4'-Fluorouridine is an oral antiviral that blocks respiratory syncytial virus and SARS-CoV-2 replication.

The COVID-19 pandemic has underscored the critical need for broad-spectrum therapeutics against respiratory viruses. Respiratory syncytial virus (RSV) is a major threat to pediatric patients and older adults. We describe 4'-fluorouridine (4'-FlU, EIDD-2749), a ribonucleoside analog that inhibits RSV, related RNA viruses, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with high selectivity index in cells and human airway epithelia organoids. Polymerase inhibition within in vitro RNA-dependent RNA polymerase assays established for RSV and SARS-CoV-2 revealed transcriptional stalling after incorporation. Once-daily oral treatment was highly efficacious at 5 milligrams per kilogram (mg/kg) in RSV-infected mice or 20 mg/kg in ferrets infected with different SARS-CoV-2 variants of concern, initiated 24 or 12 hours after infection, respectively. These properties define 4'-FlU as a broad-spectrum candidate for the treatment of RSV, SARS-CoV-2, and related RNA virus infections.

A Novel Anphevirus in Aedes albopictus Mosquitoes Is Distributed Worldwide and Interacts with the Host RNA Interference Pathway.

The Asian tiger mosquito Aedes albopictus is a competent vector for several human arboviruses including dengue, chikungunya and Zika viruses. Mosquitoes also harbor insect-specific viruses (ISVs) that may modulate host physiology and potentially affect the transmission of viruses that are pathogenic to vertebrates, thus representing a potential tool for vector control strategies. In Ae. albopictus we identified a novel anphevirus (family Xinmoviridae; order Mononegavirales) provisionally designated here as Aedes albopictus anphevirus (AealbAV). AealbAV contains a ~12.4 kb genome that is highly divergent from currently known viruses but displays gene content and genomic organization typical of known anpheviruses. We identified AealbAV in several publicly available RNA-Seq datasets from different geographical regions both in laboratory colonies and field collected mosquitoes. Coding-complete genomes of AealbAV strains are highly similar worldwide (>96% nucleotide identity) and cluster according to the geographical origin of their hosts. AealbAV appears to be present in various body compartments and mosquito life stages, including eggs. We further detected AealbAV-derived vsiRNAs and vpiRNAs in publicly available miRNA-Seq libraries of Ae. albopictus and in samples experimentally coinfected with chikungunya virus. This suggests that AealbAV is targeted by the host RNA interference (RNAi) response, consistent with persistent virus replication. The discovery and characterization of AealbAV in Ae. albopictus will now allow us to identify its infection in mosquito populations and laboratory strains, and to assess its potential impact on Ae. albopictus physiology and ability to transmit arboviruses.

Chaperoning the Mononegavirales: Current Knowledge and Future Directions.

The order Mononegavirales harbors numerous viruses of significant relevance to human health, including both established and emerging infections. Currently, vaccines are only available for a small subset of these viruses, and antiviral therapies remain limited. Being obligate cellular parasites, viruses must utilize the cellular machinery for their replication and spread. Therefore, targeting cellular pathways used by viruses can provide novel therapeutic approaches. One of the key challenges confronted by both hosts and viruses alike is the successful folding and maturation of proteins. In cells, this task is faced by cellular molecular chaperones, a group of conserved and abundant proteins that oversee protein folding and help maintain protein homeostasis. In this review, we summarize the current knowledge of how the Mononegavirales interact with cellular chaperones, highlight key gaps in our knowledge, and discuss the potential of chaperone inhibitors as antivirals.

Aedes Anphevirus: an Insect-Specific Virus Distributed Worldwide in Aedes aegypti Mosquitoes That Has Complex Interplays with Wolbachia and Dengue Virus Infection in Cells.

Insect-specific viruses (ISVs) of the yellow fever mosquito Aedes aegypti have been demonstrated to modulate transmission of arboviruses such as dengue virus (DENV) and West Nile virus by the mosquito. The diversity and composition of the virome of A. aegypti, however, remains poorly understood. In this study, we characterized Aedes anphevirus (AeAV), a negative-sense RNA virus from the order Mononegavirales AeAV identified from Aedes cell lines was infectious to both A. aegypti and Aedes albopictus cells but not to three mammalian cell lines. To understand the incidence and genetic diversity of AeAV, we assembled 17 coding-complete and two partial genomes of AeAV from available transcriptome sequencing (RNA-Seq) data. AeAV appears to transmit vertically and be present in laboratory colonies, wild-caught mosquitoes, and cell lines worldwide. Phylogenetic analysis of AeAV strains indicates that as the A. aegypti mosquito has expanded into the Americas and Asia-Pacific, AeAV has evolved into monophyletic African, American, and Asia-Pacific lineages. The endosymbiotic bacterium Wolbachia pipientis restricts positive-sense RNA viruses in A. aegypti Reanalysis of a small RNA library of A. aegypti cells coinfected with AeAV and Wolbachia produces an abundant RNA interference (RNAi) response consistent with persistent virus replication. We found Wolbachia enhances replication of AeAV compared to a tetracycline-cleared cell line, and AeAV modestly reduces DENV replication in vitro The results from our study improve understanding of the diversity and evolution of the virome of A. aegypti and adds to previous evidence that shows Wolbachia does not restrict a range of negative-strand RNA viruses.IMPORTANCE The mosquito Aedes aegypti transmits a number of arthropod-borne viruses (arboviruses), such as dengue virus and Zika virus. Mosquitoes also harbor insect-specific viruses that may affect replication of pathogenic arboviruses in their body. Currently, however, there are only a few insect-specific viruses described from A. aegypti in the literature. Here, we characterize a novel negative-strand virus, AeAV. Meta-analysis of A. aegypti samples showed that it is present in A. aegypti mosquitoes worldwide and is vertically transmitted. Wolbachia-transinfected mosquitoes are currently being used in biocontrol, as they effectively block transmission of several positive-sense RNA viruses in mosquitoes. Our results demonstrate that Wolbachia enhances the replication of AeAV and modestly reduces dengue virus replication in a cell line model. This study expands our understanding of the virome in A. aegypti as well as providing insight into the complexity of the Wolbachia virus restriction phenotype.

Proteomic Analysis of Cytoskeleton Proteins in Fish.

In this chapter, we describe laboratory protocols for rearing fish and a simple and efficient method of extracting and identifying pathogen and host proteins that may be involved in entry and replication of commercially important fish viruses. We have used the common carp (Cyprinus carpio L.) and goldfish (Cyprinus auratus) as a model system for studies of proteins involved in viral entry and replication. The chapter describes detailed protocols for maintenance of carp, cell culture, antibody purification of proteins, and use of electrospray-ionization mass spectrometry analysis to screen and identify cytoskeleton and other proteins that may be involved in viral infection and propagation in fish.

Reverse genetics of Mononegavirales: How they work, new vaccines, and new cancer therapeutics.

The order Mononegavirales includes five families: Bornaviridae, Filoviridae, Nyamaviridae, Paramyxoviridae, and Rhabdoviridae. The genome of these viruses is one molecule of negative-sense single strand RNA coding for five to ten genes in a conserved order. The RNA is not infectious until packaged by the nucleocapsid protein and transcribed by the polymerase and co-factors. Reverse genetics approaches have answered fundamental questions about the biology of Mononegavirales. The lack of icosahedral symmetry and modular organization in the genome of these viruses has facilitated engineering of viruses expressing fluorescent proteins, and these fluorescent proteins have provided important insights about the molecular and cellular basis of tissue tropism and pathogenesis. Studies have assessed the relevance for virulence of different receptors and the interactions with cellular proteins governing the innate immune responses. Research has also analyzed the mechanisms of attenuation. Based on these findings, ongoing clinical trials are exploring new live attenuated vaccines and the use of viruses re-engineered as cancer therapeutics.

Development and application of reporter-expressing mononegaviruses: current challenges and perspectives.

Reverse genetics allows the generation of recombinant viruses entirely from cDNA. One application of this technology is the creation of reporter-expressing viruses, which greatly increase the detail and ease with which these viruses can be studied. However, there are a number of challenges when working with reporter-expressing viruses. Both the reporter protein itself as well as the genetic manipulations within the viral genome required for expression of this reporter can result in altered biological properties of the recombinant virus, and lead to attenuation in vitro and/or in vivo. Further, instability of reporter expression and purging of the genetic information encoding for the reporter from the viral genome can be an issue. Finally, a practical challenge for in vivo studies lies in the attenuation of light signals when traversing tissues. Novel expression strategies and the continued development of brighter, red and far-red shifted reporters and the increased use of bioluminescent reporters for in vivo applications promise to overcome some of these limitations in future. However, a "one size fits all" approach to the design of reporter-expressing viruses has thus far not been possible. Rather, a reporter suited to the intended application must be selected and an appropriate expression strategy and location for the reporter in the viral genome chosen. Still, attenuating effects of the reporter on viral fitness are difficult to predict and have to be carefully assessed with respect to the intended application. Despite these limitations the generation of suitable reporter-expressing viruses will become more common as technology and our understanding of the intricacies of viral gene expression and regulation improves, allowing deeper insight into virus biology both in living cells and in animals.

A fusion-inhibiting peptide against Rift Valley fever virus inhibits multiple, diverse viruses.

For enveloped viruses, fusion of the viral envelope with a cellular membrane is critical for a productive infection to occur. This fusion process is mediated by at least three classes of fusion proteins (Class I, II, and III) based on the protein sequence and structure. For Rift Valley fever virus (RVFV), the glycoprotein Gc (Class II fusion protein) mediates this fusion event following entry into the endocytic pathway, allowing the viral genome access to the cell cytoplasm. Here, we show that peptides analogous to the RVFV Gc stem region inhibited RVFV infectivity in cell culture by inhibiting the fusion process. Further, we show that infectivity can be inhibited for diverse, unrelated RNA viruses that have Class I (Ebola virus), Class II (Andes virus), or Class III (vesicular stomatitis virus) fusion proteins using this single peptide. Our findings are consistent with an inhibition mechanism similar to that proposed for stem peptide fusion inhibitors of dengue virus in which the RVFV inhibitory peptide first binds to both the virion and cell membranes, allowing it to traffic with the virus into the endocytic pathway. Upon acidification and rearrangement of Gc, the peptide is then able to specifically bind to Gc and prevent fusion of the viral and endocytic membranes, thus inhibiting viral infection. These results could provide novel insights into conserved features among the three classes of viral fusion proteins and offer direction for the future development of broadly active fusion inhibitors.

Unravelling the complexities of respiratory syncytial virus RNA synthesis.

Human respiratory syncytial virus (RSV) is the leading cause of paediatric respiratory disease and is the focus of antiviral- and vaccine-development programmes. These goals have been aided by an understanding of the virus genome architecture and the mechanisms by which it is expressed and replicated. RSV is a member of the order Mononegavirales and, as such, has a genome consisting of a single strand of negative-sense RNA. At first glance, transcription and genome replication appear straightforward, requiring self-contained promoter regions at the 3' ends of the genome and antigenome RNAs, short cis-acting elements flanking each of the genes and one polymerase. However, from these minimal elements, the virus is able to generate an array of capped, methylated and polyadenylated mRNAs and encapsidated antigenome and genome RNAs, all in the appropriate ratios to facilitate virus replication. The apparent simplicity of genome expression and replication is a consequence of considerable complexity in the polymerase structure and its cognate cis-acting sequences; here, our understanding of mechanisms by which the RSV polymerase proteins interact with signals in the RNA template to produce different RNA products is reviewed.

Transcription and replication of nonsegmented negative-strand RNA viruses.

The nonsegmented negative-strand (NNS) RNA viruses of the order Mononegavirales include a wide variety of human, animal, and plant pathogens. The NNS RNA genomes of these viruses are templates for two distinct RNA synthetic processes: transcription to generate mRNAs and replication of the genome via production of a positive-sense antigenome that acts as template to generate progeny negative-strand genomes. The four virus families within the Mononegavirales all express the information encoded in their genomes by transcription of discrete subgenomic mRNAs. The key feature of transcriptional control in the NNS RNA viruses is entry of the virus-encoded RNA-dependent RNA polymerase at a single 3' proximal site followed by obligatory sequential transcription of the linear array of genes. Levels of gene expression are primarily regulated by position of each gene relative to the single promoter and also by cis-acting sequences located at the beginning and end of each gene and at the intergenic junctions. Obligatory sequential transcription dictates that termination of each upstream gene is required for initiation of downstream genes. Therefore, termination is a means to regulate expression of individual genes within the framework of a single transcriptional promoter. By engineering either whole virus genomes or subgenomic replicon derivatives, elements important for signaling transcript initiation, 5' end modification, 3' end polyadenylation, and transcription termination have been identified. Although the diverse families of NNS RNA virus use different sequences to control these processes, transcriptional termination is a common theme in controlling gene expression and overall transcriptional regulation is key in controlling the outcome of viral infection. The latest models for control of replication and transcription are discussed.

Risk of spread of penaeid shrimp viruses in the Americas by the international movement of live and frozen shrimp.

Within the past decade, viral diseases have emerged as serious economic impediments to successful shrimp farming in many of the shrimp-farming countries of the world. In the western hemisphere, the viral agents of Taura syndrome (TS) and infectious hypodermal and haematopoietic necrosis have caused serious disease epizootics throughout the shrimp-growing regions of the Americas and Hawaii, while in Asia the viral agents of white spot syndrome (WSS) and yellow head (YH) have caused pandemics with catastrophic losses. The international transfer of live shrimp for aquaculture purposes is an obvious mechanism by which the viruses have spread within and between regions in which they have occurred. Shrimp-eating gulls, other seabirds and aquatic insects may also be factors in the spread of shrimp viruses between and within regions. Another potentially important mechanism for the international spread of these pathogens is the trade in frozen commodity shrimp, which may contain viruses exotic to the importing countries. The viral agents of WSS, YH and TS have been found, and demonstrated to be infectious, in frozen shrimp imported into the United States market. Mechanisms identified for the potential transfer of virus in imported frozen products to domestic populations of cultured or wild penaeid shrimp stocks include: the release of untreated liquid or solid wastes from shrimp importing and processing plants directly into coastal waters, improper disposal of solid waste from shrimp importing and processing plants in landfills so that the waste is accessible to gulls and other seabirds, and the use of imported shrimp as bait by sports fishermen.