Knowns and unknowns of TiLV-associated neuronal disease.

Tilapia Lake Virus (TiLV) is associated with pathological changes in the brain of infected fish, but the mechanisms driving the virus's neuropathogenesis remain poorly characterized. TiLV establishes a persistent infection in the brain of infected fish even when the virus is no longer detectable in the peripheral organs, rendering therapeutic interventions and disease management challenging. Moreover, the persistence of the virus in the brain may pose a risk for viral reinfection and spread and contribute to ongoing tissue damage and neuroinflammatory processes. In this review, we explore TiLV-associated neurological disease. We discuss the possible mechanism(s) used by TiLV to enter the central nervous system (CNS) and examine TiLV-induced neuroinflammation and brain immune responses. Lastly, we discuss future research questions and knowledge gaps to be addressed to significantly advance this field.

Development of Virus-like Particle Plant-Based Vaccines against Avian H5 and H9 Influenza A Viruses.

Avian influenza A virus (AIV) is a significant cause of mortality in poultry, causing substantial economic loss, particularly in developing countries, and has zoonotic potential. For example, highly pathogenic avian influenza (HPAI) viruses of the H5 subtype have been circulating in Egypt for around two decades. In the last decade, H5N1 viruses of clade 2.2.1 have been succeeded by the antigenically distinct H5N8 clade 2.3.4.4b viruses. Furthermore, H9N2 viruses co-circulate with the H5N8 viruses in Egyptian poultry. It is widely recognised that effective vaccination against IAV requires a close antigenic match between the vaccine and viruses circulating in the field. Therefore, approaches to develop cost-effective vaccines that can be rapidly adapted to local virus strains are required for developing countries such as Egypt. In this project, the haemagglutinin (HA) proteins of Egyptian H5 and H9 viruses were expressed by transient transfection of plants (Nicotiana benthamiana). The formation of virus-like particles (VLPs) was confirmed by transmission electron microscopy. Mice were immunised with four doses of either H5 or H9 VLPs with adjuvant. Antibody and cellular immune responses were measured against the corresponding recombinant protein using ELISA and enzyme-linked immunosorbent assay (ELISpot), respectively. Chickens were immunised with one dose of H5 VLPs, eliciting HA-specific antibodies measured by ELISA and a pseudotyped virus neutralisation test using a heterologous H5 HA. In conclusion, plant-based VLP vaccines have potential for producing an effective vaccine candidate within a short time at a relatively low cost.

Combined biolistic and cell penetrating peptide delivery for the development of scalable intradermal DNA vaccines.

Physical-based gene delivery via biolistic methods (such as the Helios gene gun) involve precipitation of nucleic acids onto microparticles and direct transfection through cell membranes of exposed tissue (e.g. skin) by high velocity acceleration. The glycosaminoglycan (GAG)-binding enhanced transduction (GET) system exploits novel fusion peptides consisting of cell-binding, nucleic acid condensing, and cell-penetrating domains, which enable enhanced transfection across multiple cell types. In this study, we combined chemical (GET) and physical (gene gun) DNA delivery systems, and hypothesized the combination would generate enhanced distribution and effective uptake in cells not initially transfected by biolistic penetration. Physicochemical characterization, optimization of bullet contents and transfection experiments in vitro in cell monolayers and engineered tissue demonstrated these formulations transfected efficiently, including DC2.4 dendritic cells. We incorporated these formulations into a biolistic format for gene gun by forming fireable dry bullets obtained via lyophilization (freeze drying). This system is simple and with enhanced scalability compared to conventional methods to generate bullets. Flushed GET bullet contents retained their ability to mediate transfection (17-fold greater and 13-fold greater reporter gene expression than standard spermidine bullets in the absence and presence of serum, respectively). Fired GET bullets in vitro (in cells and collagen gels) and in vivo (mice) showed increased reporter gene transfection compared to untreated controls, whilst maintaining cell viability in vitro and having no obvious toxicity in vivo. Lastly, a SARS-CoV-2 plasmid DNA vaccine with spike (S) protein-receptor binding domain (S-RBD) was delivered by gene gun using GET bullets. Specific T cell and antibody responses comparable to the conventional system were generated. The non-physical and physical combination of GET­gold-DNA carriers using gene gun shows potential as an alternative DNA delivery method that is scalable for mass deployable vaccination and intradermal gene delivery.

Systematic Review of Equine Influenza A Virus Vaccine Studies and Meta-Analysis of Vaccine Efficacy.

Vaccines against equine influenza have been available since the late 1960s, but outbreaks continue to occur periodically, affecting both vaccinated and unvaccinated animals. The aim of this study was to systematically evaluate the efficacy of vaccines against influenza A virus in horses (equine IAV). For this, PubMed, CAB abstracts, and Web of Science were searched for controlled trials of equine IAV vaccines published up to December 2020. Forty-three articles reporting equine IAV vaccination and challenge studies in previously naïve equids using an appropriate comparison group were included in a qualitative analysis of vaccine efficacy. A value for vaccine efficacy (VE) was calculated as the percentage reduction in nasopharyngeal virus shedding detected by virus isolation in embryonated hens' eggs from 38 articles. Among 21 studies involving commercial vaccines, the mean VE was 50.03% (95% CI: 23.35-76.71%), ranging from 0 to 100%. Among 17 studies reporting the use of experimental vaccines, the mean VE was 40.37% (95% CI: 19.64-62.44), and the range was again 0-100%. Overall, complete protection from virus shedding was achieved in five studies. In conclusion, although commercially available vaccines can, in some circumstances, offer complete protection from infection, the requirement for frequent vaccination in the field to limit virus shedding and hence transmission is apparent. Although most studies were conducted by a few centres, a lack of consistent study design made comparisons difficult.

Comparative transcriptome analysis of spleen of Newcastle Disease Virus (NDV) infected chicken and Japanese quail: a potential role of NF-κß pathway activation in NDV resistance.

Newcastle disease (ND) affects a few hundred avian species including chicken and several species of domestic and wild birds. The clinical outcome of Newcastle disease virus (NDV) infection ranges from mild to severe fatal disease depending on the NDV pathotype and the host species involved. Japanese quails serve as natural reservoirs of NDV and play important role in NDV epidemiology. While infection of chicken with velogenic NDV results in severe often fatal illness, the same infection in Japanese quails results in inapparent infection. The molecular basis of this contrasting clinical outcomes of NDV infection is not yet clearly known. We compared global gene expression in spleen of chicken and Japanese quails infected with lentogenic and velogenic NDVs. We found contrasting regulation of key genes associated with NF-κB pathway and T-cell activation between chicken and Japanese quails. Our data suggests association of NDV resistance in Japanese quails to activation of NF-κB pathway and T cell proliferation. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-023-00833-y.

Mapping Polyclonal Antibody Responses to Infection Using Next-Generation Phage Display.

Peptide phage display has historically been used to epitope map monoclonal antibodies. More recently, by coupling this method with next-generation sequencing (so-called next-generation phage display, NGPD) to mass screen peptide binding events, the methodology has been successfully applied to map polyclonal antibody responses to infection. This leads to the identification of panels of mimotopes that represent the pathogen's epitopes. One potential advantage of using such an approach is that the mimotopes can represent not just linear epitopes but also conformational epitopes or those produced from post-translational modifications of proteins or from other non-protein macromolecules. The mapping of such complex immunological recognition of a pathogen can inform novel serological assay development and vaccine design. Here, we provide detailed methods for the application of NGPD to identify panels of mimotopes that are recognized specifically by antibodies from individuals with a particular infection.

Immune responses to Tilapia lake virus infection: what we know and what we don't know.

Tilapia lake virus (TiLV) is a novel contagious pathogen associated with a lethal disease affecting and decimating tilapia populations on several continents across the globe. Fish viral diseases, such as Tilapia lake virus disease (TiLVD), represent a serious threat to tilapia aquaculture. Therefore, a better understanding of the innate immune responses involved in establishing an antiviral state can help shed light on TiLV disease pathogenesis. Moreover, understanding the adaptive immune mechanisms involved in mounting protection against TiLV could greatly assist in the development of vaccination strategies aimed at controlling TiLVD. This review summarizes the current state of knowledge on the immune responses following TiLV infection. After describing the main pathological findings associated with TiLVD, both the innate and adaptive immune responses and mechanisms to TiLV infection are discussed, in both disease infection models and in vitro studies. In addition, our work, highlights research questions, knowledge gaps and research areas in the immunology of TiLV infection where further studies are needed to better understand how disease protection against TiLV is established.

Tilapia Lake Virus Vaccine Development: A Review on the Recent Advances.

Tilapia tilapinevirus (or tilapia lake virus, TiLV) is a recently emerging virus associated with a novel disease affecting and decimating tilapia populations around the world. Since its initial identification, TiLV has been reported in 17 countries, often causing mortalities as high as 90% in the affected populations. To date, no therapeutics or commercial vaccines exist for TiLV disease control. Tilapia exposed to TiLV can develop protective immunity, suggesting that vaccination is achievable. Given the important role of vaccination in fish farming, several vaccine strategies are currently being explored and put forward against TiLV but, a comprehensive overview on the efficacy of these platforms is lacking. We here present these approaches in relation with previously developed fish vaccines and discuss their efficacy, vaccine administration routes, and the various factors that can impact vaccine efficacy. The overall recent advances in TiLV vaccine development show different but promising levels of protection. The field is however hampered by the lack of knowledge of the biology of TiLV, notably the function of its genes. Further research and the incorporation of several approaches including prime-boost vaccine regimens, codon optimization, or reverse vaccinology would be beneficial to increase the effectiveness of vaccines targeting TiLV and are further discussed in this review.

Therapeutic Phage Display-Derived Single-Domain Antibodies for Pandemic Preparedness.

Driven by necessity, the COVID-19 pandemic caused by SARS-CoV-2 has accelerated the development and implementation of new vaccine platforms and other viral therapeutics. Among these is the therapeutic use of antibodies including single-domain antibodies, in particular the camelid variable heavy-chain fragment (VHH). Such therapies can provide a critical interim intervention when vaccines have not yet been developed for an emerging virus. It is evident that an increasing number of different viruses are emerging and causing epidemics and pandemics with increasing frequency. It is therefore imperative that we capitalize on the experience and knowledge gained from combatting COVID-19 to be better prepared for the next pandemic.

Seroprevalence of Equine Influenza and Its Associated Risk Factors in Northwest Nigeria.

Equine influenza (EI) is a fast-spreading respiratory disease of equids caused by equine influenza A virus (EIV), often resulting in high morbidity and a huge economic impact on the equine industry globally. In this cross-sectional study to determine the seroprevalence of EI and its associated risk factors, sera from 830 horses bled on a single occasion in Northwest Nigeria between October 2019 and January 2020 were screened for antibodies to A/equine/Richmond/1/2007 (H3N8) using the single radial haemolysis (SRH) assay. Antibodies were detected in 71.3% (592/830, 95% CI: 68−74%) of horses (SRH area ≥ 0.5 mm2). Although there were statistically significant univariable associations between seropositivity and age, sex, breed, purpose and coat colour, only age remained significant when included with each of the other variables in bivariable analyses. There was a clear trend for increasing odds of seropositivity with increasing age: OR 1.6, 95% CI: 1.05−2.40 (p = 0.03) for 5−14-year-olds and OR 8.13, 95% CI: 2.75−24.1 (p < 0.001) for ≥15-year-olds compared to horses <5 years old. The mean SRH value was 78.2 mm2 (median = 88 mm2, interquartile range = 0−121 mm2) with only 9% of the horses having an SRH value > 150 mm2, considered sufficient to protect against clinical disease and virus shedding. Comparative screening of a subset of the horses (n = 118) with a 2019 H3N8 virus (A/equine/Worcestershire/2019) revealed a significantly greater seropositivity (p = 0.0001) than A/equine/Richmond/1/2007 consistent with exposure of the population during a widespread outbreak of EI in the region in 2019. In conclusion, there was an insufficient level of protection against EI in the region and introduction of a vaccination programme with vaccines containing recently circulating virus is recommended to mitigate against further outbreaks of EI in Nigeria.

Epidemiology of equine influenza in the Maghreb area.

Equine influenza (EI) is one of the most contagious respiratory infections in horses, donkeys and mules, caused by equine influenza A virus (EIV). It remains a disease with a strong economic stake for the equine industry. This review focuses on the epidemiological situation of EIV in the Maghreb area, which includes Algeria, Morocco and Tunisia. There is serological evidence for extensive circulation of EIV in the Maghreb area since the early 1970s, but reports of detailed investigation of outbreaks are scarce with no documented isolation or molecular characterization of EIV from Tunisia. Isolates of EIV were obtained from outbreaks in Algeria in 1971/1972 and 2011. Similarly, in Morocco, isolates were obtained from outbreaks in 1997 and 2004. The viruses isolated in 2004 showed evidence of 'evolutionary stasis', with haemagglutinin and non-structural protein 1 sequences most similar to those of viruses isolated decades earlier. In conclusion, effective surveillance of equids in the Maghreb region, where there is potential for virus re-emergence, should be encouraged.

An Equine Model for Vaccination against a Hepacivirus: Insights into Host Responses to E2 Recombinant Protein Vaccination and Subsequent Equine Hepacivirus Inoculation.

Equine hepacivirus (EqHV) is the closest known genetic homologue of hepatitis C virus. An effective prophylactic vaccine is currently not available for either of these hepaciviruses. The equine as potential surrogate model for hepacivirus vaccine studies was investigated, while equine host responses following vaccination with EqHV E2 recombinant protein and subsequent EqHV inoculation were elucidated. Four ponies received prime and booster vaccinations (recombinant protein, adjuvant) four weeks apart (day -55 and -27). Two control ponies received adjuvant only. Ponies were inoculated with EqHV RNA-positive plasma on day 0. Blood samples and liver biopsies were collected over 26 weeks (day -70 to +112). Serum analyses included detection of EqHV RNA, isotypes of E2-specific immunoglobulin G (IgG), nonstructural protein 3-specific IgG, haematology, serum biochemistry, and metabolomics. Liver tissue analyses included EqHV RNA detection, RNA sequencing, histopathology, immunohistochemistry, and fluorescent in situ hybridization. Al-though vaccination did not result in complete protective immunity against experimental EqHV inoculation, the majority of vaccinated ponies cleared the serum EqHV RNA earlier than the control ponies. The majority of vaccinated ponies appeared to recover from the EqHV-associated liver insult earlier than the control ponies. The equine model shows promise as a surrogate model for future hepacivirus vaccine research.

Inference for a spatio-temporal model with partial spatial data: African horse sickness virus in Morocco.

African horse sickness virus (AHSV) is a vector-borne virus spread by midges (Culicoides spp.). The virus causes African horse sickness (AHS) disease in some species of equid. AHS is endemic in parts of Africa, previously emerged in Europe and in 2020 caused outbreaks for the first time in parts of Eastern Asia. Here we analyse a unique historic dataset from the 1989-1991 emergence of AHS in Morocco in a naïve population of equids. Sequential Monte Carlo and Markov chain Monte Carlo techniques are used to estimate parameters for a spatial-temporal model using a transmission kernel. These parameters allow us to observe how the transmissibility of AHSV changes according to the distance between premises. We observe how the spatial specificity of the dataset giving the locations of premises on which any infected equids were reported affects parameter estimates. Estimations of transmissibility were similar at the scales of village (location to the nearest 1.3 km) and region (median area 99 km2), but not province (median area 3000 km2). This data-driven result could help inform decisions by policy makers on collecting data during future equine disease outbreaks, as well as policies for AHS control.

Gene Expression Profile Induced by Two Different Variants of Street Rabies Virus in Mice.

Pathogenicity and pathology of rabies virus (RABV) varies according to the variant, but the mechanisms are not completely known. In this study, gene expression profile in brains of mice experimentally infected with RABV isolated from a human case of dog rabies (V2) or vampire bat-acquired rabies (V3) were analyzed. In total, 138 array probes associated with 120 genes were expressed differentially between mice inoculated with V2 and sham-inoculated control mice at day 10 post-inoculation. A single probe corresponding to an unannotated gene was identified in V3 versus control mice. Gene ontology (GO) analysis revealed that all of the genes upregulated in mice inoculated with V2 RABV were involved in the biological process of immune defense against pathogens. Although both variants are considered pathogenic, inoculation by the same conditions generated different gene expression results, which is likely due to differences in pathogenesis between the dog and bat RABV variants. This study demonstrated the global gene expression in experimental infection due to V3 wild-type RABV, from the vampire bat Desmodus rotundus, an important source of infection for humans, domestic animals and wildlife in Latin America.

Development of Plant-Based Vaccines for Prevention of Avian Influenza and Newcastle Disease in Poultry.

Viral diseases, including avian influenza (AI) and Newcastle disease (ND), are an important cause of morbidity and mortality in poultry, resulting in significant economic losses. Despite the availability of commercial vaccines for the major viral diseases of poultry, these diseases continue to pose a significant risk to global food security. There are multiple factors for this: vaccine costs may be prohibitive, cold chain storage for attenuated live-virus vaccines may not be achievable, and commercial vaccines may protect poorly against local emerging strains. The development of transient gene expression systems in plants provides a versatile and robust tool to generate a high yield of recombinant proteins with superior speed while managing to achieve cost-efficient production. Plant-derived vaccines offer good stability and safety these include both subunit and virus-like particle (VLP) vaccines. VLPs offer potential benefits compared to currently available traditional vaccines, including significant reductions in virus shedding and the ability to differentiate between infected and vaccinated birds (DIVA). This review discusses the current state of plant-based vaccines for prevention of the AI and ND in poultry, challenges in their development, and potential for expanding their use in low- and middle-income countries.

Pentacyclic and hexacyclic cucurbitacins from Elaeocarpuspetiolatus.

Four undescribed cucurbitacins, designated as petiolaticins A-D, and four known cucurbitacins were isolated from the bark and leaves of Elaeocarpus petiolatus (Jack) Wall. Their chemical structures were elucidated based on detailed analyses of the NMR and MS data. The absolute configuration of petiolaticin A was also determined by X-ray diffraction analysis. Petiolaticin A represents a cucurbitacin derivative incorporating a 3,4-epoxyfuranyl-bearing side chain, while petiolaticin B possesses a furopyranyl unit fused to the tetracyclic cucurbitane core structure. Petiolaticins A, B, and D were evaluated in vitro against a panel of human breast, pancreatic, and colorectal cancer cell lines. Petiolaticin A exhibited the greatest cytotoxicity against the MDA-MB-468, MDA-MB-231, MCF-7, and SW48 cell lines (IC50 7.4, 9.2, 9.3, and 4.6 µM, respectively). Additionally, petiolaticin D, 16α,23α-epoxy-3ß,20ß-dihydroxy-10αH,23ßH-cucurbit-5,24-dien-11-one, and 16α,23α-epoxy-3ß,20ß-dihydroxy-10αH,23ßH-cucurbit-5,24-dien-11-one 3-O-ß-D-glucopyranoside were tested for their ability to inhibit cell entry of a pseudotyped virus bearing the hemagglutinin envelope protein of a highly pathogenic avian influenza virus. Petiolaticin D showed the highest inhibition (44.3%), followed by 16α,23α-epoxy-3ß,20ß-dihydroxy-10αH,23ßH-cucurbit-5,24-dien-11-one (21.0%), and 16α,23α-epoxy-3ß,20ß-dihydroxy-10αH,23ßH-cucurbit-5,24-dien-11-one 3-O-ß-D-glucopyranoside showed limited inhibition (9.0%). These preliminary biological assays have demonstrated that petiolaticins A and D possess anticancer and antiviral properties, respectively, which warrant for further investigations.

Re-parameterization of a mathematical model of African horse sickness virus using data from a systematic literature search.

African horse sickness (AHS) is a vector-borne disease transmitted by Culicoides spp., endemic to sub-Saharan Africa. There have been many examples of historic and recent outbreaks in the Middle East, Asia and Europe. However, not much is known about infection dynamics and outbreak potential in these naive populations. In order to better inform a previously published ordinary differential equation model, we performed a systematic literature search to identify studies documenting experimental infection of naive (control) equids in vaccination trials. Data on the time until the onset of viraemia, clinical signs and death after experimental infection of a naive equid and duration of viraemia were extracted. The time to viraemia was 4.6 days and the time to clinical signs was 4.9 days, longer than the previously estimated latent period of 3.7 days. The infectious periods of animals that died/were euthanized or survived were found to be 3.9 and 8.7 days, whereas previous estimations were 4.4 and 6 days, respectively. The case fatality was also found to be higher than previous estimations. The updated parameter values (along with other more recently published estimates from literature) resulted in an increase in the number of host deaths, decrease in the duration of the outbreak and greater prevalence in vectors.

A Scoping Review of West Nile Virus Seroprevalence Studies among African Equids.

West Nile virus (WNV) is an emerging and re-emerging zoonotic flavivirus first identified in and endemic to Africa. The virus is transmitted between birds by biting mosquitoes, with equids and humans being incidental hosts. The majority of infected incidental hosts display no or only mild clinical signs, but a fraction develop encephalitis. The aim of this scoping review was to identify and evaluate primary research on the presence of antibodies to WNV among African equids. Three bibliographic databases and the grey literature were searched. Of 283 articles identified, only 16 satisfied all the inclusion criteria. Data were collated on study design and outcomes. The overall seroprevalence reported ranged from 17.4 to 90.3%, with 1998 (35%) of the 5746 horses, donkeys and mules having screened positive for WNV antibodies. Several articles determined that seroprevalence increased significantly with age. Due to co-circulation of other flaviviruses in Africa, in the majority of studies that screened samples by ELISA, positive results were confirmed using a more specific neutralization test. However, only eight studies tested against other flaviviruses, including Potiskum, Uganda S, Wesselsbron and yellow fever virus in one, Japanese encephalitis and Usutu virus (USUV) in one, tick-borne encephalitis and USUV in one and USUV only in three. Equids are regarded as useful sentinel animals for WNV, but variation in study design poses challenges when trying to determine risk factors for, and trends in, WNV seroprevalence.

Serological Cross-Reactions between Expressed VP2 Proteins from Different Bluetongue Virus Serotypes.

Bluetongue (BT) is a severe and economically important disease of ruminants that is widely distributed around the world, caused by the bluetongue virus (BTV). More than 28 different BTV serotypes have been identified in serum neutralisation tests (SNT), which, along with geographic variants (topotypes) within each serotype, reflect differences in BTV outer-capsid protein VP2. VP2 is the primary target for neutralising antibodies, although the basis for cross-reactions and serological variations between and within BTV serotypes is poorly understood. Recombinant BTV VP2 proteins (rVP2) were expressed in Nicotiana benthamiana, based on sequence data for isolates of thirteen BTV serotypes (primarily from Europe), including three 'novel' serotypes (BTV-25, -26 and -27) and alternative topotypes of four serotypes. Cross-reactions within and between these viruses were explored using rabbit anti-rVP2 sera and post BTV-infection sheep reference-antisera, in I-ELISA (with rVP2 target antigens) and SNT (with reference strains of BTV-1 to -24, -26 and -27). Strong reactions were generally detected with homologous rVP2 proteins or virus strains/serotypes. The sheep antisera were largely serotype-specific in SNT, but more cross-reactive by ELISA. Rabbit antisera were more cross-reactive in SNT, and showed widespread, high titre cross-reactions against homologous and heterologous rVP2 proteins in ELISA. Results were analysed and visualised by antigenic cartography, showing closer relationships in some, but not all cases, between VP2 topotypes within the same serotype, and between serotypes belonging to the same 'VP2 nucleotype'.

Systematic review and meta-analysis of seroprevalence studies of West Nile virus in equids in Europe between 2001 and 2018.

There is some evidence that West Nile virus (WNV), which causes encephalomyelitis in equids, is an emerging disease in Europe. The aim of this study was to perform a systematic review and meta-analysis to analyse seroprevalence studies of WNV in equids in European countries between 2001 and 2018. Two electronic databases, PubMed and Scopus, were searched for relevant publications published from 2001 to 2018 using predetermined keywords. A total of 1,484 papers were initially found. After applying the eligibility criteria, 39 papers were finally included in the systematic review. Analysis of 28,089 equids from 16 European countries revealed a pooled seroprevalence of 8% (95% CI 5%-12%, p < .001, I2  = 99.3%) in Europe. The pooled seroprevalence was slightly higher in Mediterranean basin countries than other countries and when calculated for samples collected between 2001 and 2009 compared to 2010 to 2018. Differences in study design (e.g. sampling associated with recent outbreaks of WNV) contributed to a high degree of variability among studies. Further studies with harmonized study design and reporting of the results are recommended to better estimate and monitor European seroprevalence of WNV in equids.