We describe the optimisation of a simplified sample preparation method which permits rapid and direct detection of SARS-CoV-2 RNA within saliva, using reverse-transcription loop-mediated isothermal amplification (RT-LAMP). Treatment of saliva samples prior to RT-LAMP by dilution 1:1 in Mucolyse^TM, followed by dilution in 10 % (w/v) Chelex© 100 Resin and a 98 °C heat step for 2 min enabled detection of SARS-CoV-2 RNA in positive saliva samples. Using RT-LAMP, SARS-CoV-2 RNA was detected in as little as 05:43 min, with no amplification detected in 3097 real-time reverse transcription PCR (rRT-PCR) negative saliva samples from staff tested within a service evaluation study, or for other respiratory pathogens tested (n = 22). Saliva samples can be collected non-invasively, without the need for skilled staff and can be obtained from both healthcare and home settings. Critically, this approach overcomes the requirement for, and validation of, different swabs and the global bottleneck in obtaining access to extraction robots and reagents to enable molecular testing by rRT-PCR. Such testing opens the possibility of public health approaches for effective intervention during the COVID-19 pandemic through regular SARS-CoV-2 testing at a population scale, combined with isolation and contact tracing.

Peste des petits ruminants (PPR) is a transboundary viral disease that threatens more than 1.74 billion goats and sheep in approximately 70 countries globally. In 2015, the international community set the goal of eradicating PPR by 2030, and, since then, Food and Agriculture Organization of the United Nations (FAO) and World Organization for Animal Health (OIE) have jointly developed and implemented the Global Control and Eradication Strategy for PPR. Here, data from the United Nations Food and Agriculture Organization Statistical Database (FAOSTAT), the OIE World Animal Health Information System (WAHIS), Regional Roadmap Meetings, and countries’ responses to PPR Monitoring and Assessment Tool (PMAT) questionnaires were analyzed to inform on current progress towards PPR eradication. OIE recorded the use of over 333 million doses of vaccine in 12 countries from 2015 to 2018, 41.8% of which were used in Asia and 58.2% in Africa. Between 2015 and 2019, a total of 12,757 PPR outbreaks were reported to OIE: 75.1% in Asia, 24.8% in Africa, and 0.1% in Europe. The number of global outbreaks in 2019 fell to 1218, compared with 3688 in 2015. Analysis of vaccine use and PPR outbreaks in countries indicates that disease control strategies, particularly vaccination campaigns and vaccine distribution strategies, still require scientific evaluation. It is imperative that vaccination is undertaken based on the epidemiology of the disease in a region and is coordinated between neighboring countries to restrict transboundary movements. Strengthening surveillance and post-vaccination sero-monitoring at the national level is also essential. The PPR vaccine stock/bank established by FAO, OIE, and other partners have improved the quality assurance and supply of vaccines. However, to achieve PPR eradication, filling the funding gap for vaccination campaigns and other program activities will be critical.

While wing form is known to differ between males and females of the genus Culicoides (Latreille, 1809), detailed studies of sexual dimorphism are lacking. In this study we analyze sex-specific differences in the wing form of five species of the subgenus Avaritia (Fox, 1955) using geometric morphometrics and comparative phylogenetic methods. Our results confirm the existence of marked sexual dimorphism in the wing form of the studied species and reveal for the first time that while there is a shared general pattern of sexual shape dimorphism within the subgenus, sexual size dimorphism and particular features of sexual shape dimorphism differ among species. Sexual shape dimorphism was found to be poorly associated to size and the evolutionary history of the species. The tight association of sexual shape dimorphism with aspect ratio suggests that the shape of the wing is optimized for the type of flight of each sex, i.e. dispersal flight in females vs aerobatic flight in males. Moreover, the fact that interspecific shape differences are greater and more strongly associated to aspect ratio in males than in females might be indicating that in males the selective pressures affecting flight performance characteristics are more heterogeneous and/or stronger than in females among the studied species. 

Genetic pest management (GPM) methods involve releasing modified versions of a pest species to mate with wild pests in the target area. Proposed for a wide range of applications in public health, agriculture and conservation, most progress has been made with pest insects. Offspring of the released modified insects and wild pests carry the modification-which might be transgenes, artificially introduced Wolbachia or genetic damage from radiation, for example-but they also carry a complete haploid genome from their laboratory-reared parent, as well as one from their wild parent. Unless these F₁ hybrids are completely unable to reproduce, further mating will lead to introgression of DNA sequences from the release strain into the wild population. We discuss issues around strain selection and the potential consequences of such introgression. We conclude that such introgression is probably harmless in almost all circumstances, and could, in theory, provide specific additional benefits to the release programme. We outline population monitoring approaches that could be used, going forward, to determine how background genetics may affect GPM.

This article is part of the theme issue 'Novel control strategies for mosquito-borne diseases'.

The insect sex determination and the intimately linked dosage compensation pathways represent a challenging evolutionary puzzle that has been solved only in Drosophila melanogaster. Analyses of orthologs of the Drosophila genes identified in non-drosophilid taxa^1,2  revealed that evolution of sex determination pathways is consistent with a bottom-up mode³, where only the terminal genes within the pathway are well conserved. doublesex (dsx), occupying a bottom-most position and encoding sex-specific proteins orchestrating downstream sexual differentiation processes, is an ancient sex-determining gene present in all studied species^2,4,5. With the exception of lepidopterans, its female-specific splicing is known to be regulated by transformer (tra) and its co-factor transformer-2 (tra2)^6-20. Here we show that in the African malaria mosquito Anopheles gambiae, a gene, which likely arose in the Anopheles lineage and which we call femaleless (fle), controls sex determination in females by regulating splicing of dsx and fruitless (fru; another terminal gene within a branch of the sex determination pathway). Moreover, fle represents a novel molecular link between the sex determination and dosage compensation pathways. It is necessary to suppress activation of dosage compensation in females, as demonstrated by the significant upregulation of the female X chromosome genes and a correlated female-specific lethality, but no negative effect on males, in response to fle knockdown. This unexpected property, combined with a high level of conservation in sequence and function in anopheline mosquitoes, makes fle an excellent target for genetic control of all major vectors of human malaria.

Multiple national and international trends and drivers are radically changing what biological security means for the United Kingdom (UK). New technologies present novel opportunities and challenges, and globalisation has created new pathways and increased the speed, volume and routes by which organisms can spread. The UK Biological Security Strategy (2018) acknowledges the importance of research on biological security in the UK. Given the breadth of potential research, a targeted agenda identifying the questions most critical to effective and coordinated progress in different disciplines of biological security is required. We used expert elicitation to generate 80 policy-relevant research questions considered by participants to have the greatest impact on UK biological security. Drawing on a collaboratively-developed set of 450 questions, proposed by 41 experts from academia, industry and the UK government (consulting 168 additional experts) we subdivided the final 80 questions into six categories: bioengineering; communication and behaviour; disease threats (including pandemics); governance and policy; invasive alien species; and securing biological materials and securing against misuse. Initially, the questions were ranked through a voting process and then reduced and refined to 80 during a one-day workshop with 35 participants from a variety of disciplines. Consistently emerging themes included: the nature of current and potential biological security threats, the efficacy of existing management actions, and the most appropriate future options. The resulting questions offer a research agenda for biological security in the UK that can assist the targeting of research resources and inform the implementation of the UK Biological Security Strategy. These questions include research that could aid with the mitigation of Covid-19, and preparation for the next pandemic. We hope that our structured and rigorous approach to creating a biological security research agenda will be replicated in other countries and regions. The world, not just the UK, is in need of a thoughtful approach to directing biological security research to tackle the emerging issues.

Global vaccination programs using live-attenuated oral and inactivated polio vaccine (OPV and IPV) have almost eradicated poliovirus (PV) but these vaccines or their production pose significant risk in a polio-free world. Recombinant PV virus-like particles (VLPs), lacking the viral genome, represent safe next-generation vaccines, however their production requires optimisation. Here we present an efficient mammalian expression strategy producing good yields of wild-type PV VLPs for all three serotypes and a thermostabilised variant for PV3. Whilst the wild-type VLPs were predominantly in the non-native C-antigenic form, the thermostabilised PV3 VLPs adopted the native D-antigenic conformation eliciting neutralising antibody titres equivalent to the current IPV and were indistinguishable from natural empty particles by cryo-electron microscopy with a similar stabilising lipidic pocket-factor in the VP1 β-barrel. This factor may not be available in alternative expression systems, which may require synthetic pocket-binding factors. VLPs equivalent to these mammalian expressed thermostabilized particles, represent safer non-infectious vaccine candidates for the post-eradication era.

Avian pathogenic Escherichia coli (APEC) cause colibacillosis in birds, a syndrome of severe respiratory and systemic disease that constitutes a major threat due to early mortality, condemnation of carcasses and reduced productivity. APEC can infect different types of birds in all commercial settings, and birds of all ages although disease tends to be more severe in younger birds likely a consequence of an immature immune system. APEC can act as both primary and secondary pathogens, with predisposing factors for secondary infections including poor housing conditions, respiratory viral and Mycoplasma spp. infections or vaccinations. Controlled studies with APEC as a primary pathogen have been used to study the bird's immune response to APEC, although it may not always be representative of natural infections which may be more complex due to the presence of secondary agents, stress and environmental factors. Under controlled experimental conditions, a strong early innate immune response is induced which includes host defence peptides in mucus and a cellular response driven by heterophils and macrophages. Both antibody and T-cell mediated adaptive responses have been demonstrated after vaccination. In this review we will discuss the bird's immune response to APEC as primary pathogen with a bias towards the innate immune response as mechanistic adaptive studies clearly form a much more limited body of work despite numerous vaccine trials.

African swine fever virus (ASFV) is a highly contagious pathogen which causes a lethal haemorrhagic fever in domestic pigs and wild boar. The large, double-stranded DNA virus replicates in perinuclear cytoplasmic replication sites known as viral factories. These factories are complex, multi-dimensional structures. Here we investigated the protein and membrane compartments of the factory using super-resolution and electron tomography. Click IT chemistry in combination with stimulated emission depletion (STED) microscopy revealed a reticular network of newly synthesized viral proteins, including the structural proteins p54 and p34, previously seen as a pleomorphic ribbon by confocal microscopy. Electron microscopy and tomography confirmed that this network is an accumulation of membrane assembly intermediates which take several forms. At early time points in the factory formation, these intermediates present as small, individual membrane fragments which appear to grow and link together, in a continuous progression towards new, icosahedral virions. It remains unknown how these membranes form and how they traffic to the factory during virus morphogenesis.