Edible insects unlikely to contribute to transmission of coronavirus SARS-CoV-2

In the context of food safety, edible insects are evaluated for biological hazards such as microbial pathogens according to regulations currently in place. When the European Food Safety Authority evaluated the hazards of edible insects as a potential source of pathogenic viruses for humans and livestock, the novel zoonotic coronavirus SARS-CoV-2 had not yet emerged but other pathogenic coronaviruses such as SARS (SARS-CoV) and MERS (MERS-CoV) were known. As a result of the COVID-19 pandemic, animal sources of protein for human consumption are being evaluated for the risks of being a transmission vector of coronaviruses, like SARS-CoV-2. Insects lack a receptor that can bind SARS-CoV-2, thus preventing the virus from replicating in insects, unlike some vertebrate livestock species and companion animals. Despite extensive monitoring, coronaviruses have never been recorded in insect microbiomes. Contamination of insects produced for food or feed may occur during the production process, resulting from rearing substrate or from insect farmers. However, the currently permitted rearing substrates do not include animal products and the farming process is highly automated, thus limiting interactions between farmers and insects. If contamination would still occur, the fact that the insects in production are not hosts to SARS-CoV-2 precludes virus replication and the further processing of the insects will destroy the contamination. We conclude that the hazard of edible insects being a transmission vector of SARS-CoV-2 is extremely low.</p

Chikungunya Virus nsP3 Blocks Stress Granule Assembly by Recruitment of G3BP into Cytoplasmic Foci

Chikungunya virus nonstructural protein nsP3 has an essential but unknown role in alphavirus replication and interacts with Ras-GAP SH3 domain-binding protein (G3BP). Here we describe the first known function of nsP3, to inhibit stress granule assembly by recruiting G3BP into cytoplasmic foci. A conserved SH3 domain-binding motif in nsP3 is essential for both nsP3-G3BP interactions and viral RNA replication. This study reveals a novel role for nsP3 as a regulator of the cellular stress respons

A heritable antiviral RNAi response limits Orsay virus infection in Caenorhabditis elegans N2

Orsay virus (OrV) is the first virus known to be able to complete a full infection cycle in the model nematode species Caenorhabditis elegans. OrV is transmitted horizontally and its infection is limited by antiviral RNA interference (RNAi). However, we have no insight into the kinetics of OrV replication in C. elegans. We developed an assay that infects worms in liquid, allowing precise monitoring of the infection. The assay revealed a dual role for the RNAi response in limiting Orsay virus infection in C. elegans. Firstly, it limits the progression of the initial infection at the step of recognition of dsRNA. Secondly, it provides an inherited protection against infection in the offspring. This establishes the heritable RNAi response as anti-viral mechanism during OrV infections in C. elegans. Our results further illustrate that the inheritance of the anti-viral response is important in controlling the infection in the canonical wild type Bristol N2. The OrV replication kinetics were established throughout the worm life-cycle, setting a standard for further quantitative assays with the OrV-C. elegans infection model

Novel approaches for the rapid development of rationally designed arbovirus vaccines

Vector-borne diseases, including those transmitted by mosquitoes, account for more than 17% of infectious diseases worldwide. This number is expected to rise with an increased spread of vector mosquitoes and viruses due to climate change and man-made alterations to ecosystems. Among the most common, medically relevant mosquito-borne infections are those caused by arthropod-borne viruses (arboviruses), especially members of the genera Flavivirus and Alphavirus. Arbovirus infections can cause severe disease in humans, livestock and wildlife. Severe consequences from infections include congenital malformations as well as arthritogenic, haemorrhagic or neuroinvasive disease. Inactivated or live-attenuated vaccines (LAVs) are available for a small number of arboviruses; however there are no licensed vaccines for the majority of these infections. Here we discuss recent developments in pan-arbovirus LAV approaches, from site-directed attenuation strategies targeting conserved determinants of virulence to universal strategies that utilize genome-wide re-coding of viral genomes. In addition to these approaches, we discuss novel strategies targeting mosquito saliva proteins that play an important role in virus transmission and pathogenesis in vertebrate hosts. For rapid pre-clinical evaluations of novel arbovirus vaccine candidates, representative in vitro and in vivo experimental systems are required to assess the desired specific immune responses. Here we discuss promising models to study attenuation of neuroinvasion, neurovirulence and virus transmission, as well as antibody induction and potential for cross-reactivity. Investigating broadly applicable vaccination strategies to target the direct interface of the vertebrate host, the mosquito vector and the viral pathogen is a prime example of a One Health strategy to tackle human and animal diseases.Molecular basis of virus replication, viral pathogenesis and antiviral strategie

Mosquito and Drosophila entomobirnaviruses suppress dsRNA- and siRNA-induced RNAi

RNA interference (RNAi) is a crucial antiviral defense mechanism in insects, including the major mosquito species that transmit important human viruses. To counteract the potent antiviral RNAi pathway, insect viruses encode RNAi suppressors. However, whether mosquito-specific viruses suppress RNAi remains unclear. We therefore set out to study RNAi suppression by Culex Y virus (CYV), a mosquito-specific virus of the Birnaviridae family that was recently isolated from Culex pipiens mosquitoes. We found that the Culex RNAi machinery processes CYV double-stranded RNA (dsRNA) into viral small interfering RNAs (vsiRNAs). Furthermore, we show that RNAi is suppressed in CYV-infected cells and that the viral VP3 protein is responsible for RNAi antagonism. We demonstrate that VP3 can functionally replace B2, the well-characterized RNAi suppressor of Flock House virus. VP3 was found to bind long dsRNA as well as siRNAs and interfered with Dicer-2-mediated cleavage of long dsRNA into siRNAs. Slicing of target RNAs by pre-assembled RNA-induced silencing complexes was not affected by VP3. Finally, we show that the RNAi-suppressive activity of VP3 is conserved in Drosophila X virus, a birnavirus that persistently infects Drosophila cell cultures. Together, our data indicate that mosquito-specific viruses may encode RNAi antagonists to suppress antiviral RNAi

Comparative Usutu and West Nile virus transmission potential by local Culex pipiens mosquitoes in north-western Europe

Originating from Africa, Usutu virus (USUV) first emerged in Europe in 2001. This mosquito-borne flavivirus caused high mortality rates in its bird reservoirs, which strongly resembled the introduction of West Nile virus (WNV) in 1999 in the United States. Mosquitoes infected with USUV incidentally transmit the virus to other vertebrates, including humans, which can result in neuroinvasive disease. USUV and WNV co-circulate in parts of southern Europe, but the distribution of USUV extends into central and northwestern Europe. In the field, both viruses have been detected in the northern house mosquito Culex pipiens, of which the potential for USUV transmission is unknown. To understand the transmission dynamics and assess the potential spread of USUV, we determined the vector competence of C. pipiens for USUV and compared it with the well characterized WNV. We show for the first time that northwestern European mosquitoes are highly effective vectors for USUV, with infection rates of 11% at 18. °C and 53% at 23. °C, which are comparable with values obtained for WNV. Interestingly, at a high temperature of 28. °C, mosquitoes became more effectively infected with USUV (90%) than with WNV (58%), which could be attributed to barriers in the mosquito midgut. Small RNA deep sequencing of infected mosquitoes showed for both viruses a strong bias for 21-nucleotide small interfering (si)RNAs, which map across the entire viral genome both on the sense and antisense strand. No evidence for viral PIWI-associated RNA (piRNA) was found, suggesting that the siRNA pathway is the major small RNA pathway that targets USUV and WNV infection in C. pipiens mosquitoes

Dengue Non-coding RNA: TRIMmed for Transmission

Dengue virus RNA is trimmed by the 5'¿3' exoribonuclease XRN1 to produce an abundant, non-coding subgenomic flavivirus RNA (sfRNA) in infected cells. In a recent paper in Science, Manokaran et al. (2015) report that sfRNA binds TRIM25 to evade innate immune sensing of viral RNA by RIG-I

Flavivirus RNAi suppression: decoding non-coding RNA

Flaviviruses are important human pathogens that are transmitted by invertebrate vectors, mostly mosquitoes and ticks. During replication in their vector, flaviviruses are subject to a potent innate immune response known as antiviral RNA interference (RNAi). This defense mechanism is associated with the production of small interfering (si)RNA that lead to degradation of viral RNA. To what extent flaviviruses would benefit from counteracting antiviral RNAi is subject of debate. Here, the experimental evidence to suggest the existence of flavivirus RNAi suppressors is discussed. I will highlight the putative role of non-coding, subgenomic flavivirus RNA in suppression of RNAi in insect and mammalian cells. Novel insights from ongoing research will reveal how arthropod-borne viruses modulate innate immunity including antiviral RNAi

Arbovirus vaccines: opportunities for the baculovirus-insect cell expression system

The baculovirus-insect cell expression system is a well-established technology for the production of heterologous viral (glyco)proteins in cultured cells, applicable for basic scientific research as well as for the development and production of vaccines and diagnostics. Arboviruses form an emerging group of medically important viral pathogens that are transmitted to humans and animals via arthropod vectors, mostly mosquitoes, ticks or midges. Few arboviral vaccines are currently available, but there is a growing need for safe and effective vaccines against some highly pathogenic arboviruses such as Chikungunya, dengue, West Nile, Rift Valley fever and Bluetongue viruses. This comprehensive review discusses the biology and current state of the art in vaccine development for arboviruses belonging to the families Togaviridae, Flaviviridae, Bunyaviridae and Reoviridae and the potential of the baculovirus-insect cell expression system for vaccine antigen production The members of three of these four arbovirus families have enveloped virions and display immunodominant glycoproteins with a complex structure at their surface. Baculovirus expression of viral antigens often leads to correctly folded and processed (glyco)proteins able to induce protective immunity in animal models and humans. As arboviruses occupy a unique position in the virosphere in that they also actively replicate in arthropod cells, the baculovirus-insect cell expression system is well suited to produce arboviral proteins with correct folding and post-translational processing. The opportunities for recombinant baculoviruses to aid in the development of safe and effective subunit and virus-like particle vaccines against arboviral diseases are discussed