BACKGROUND:
Culicoides biting midges (Diptera: Ceratopogonidae) are biological vectors of internationally important arboviruses and inflict biting nuisance on humans, companion animals and livestock. In temperate regions, transmission of arboviruses is limited by temperature thresholds, in both replication and dissemination of arboviruses within the vector and in the flight activity of adult Culicoides. This study aims to determine the cold-temperature thresholds for flight activity of Culicoides from the UK under laboratory conditions.

METHODS:
Over 18,000 Culicoides adults were collected from the field using 4 W down-draught miniature ultraviolet Centers for Disease Control traps. Populations of Culicoides were sampled at three different geographical locations within the UK during the summer months and again in the autumn at one geographical location. Activity at constant temperatures was assessed using a bioassay that detected movement of adult Culicoides towards an ultraviolet light source over a 24-h period.

RESULTS:
The proportion of active adult Culicoides increased with temperature but cold temperature thresholds for activity varied significantly according to collection season and location. Populations dominated by the subgenus Avaritia collected in South East England had a lower activity threshold temperature in the autumn (4 °C) compared with populations collected in the summer (10 °C). Within the subgenus Avaritia, Culicoides scoticus was significantly more active across all temperatures tested than Culicoides obsoletus within the experimental setup. Populations of Culicoides impunctatus collected in the North East of England were only active once temperatures reached 14 °C. Preliminary data suggested flight activity of the subgenus Avaritia does not differ between populations in South East England and those in the Scottish Borders.

CONCLUSIONS:
These findings demonstrate seasonal changes in temperature thresholds for flight and across different populations of Culicoides. These data, alongside that defining thresholds for virus replication within Culicoides, provide a primary tool for risk assessment of arbovirus transmission in temperate regions. In addition, the study also provides a comparison with thermal limits derived directly from light-suction trapping data, which is currently used as the main method to define adult Culicoides activity during surveillance.

Peste des petits ruminants (PPR) is a severe disease of goats and sheep that is widespread in Africa, the Middle East and Asia. The disease is caused by peste des petits ruminants virus (PPRV); cell culture‐attenuated strains of PPRV have been shown, both experimentally and by extensive use in the field, to be effective vaccines and are widely used. We have previously demonstrated that these vaccines elicit both serological (PPRV‐specific antibody) and cell‐based (PPRV‐specific CD4⁺ and CD8⁺ T cells) immune responses. However, it is not known which of these responses are required for protection from PPRV, information that would be useful in the evaluation of new vaccines that are being developed to provide the capability to differentiate infected and vaccinated animals (DIVA capability). To begin to address this issue, we have used a complement‐fixing monoclonal antibody recognizing caprine CD8 to deplete >99.9% of circulating CD8⁺ T cells from vaccinated goats. Animals were then infected with wild‐type PPRV. Despite the absence of the CD8⁺ T‐cell component of the vaccine‐induced immune response, the vaccinated animals were almost fully protected, showing no pyrexia or viraemia, and almost no clinical signs. These data suggest that a virus‐specific CD8⁺ T‐cell response is not critical for protection against PPRV and that virus‐specific antibody and/or CD4⁺ T cells are the main mediators of protection. We have also shown that the leucopenia caused by infection with wild‐type PPRV affects all major classes of circulating leucocytes.

Dengue virus (DENV) is the most prevalent mosquito-borne virus causing human disease. Of the 4 DENV serotypes, epidemiological data suggest that DENV-2 secondary infections are associated with more severe disease than DENV-4 infections. Mass cytometry by time-of-flight (CyTOF) was used to dissect immune changes induced by DENV-2 and DENV-4 in human DCs, the initial targets of primary infections that likely affect infection outcomes. Strikingly, DENV-4 replication peaked earlier and promoted stronger innate immune responses, with increased expression of DC activation and migration markers and increased cytokine production, compared with DENV-2. In addition, infected DCs produced higher levels of inflammatory cytokines compared with bystander DCs, which mainly produced IFN-induced cytokines. These high-dimensional analyses during DENV-2 and DENV-4 infections revealed distinct viral signatures marked by different replication strategies and antiviral innate immune induction in DCs, which may result in different viral fitness, transmission, and pathogenesis.

RNA interference (RNAi) controls arbovirus infections in mosquitoes. Two different RNAi pathways are involved in antiviral responses: the PIWI-interacting RNA (piRNA) and exogenous short interfering RNA (exo-siRNA) pathways, which are characterized by the production of virus-derived small RNAs of 25-29 and 21 nucleotides, respectively. The exo-siRNA pathway is considered to be the key mosquito antiviral response mechanism. In Aedes aegypti-derived cells, Zika virus (ZIKV)-specific siRNAs were produced and loaded into the exo-siRNA pathway effector protein Argonaute 2 (Ago2); although the knockdown of Ago2 did not enhance virus replication. Enhanced ZIKV replication was observed in a Dcr2-knockout cell line suggesting that the exo-siRNA pathway is implicated in the antiviral response. Although ZIKV-specific piRNA-sized small RNAs were detected, these lacked the characteristic piRNA ping-pong signature motif and were bound to Ago3 but not Piwi5 or Piwi6. Silencing of PIWI proteins indicated that the knockdown of Ago3, Piwi5 or Piwi6 did not enhance ZIKV replication and only Piwi4 displayed antiviral activity. We also report that the expression of ZIKV capsid (C) protein amplified the replication of a reporter alphavirus; although, unlike yellow fever virus C protein, it does not inhibit the exo-siRNA pathway. Our findings elucidate ZIKV-mosquito RNAi interactions that are important for understanding its spread.

The mechanism by which herpesviruses acquire their tegument is not yet clear. One model is that outer tegument proteins are recruited by the cytoplasmic tails of viral glycoproteins. In the case of herpes simplex virus tegument protein VP22, interactions with the glycoproteins gE and gD have been shown. We have previously shown that the C-terminal half of VP22 contains the necessary signal for assembly into the virus. Here, we show that during infection VP22 interacts with gE and gM, as well as its tegument partner VP16. However, by using a range of techniques we were unable to demonstrate VP22 binding to gD. By using pulldown assays, we show that while the cytoplasmic tails of both gE and gM interact with VP22, only gE interacts efficiently with the C-terminal packaging domain of VP22. Furthermore, gE but not gM can recruit VP22 to the Golgi/trans-Golgi network region of the cell in the absence of other virus proteins. To examine the role of the gE-VP22 interaction in infection, we constructed a recombinant virus expressing a mutant VP22 protein with a 14-residue deletion that is unable to bind gE (ΔgEbind). Coimmunoprecipitation assays confirmed that this variant of VP22 was unable to complex with gE. Moreover, VP22 was no longer recruited to its characteristic cytoplasmic trafficking complexes but exhibited a diffuse localization. Importantly, packaging of this variant into virions was abrogated. The mutant virus exhibited poor growth in epithelial cells, similar to the defect we have observed for a VP22 knockout virus. These results suggest that deletion of just 14 residues from the VP22 protein is sufficient to inhibit binding to gE and hence recruitment to the viral envelope and assembly into the virus, resulting in a growth phenotype equivalent to that produced by deleting the entire reading frame.

Although the herpes simplex virus type 1 (HSV-1) tegument is comprised of a large number of viral and cellular proteins, how and where in the cell these proteins are recruited into the virus structure is poorly understood. We have shown previously that the immediate-early gene product ICP0 is packaged by a mechanism dependent on the major tegument protein VP22, while others have shown a requirement for ICP27. We now extend our studies to show that ICP0 packaging correlates directly with the ability of ICP0 to complex with VP22 in infected cells. ICP27 is not, however, present in this VP22-ICP0 complex but is packaged into the virion in a VP22- and ICP0-independent manner. Biochemical fractionation of virions indicated that ICP0 associates tightly with the virus capsid, but intranuclear capsids contained no detectable ICP0. The RING finger domain of ICP0 and the N terminus of VP22 were both shown to be essential but not sufficient for ICP0 packaging and complex formation. Strikingly, however, the N-terminal region of VP22, while unable to form a complex with ICP0, inhibited its translocation from the nucleus to the cytoplasm. PML degradation by ICP0 was efficient in cells infected with this VP22 mutant virus, confirming that ICP0 retains activity. Hence, we would suggest that VP22 is an important molecular partner of ICP0 that controls at least one of its activities: its assembly into the virion. Moreover, we propose that the pathway by which VP22 recruits ICP0 to the virion may begin in the nucleus prior to ICP0 translocation to its final site of assembly in the cytoplasm.

A large proportion of the genome of most eukaryotic organisms consists of highly repetitive mobile genetic elements. The sum of these elements is called the "mobilome," which in eukaryotes is made up mostly of transposons. Transposable elements contribute to disease, evolution, and normal physiology by mediating genetic rearrangement, and through the "domestication" of transposon proteins for cellular functions. Although 'omics studies of mobilome genomes and transcriptomes are common, technical challenges have hampered high-throughput global proteomics analyses of transposons. In a recent paper, we overcame these technical hurdles using a technique called "proteomics informed by transcriptomics" (PIT), and thus published the first unbiased global mobilome-derived proteome for any organism (using cell lines derived from the mosquito Aedes aegypti). In this commentary, we describe our methods in more detail, and summarise our major findings. We also use new genome sequencing data to show that, in many cases, the specific genomic element expressing a given protein can be identified using PIT. This proteomic technique therefore represents an important technological advance that will open new avenues of research into the role that proteins derived from transposons and other repetitive and sequence diverse genetic elements, such as endogenous retroviruses, play in health and disease.

BACKGROUND: Aedes aegypti is a vector for the (re-)emerging human pathogens dengue, chikungunya, yellow fever and Zika viruses. Almost half of the Ae. aegypti genome is comprised of transposable elements (TEs). Transposons have been linked to diverse cellular processes, including the establishment of viral persistence in insects, an essential step in the transmission of vector-borne viruses. However, up until now it has not been possible to study the overall proteome derived from an organism's mobile genetic elements, partly due to the highly divergent nature of TEs. Furthermore, as for many non-model organisms, incomplete genome annotation has hampered proteomic studies on Ae. aegypti.

RESULTS: We analysed the Ae. aegypti proteome using our new proteomics informed by transcriptomics (PIT) technique, which bypasses the need for genome annotation by identifying proteins through matched transcriptomic (rather than genomic) data. Our data vastly increase the number of experimentally confirmed Ae. aegypti proteins. The PIT analysis also identified hotspots of incomplete genome annotation, and showed that poor sequence and assembly quality do not explain all annotation gaps. Finally, in a proof-of-principle study, we developed criteria for the characterisation of proteomically active TEs. Protein expression did not correlate with a TE's genomic abundance at different levels of classification. Most notably, long terminal repeat (LTR) retrotransposons were markedly enriched compared to other elements. PIT was superior to 'conventional' proteomic approaches in both our transposon and genome annotation analyses.

CONCLUSIONS: We present the first proteomic characterisation of an organism's repertoire of mobile genetic elements, which will open new avenues of research into the function of transposon proteins in health and disease. Furthermore, our study provides a proof-of-concept that PIT can be used to evaluate a genome's annotation to guide annotation efforts which has the potential to improve the efficiency of annotation projects in non-model organisms. PIT therefore represents a valuable new tool to study the biology of the important vector species Ae. aegypti, including its role in transmitting emerging viruses of global public health concern.

The global deployment of an effective and safe vaccine is currently a public health priority to curtail the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we evaluated a Newcastle disease virus (NDV)-based intranasal vectored-vaccine in mice and hamsters for its immunogenicity, safety and protective efficacy in challenge studies with SARS-CoV-2. The recombinant (r)NDV-S vaccine expressing spike (S) protein of SARS-CoV-2 administrated via intranasal route in mice induced high levels of SARS-CoV-2-specific neutralizing immunoglobulin A (IgA) and IgG2a antibodies and T cell-mediated immunity. Hamsters vaccinated with two doses of vaccine showed complete protection from clinical disease including lung infection, inflammation, and pathological lesions after SARS-CoV-2 challenge. Importantly, a single or double dose of intranasal rNDV-S vaccine completely blocked SARS-CoV-2 shedding in nasal turbinate and lungs within 4 days of vaccine administration in hamsters. Taken together, intranasal administration of rNDV-S has the potential to control infection at the site of inoculation, which should prevent both the clinical disease and transmission to halt the spread of the COVID-19 pandemic.