Peste des petits ruminants virus (PPRV) causes a highly devastating disease, peste des petits ruminants (PPR) of sheep and goats, that threatens food security, small ruminant production, and the conservation of wild small ruminants in many developing countries, especially in Africa. Robust serological and molecular diagnostic tools are available to detect PPRV infection, but they were mainly developed for domestic sheep and goats. The presence of a wide host range for PPRV does present serological diagnostic challenges. New innovative diagnostic tools are needed to detect PPRV in atypical hosts (e.g., Camelidae, Suidae, and Bovinae), in wildlife ecosystems and in complex field situations. Interestingly, single-domain antigen binding fragments (nanobodies) derived from heavy-chain-only camelid antibodies have emerged as a new hope in the development of accurate, rapid, and cost-effective diagnostic tools in veterinary and biomedical fields that are suitable for low-income countries. The main objective of this study was to construct an immune nanobody library to retrieve PPRV-reactive nanobodies that enable the development of diagnostic and therapeutic nanobodies in the future. Here, a strategy was developed whereby an alpaca (Vicugna pacos) was immunized with a live attenuated vaccine strain (PPRV/N/75/1) to raise an affinity-matured immune response in the heavy-chain-only antibody classes. The nanobody gene repertoire was engineered in pMECS-GG phagemid, whereby a ccdB gene (encoding a lethal protein) was substituted by the nanobody gene. An immune nanobody library with approximately sixty-four million independent transformants was constructed, of which 100% contained an insert with the proper size of nanobody gene. Following phage display and biopanning, nine nanobodies that specifically recognise completely inactivated PPRV were identified on enzyme-linked immunosorbent assay. They showed superb potency in rapidly identifying PPRV, which is likely to open a new perspective in the diagnosis and possible treatment of PPR infection.

Mosquito-borne diseases, such as dengue and malaria, pose significant global health burdens. Unfortunately, current control methods based on insecticides and environmental maintenance have fallen short of eliminating the disease burden. Scalable, deployable, genetic-based solutions are sought to reduce the transmission risk of these diseases. Pathogen-blocking Wolbachia bacteria, or genome engineering-based mosquito control strategies including gene drives have been developed to address these problems, both requiring the release of modified mosquitoes into the environment. Here, we review the latest developments, notable similarities, and critical distinctions between these promising technologies and discuss their future applications for mosquito-borne disease control.

This article reports the changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses (ICTV) in March 2021. The entire ICTV was invited to vote on 290 taxonomic proposals approved by the ICTV Executive Committee at its meeting in October 2020, as well as on the proposed revision of the International Code of Virus Classification and Nomenclature (ICVCN). All proposals and the revision were ratified by an absolute majority of the ICTV members. Of note, ICTV mandated a uniform rule for virus species naming, which will follow the binomial 'genus-species' format with or without Latinized species epithets. The Study Groups are requested to convert all previously established species names to the new format. ICTV has also abolished the notion of a type species, i.e., a species chosen to serve as a name-bearing type of a virus genus. The remit of ICTV has been clarified through an official definition of 'virus' and several other types of mobile genetic elements. The ICVCN and ICTV Statutes have been amended to reflect these changes.

The immunogenicity and protective efficacy of vaccines can be enhanced by the selective delivery of antigens to the antigen-presenting cells (APCs). In this study, H9N2 avian influenza virus haemagglutinin (HA) antigen, was targeted by fusing it to single-chain fragment variable (scFv) antibodies specific to CD83 receptor expressed on chicken APCs. We observed an increased level of IFNγ, IL6, IL1β, IL4, and CxCLi2 mRNA upon stimulation of chicken splenocytes ex vivo by CD83 scFv targeted H9HA. In addition, CD83 scFv targeted H9HA induced higher serum haemagglutinin inhibition activity and virus neutralising antibodies compared to untargeted H9HA, with induction of antibodies as early as day 6 post primary vaccination. Furthermore, chickens vaccinated with CD83 scFv targeted H9HA showed reduced H9N2 challenge virus shedding compared to untargeted H9HA. These results suggest that targeting antigens to CD83 receptors could improve the efficacy of poultry vaccines.

Culex quinquefasciatus Say is a mosquito distributed in both tropical and subtropical regions of the world. It is a night-active, opportunistic blood-feeder and vectors many animal and human diseases, including West Nile Virus and avian malaria. Current vector control methods (e.g. physical/chemical) are increasingly ineffective; use of insecticides also imposes hazards to both human and ecosystem health. Advances in genome editing have allowed the development of genetic insect control methods, which are species-specific and, theoretically, highly effective. CRISPR/Cas9 is a bacteria-derived programmable gene editing tool that is functional in a range of species. We describe the first successful germline gene knock-in by homology dependent repair in C. quinquefasciatus. Using CRISPR/Cas9, we integrated an sgRNA expression cassette and marker gene encoding a fluorescent protein fluorophore (Hr5/IE1-DsRed, Cq7SK-sgRNA) into the kynurenine 3-monooxygenase (kmo) gene. We achieved a minimum transformation rate of 2.8%, similar to rates in other mosquito species. Precise knock-in at the intended locus was confirmed. Insertion homozygotes displayed a white eye phenotype in early-mid larvae and a recessive lethal phenotype by pupation. This work provides an efficient method for engineering C. quinquefasciatus, providing a new tool for developing genetic control tools for this vector.

RNA structures can form functional elements that play crucial roles in the replication of positive-sense RNA viruses. While RNA structures in the untranslated regions (UTRs) of several picornaviruses have been functionally characterized, the roles of putative RNA structures predicted for protein coding sequences (or open reading frames [ORFs]) remain largely undefined. Here, we have undertaken a bioinformatic analysis of the foot-and-mouth disease virus (FMDV) genome to predict 53 conserved RNA structures within the ORF. Forty-six of these structures were located in the regions encoding the nonstructural proteins (nsps). To investigate whether structures located in the regions encoding the nsps are required for FMDV replication, we used a mutagenesis method, CDLR mapping, where sequential coding segments were shuffled to minimize RNA secondary structures while preserving protein coding, native dinucleotide frequencies, and codon usage. To examine the impact of these changes on replicative fitness, mutated sequences were inserted into an FMDV subgenomic replicon. We found that three of the RNA structures, all at the 3' termini of the FMDV ORF, were critical for replicon replication. In contrast, disruption of the other 43 conserved RNA structures that lie within the regions encoding the nsps had no effect on replicon replication, suggesting that these structures are not required for initiating translation or replication of viral RNA. Conserved RNA structures that are not essential for virus replication could provide ideal targets for the rational attenuation of a wide range of FMDV strains.

IMPORTANCE Some RNA structures formed by the genomes of RNA viruses are critical for viral replication. Our study shows that of 46 conserved RNA structures located within the regions of the foot-and-mouth disease virus (FMDV) genome that encode the nonstructural proteins, only three are essential for replication of an FMDV subgenomic replicon. Replicon replication is dependent on RNA translation and synthesis; thus, our results suggest that the three RNA structures are critical for either initiation of viral RNA translation and/or viral RNA synthesis. Although further studies are required to identify whether the remaining 43 RNA structures have other roles in virus replication, they may provide targets for the rational large-scale attenuation of a wide range of FMDV strains. FMDV causes a highly contagious disease, posing a constant threat to global livestock industries. Such weakened FMDV strains could be investigated as live-attenuated vaccines or could enhance biosecurity of conventional inactivated vaccine production.

Foot-and-mouth disease (FMD) is a global burden on the livestock industry. The causative agent, FMD virus (FMDV), is highly infectious and exists in seven distinct serotypes. Vaccination remains the most effective control strategy in endemic regions and current FMD vaccines are made from inactivated preparations of whole virus. The inherent instability of FMDV and the emergence of new strains presents challenges to efficacious vaccine development. Currently, vaccines available in East Africa are comprised of relatively historic strains with unreported stabilities. As an initial step to produce an improved multivalent FMD vaccine we have identified naturally stable East African FMDV strains for each of the A, O, SAT1 and SAT2 serotypes and investigated their potential for protecting ruminants against strains that have recently circulated in East Africa. Interestingly, high diversity in stability between and within serotypes was observed, and in comparison to non-African A serotype viruses reported to date, the East African strains tested in this study are less stable. Candidate vaccine strains were adapted to propagation in BHK-21 cells with minimal capsid changes and used to generate vaccinate sera that effectively neutralised a panel of FMDV strains selected to improve FMD vaccines used in East Africa. This work highlights the importance of combining tools to predict and assess FMDV vaccine stability, with cell culture adaptation and serological tests in the development of FMD vaccines.

Vaccinia virus produces two types of virions known as single-membraned intracellular mature virus (MV) and double-membraned extracellular enveloped virus (EV). EV production peaks earlier when initial MV are further wrapped and secreted to spread infection within the host. However, late during infection MV accumulate intracellularly and become important for host-to-host transmission. The process that regulates this switch remains elusive and is thought to be influenced by host factors. Here we examined the hypothesis that EV and MV production are regulated by the virus through expression of F13 and the MV-specific protein A26. By switching the promoters and altering the expression kinetics of F13 and A26, we demonstrate that A26 expression downregulates EV production and plaque size, thus limiting viral spread. This process correlates with A26 association with the MV surface protein A27 and exclusion of F13, thus reducing EV titres. Thus, MV maturation is controlled by the abundance of the viral A26 protein, independently of other factors, and is rate-limiting for EV production. The A26 gene is conserved within vertebrate poxviruses, but strikingly lost in poxviruses known to be transmitted exclusively by biting arthropods. A26-mediated virus maturation thus has the appearance to be an ancient evolutionary adaptation to enhance transmission of poxviruses that has subsequently been lost from vector-adapted species, for which it may serve as a genetic signature. The existence of virus-regulated mechanisms to produce virions adapted to fulfil different functions represents a novel level of complexity in mammalian viruses with major impact on evolution, adaptation and transmission. IMPORTANCE Chordopoxviruses are mammalian viruses that uniquely produce a first type of virion adapted to spread within the host and a second type that enhances transmission between hosts, which can take place by multiple ways including direct contact, respiratory droplets, oral/fecal routes, or via vectors. Both virion types are important to balance intra-host dissemination and inter-host transmission, so virus maturation pathways must be tightly controlled. Here we provide evidence that the abundance and kinetics of expression of the viral protein A26 regulates this process by preventing formation of the first form and shifting maturation towards the second form. A26 is expressed late after the initial wave of progeny virions is produced, so sufficient viral dissemination is ensured, and provides virions with enhanced environmental stability. Conservation of A26 in all vertebrate poxviruses but those transmitted exclusively via biting arthropods reveals the importance of A26-controlled virus maturation for transmission routes involving environmental exposure.

Improving the immunogenicity and protective efficacy of vaccines is critical to reducing disease impacts. One strategy used to enhance the immunogenicity of vaccines is the selective delivery of protective antigens to the antigen presenting cells (APCs). In this study, we have developed a targeted antigen delivery vaccine (TADV) system by recombinantly fusing the ectodomain of hemagglutinin (HA) antigen of H9N2 influenza A virus to single chain fragment variable (scFv) antibodies specific for the receptors expressed on chicken APCs; Dec205 and CD11c. Vaccination of chickens with TADV containing recombinant H9HA Foldon-Dec205 scFv or H9HA Foldon-CD11c scFv proteins elicited faster (as early as day 6 post primary vaccination) and higher anti-H9HA IgM and IgY, haemagglutination inhibition, and virus neutralisation antibodies compared to the untargeted H9HA protein. Comparatively, CD11c scFv conjugated H9HA protein showed higher immunogenic potency compared to Dec205 scFv conjugated H9HA protein. The higher immune potentiating ability of CD11c scFv was also reflected in ex-vivo chicken splenocyte stimulation assay, whereby H9HA Foldon-CD11c scFv induced higher levels of cytokines (IFNγ, IL6, IL1β, and IL4) compared to H9HA Foldon-Dec205 scFv. Overall, the results conclude that TADV could be a better alternative to the currently available inactivated virus vaccines.