood. In children with acute gastroenteritis, this study used next-generation sequencing to recognize unknown sources of viruses. In particular, 251 fecal samples obtained from individuals were sequenced in southern, northeastern, and northern Brazil. all samples were also analyzed using culture methods and parasitological tests to classify other enteric pathogens such as bacteria, parasites, and viruses. 1.9% of the samples tested positive for HuV, for a total of 5 positive children, with a mean age of 2 year, with three males and two females. Detailed molecular characterization of full genomes showed that Brazilian HuVs' nucleotide divergence is less than 11%. The genetic gap between Brazilian sequences and the closest HuV reported previously, on the other hand, is 18%. The study showed that Brazilian sequences are closely related to the HuV defined in Viet Nam in 2013, further characterization based on phylogenetics. At least two divergent clades of HuV in South America were also seen in the phylogenetic study.

Comparative viromes of Culicoides and mosquitoes reveal their consistency and diversity in viral profiles.

The genus Culicoides includes biting midges, some of which are vectors for viruses that cause diseases in humans and animals. Knowledge of the roles of Culicoides in viral ecology is inadequate. We collected ~300 000 samples of Culicoides and mosquitoes in 15 representative regions within Yunnan, China. Using mosquitoes as reference vectors, we designed a comparative virome strategy to study the viral composition, diversity, hosts and spatiotemporal distribution of Culicoides. A map of viromes in Culicoides and mosquitoes in Yunan province, China, was constructed. At the same locations, Culicoides and mosquitoes usually share a similar viral diversity. At least 10 important pathogenic viruses were detected from Culicoides. Many novel viruses were discovered, including 21 segmented viruses of Flaviviridae, 180 viruses of Monjiviricetes and 130 viruses of Bunyavirales. The findings demonstrate that Culicoides is an important part of viral ecology and should be studied and monitored for potentially emerging viruses.

Transmembrane redox regulation of genome replication functions in positive-strand RNA viruses.

Positive-strand RNA virus genome replication takes place on intracellular membranes that separate the reduced cytosol from the oxidized extracellular/luminal milieu. Ongoing studies of these membrane-bounded genome replication complexes have revealed underlying common principles in their structure, assembly and functionalization, including transmembrane features and redox dependencies. Among these, members of the alphavirus, flavivirus, and picornavirus supergroups all encode membrane-permeabilizing viroporins required for efficient RNA replication. For flaviviruses and particularly alphavirus supergroup members, these viroporins are linked to activating viral RNA capping and potentially other later-stage RNA replication functions, and to local transmembrane release of oxidizing potential to trigger these changes in cytoplasmic RNA replication complexes. Further exploration of these emerging shared principles could spur development of broad-spectrum antivirals.

A Novel RNA Virus in the Parasitoid Wasp Lysiphlebus fabarum: Genomic Structure, Prevalence, and Transmission.

We report on a novel RNA virus infecting the wasp Lysiphlebus fabarum, a parasitoid of aphids. This virus, tentatively named "Lysiphlebus fabarum virus" (LysV), was discovered in transcriptome sequences of wasps from an experimental evolution study in which the parasitoids were allowed to adapt to aphid hosts (Aphis fabae) with or without resistance-conferring endosymbionts. Based on phylogenetic analyses of the viral RNA-dependent RNA polymerase (RdRp), LysV belongs to the Iflaviridae family in the order of the Picornavirales, with the closest known relatives all being parasitoid wasp-infecting viruses. We developed an endpoint PCR and a more sensitive qPCR assay to screen for LysV in field samples and laboratory lines. These screens verified the occurrence of LysV in wild parasitoids and identified the likely wild-source population for lab infections in Western Switzerland. Three viral haplotypes could be distinguished in wild populations, of which two were found in the laboratory. Both vertical and horizontal transmission of LysV were demonstrated experimentally, and repeated sampling of laboratory populations suggests that the virus can form persistent infections without obvious symptoms in infected wasps.