The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive-strand RNA viruses.

Genome-wide screens are powerful approaches to unravel regulators of viral infections. Here, a CRISPR screen identifies the RNA helicase DDX42 as an intrinsic antiviral inhibitor of HIV-1. Depletion of endogenous DDX42 increases HIV-1 DNA accumulation and infection in cell lines and primary cells. DDX42 overexpression inhibits HIV-1 infection, whereas expression of a dominant-negative mutant increases infection. Importantly, DDX42 also restricts LINE-1 retrotransposition and infection with other retroviruses and positive-strand RNA viruses, including CHIKV and SARS-CoV-2. However, DDX42 does not impact the replication of several negative-strand RNA viruses, arguing against an unspecific effect on target cells, which is confirmed by RNA-seq analysis. Proximity ligation assays show DDX42 in the vicinity of viral elements, and cross-linking RNA immunoprecipitation confirms a specific interaction of DDX42 with RNAs from sensitive viruses. Moreover, recombinant DDX42 inhibits HIV-1 reverse transcription in vitro. Together, our data strongly suggest a direct mode of action of DDX42 on viral ribonucleoprotein complexes. Our results identify DDX42 as an intrinsic viral inhibitor, opening new perspectives to target the life cycle of numerous RNA viruses.

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.

Honey bee viruses are highly prevalent but at low intensities in wild pollinators of cucurbit agroecosystems.

Managed and wild bee populations are in decline around the globe due to several biotic and abiotic stressors. Pathogenic viruses associated with the Western honey bee (Apis mellifera) have been identified as key contributors to losses of managed honey bee colonies, and are known to be transmitted to wild bee populations through shared floral resources. However, little is known about the prevalence and intensity of these viruses in wild bee populations, or how bee visitation to flowers impacts viral transmission in agroecosystems. This study surveyed honey bee, bumble bee (Bombus impatiens) and wild squash bee (Eucera (Peponapis) pruinosa) populations in Cucurbita agroecosystems across Pennsylvania (USA) for the prevalence and intensity of five honey bee viruses: acute bee paralysis virus (ABPV), deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV), and slow bee paralysis virus (SBPV). We investigated the potential role of bee visitation rate to flowers on DWV intensity among species in the pollinator community, with the expectation that increased bee visitation to flowers would increase the opportunity for transmission events between host species. We found that honey bee viruses are highly prevalent but in lower titers in wild E. pruinosa and B. impatiens than in A. mellifera populations throughout Pennsylvania (USA). DWV was detected in 88% of B. impatiens, 48% of E. pruinosa, and 95% of A. mellifera. IAPV was detected in 5% of B. impatiens and 4% of E. pruinosa, compared to 9% in A. mellifera. KBV was detected in 1% of B. impatiens and 5% of E. pruinosa, compared to 32% in A. mellifera. Our results indicate that DWV titers are not correlated with bee visitation in Cucurbita fields. The potential fitness impacts of these low viral titers detected in E. pruinosa remain to be investigated.

Antiviral strategies targeting host factors and mechanisms obliging +ssRNA viral pathogens.

The ongoing COVID-19 pandemic, periodic recurrence of viral infections, and the emergence of challenging variants has created an urgent need of alternative therapeutic approaches to combat the spread of viral infections, failing to which may pose a greater risk to mankind in future. Resilience against antiviral drugs or fast evolutionary rate of viruses is stressing the scientific community to identify new therapeutic approaches for timely control of disease. Host metabolic pathways are exquisite reservoir of energy to viruses and contribute a diverse array of functions for successful replication and pathogenesis of virus. Targeting the host factors rather than viral enzymes to cease viral infection, has emerged as an alternative antiviral strategy. This approach offers advantage in terms of increased threshold to viral resistance and can provide broad-spectrum antiviral action against different viruses. The article here provides substantial review of literature illuminating the host factors and molecular mechanisms involved in innate/adaptive responses to viral infection, hijacking of signalling pathways by viruses and the intracellular metabolic pathways required for viral replication. Host-targeted drugs acting on the pathways usurped by viruses are also addressed in this study. Host-directed antiviral therapeutics might prove to be a rewarding approach in controlling the unprecedented spread of viral infection, however the probability of cellular side effects or cytotoxicity on host cell should not be ignored at the time of clinical investigations.

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.

Infection of cotton bollworm by Helicoverpa armigera iflavirus decreases larval fitness.

Previously, we reported a novel iflavirus in Helicoverpa armigera (helicoverpa armigera iflavirus, HaIV) and here we report the effects of HaIV on its host. In a laboratory bioassay, HaIV-positive larvae and pupae developed more slowly and had higher mortality than HaIV-negative larvae, suggesting that the virus is pathogenic. The relative fitness of H. armigera decreased with HaIV infection by a ratio of 0.65. Transcriptional analysis indicated that infection significantly changed the expression levels of host genes, with more genes affected at 72 h after inoculation than at 48 h (138 up- and 229 downregulated at 48 h; 185 up- and 299 downregulated at 72 h). Interestingly, pathways related to digestion and absorption were significantly enriched, e.g., protein digestion and absorption, suggesting developmental regulation of the host by HaIV via these pathways. HaIV-infected H. armigera showed significantly downregulated expression of genes encoding cuticular proteins (CPs), essential for structural and protective functions, at 48 h and 72 h, suggesting that HaIV increased larval mortality by downregulating CP gene expression.

Biological characterization of Euscelidius variegatus iflavirus 1.

Virus-based biocontrol technologies represent sustainable alternatives to pesticides and insecticides. Phytoplasmas are prokaryotic plant pathogens causing severe losses to crops worldwide. Novel approaches are needed since insecticides against their insect vectors and rogueing of infected plants are the only available strategies to counteract phytoplasma diseases. A new iflavirus, named EVV-1, has been described in the leafhopper phytoplasma vector Euscelidius variegatus, raising the potential to use virus-based application strategies against phytoplasma disease. Here transmission routes of EVV-1 are characterized, and localization within the host reveals the mechanism of insect tolerance to virus infection. Both vertical and horizontal transmission of EVV-1 occur and vertical transmission was more efficient. The virus is systemic and occurs in all life-stages, with the highest loads measured in ovaries and first to third instar nymphs. The basic knowledge gained here on the biology of the virus is crucial for possible future application of iflaviruses as biocontrol agents.

DDX3 inhibitors show antiviral activity against positive-sense single-stranded RNA viruses but not against negative-sense single-stranded RNA viruses: The coxsackie B model.

Picornaviridae are positive-sense single stranded RNA viruses with a similar genomic structure lacking a cap at the 5' end, but with a highly structured 5'-untranslated region (UTR) containing an internal ribosome entry site (IRES). IRES allows ribosomes to be recruited by the viral RNA and initiate translation in a cap-independent manner. Coxsackie virus type B (CV-B) belong to Picornaviridae and are widespread in human population. They usually cause subclinical infections but, occasionally, also severe diseases with various clinical manifestations. CV-B have no specific therapy. DEAD-box polypeptide 3 (DDX3) is a member of the Asp-Glu-Ala-Asp (DEAD)-box family with an ATP-dependent RNA unwinding helicase activity. Recently, several positive-sense single strand RNA viruses have been shown to need DDX3 for their translation. Here, we show that several DDX3 inhibitors reduced CV-B replication and production of viral protein, particularly when added within 12 h of infection. Based on in vitro and in silico data, we hypothesized that DDX3 inhibitors hamper interaction between DDX3 and viral IRES in a stereodynamic fashion. Accordingly, the DDX3 inhibitors tested have no activity against the Vesicular Stomatitis virus and Measles virus, which are negative-sense single stranded RNA viruses and use cap-dependent translation. This study suggests that DDX3 is required by RNA viruses lacking a cap and show that this enzyme is a valuable target to design antiviral molecules against CV-B. Thus, DDX3 is dispensable for cap-dependent translation, but required for translation of transcripts containing secondary structure in their UTRs.

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.