IFNAR(-/-) Mice Constitute a Suitable Animal Model for Epizootic Hemorrhagic Disease Virus Study and Vaccine Evaluation.

Epizootic hemorrhagic disease (EHD), caused by Epizootic hemorrhagic disease virus (EHDV), is an emerging and severe livestock disease. Recent incursion and distribution of EHDV in Europe have outlined the emerging character of EHD. Despite its worldwide impact, numerous knowledge gaps exist. A range of inconveniences restricts utilization of natural hosts of EHDV. Here, we show that adult mice deficient in type I IFN receptor (IFNAR(-/-)) are highly susceptible to EHDV-6 and EHDV-8 infection when the virus is administered subcutaneously. Disease was characterized by ruffled hair, reluctance to move, dehydration and conjunctivitis, with viraemia detected from day 5 post-infection. A deeper characterization of EHDV-8 infection showed viral replication in the lung, liver, spleen, kidney, testis and ovaries. Importantly, increased expression levels of pro-inflammatory cytokines IL-1ß, IL-6 and CXCL2 were observed in spleen after EHDV-8 infection. Furthermore, IFNAR(-/-) adult mice immunized with a EHDV-8 inactivated vaccine elicited neutralizing antibodies specific of EHDV-8 and full protection against challenge with a lethal dose of this virus. This study also explores the possibilities of this animal model for study of BTV and EHDV coinfection. In summary, the IFNAR(-/-) mouse model faithfully recapitulates EHD and can be applied for vaccine testing, which can facilitate progress in addressing the animal health challenge posed by this virus.

Cytokine mRNA Expression Profile in Target Organs of IFNAR (-/-) Mice Infected with African Horse Sickness Virus.

African horse sickness (AHS) is a highly severe disease caused by a viral etiological agent, African horse sickness virus (AHSV). It is endemic in sub-Saharan Africa, while sporadic outbreaks have occurred in North Africa, Asia, and Europe, with the most recent cases in Thailand. AHSV transmission between equines occurs primarily by biting midges of the genus Culicoides, especially C. imicola, with a wide distribution globally. As research in horses is highly restricted due to a variety of factors, small laboratory animal models that reproduce clinical signs and pathology observed in natural infection of AHSV are highly needed. Here, we investigated the expression profile of several pro-inflammatory cytokines in target organs and serum of IFNAR (-/-) mice, to continue characterizing this established animal model and to go deep into the innate immune responses that are still needed.

Engineering recombinant replication-competent bluetongue viruses expressing reporter genes for in vitro and non-invasive in vivo studies.

Bluetongue virus (BTV) is the causative agent of the important livestock disease bluetongue (BT), which is transmitted via Culicoides bites. BT causes severe economic losses associated with its considerable impact on health and trade of animals. By reverse genetics, we have designed and rescued reporter-expressing recombinant (r)BTV expressing NanoLuc luciferase (NLuc) or Venus fluorescent protein. To generate these viruses, we custom synthesized a modified viral segment 5 encoding NS1 protein with the reporter genes located downstream and linked by the Porcine teschovirus-1 (PTV-1) 2A autoproteolytic cleavage site. Therefore, fluorescent signal or luciferase activity is only detected after virus replication and expression of non-structural proteins. Fluorescence or luminescence signals were detected in cells infected with rBTV/Venus or rBTV/NLuc, respectively. Moreover, the marking of NS2 protein confirmed that reporter genes were only expressed in BTV-infected cells. Growth kinetics of rBTV/NLuc and rBTV/Venus in Vero cells showed replication rates similar to those of wild-type and rBTV. Infectivity studies of these recombinant viruses in IFNAR(-/-) mice showed a higher lethal dose for rBTV/NLuc and rBTV/Venus than for rBTV indicating that viruses expressing the reporter genes are attenuated in vivo. Interestingly, luciferase activity was detected in the plasma of viraemic mice infected with rBTV/NLuc. Furthermore, luciferase activity quantitatively correlated with RNAemia levels of infected mice throughout the infection. In addition, we have investigated the in vivo replication and dissemination of BTV in IFNAR (-/-) mice using BTV/NLuc and non-invasive in vivo imaging systems.IMPORTANCEThe use of replication-competent viruses that encode a traceable fluorescent or luciferase reporter protein has significantly contributed to the in vitro and in vivo study of viral infections and the development of novel therapeutic approaches. In this work, we have generated rBTV that express fluorescent or luminescence proteins to track BTV infection both in vitro and in vivo. Despite the availability of vaccines, BTV and other related orbivirus are still associated with a significant impact on animal health and have important economic consequences worldwide. Our studies may contribute to the advance in orbivirus research and pave the way for the rapid development of new treatments, including vaccines.

Epizootic Hemorrhagic Disease Virus: Current Knowledge and Emerging Perspectives.

Epizootic Hemorrhagic Disease (EHD) of ruminants is a viral pathology that has significant welfare, social, and economic implications. The causative agent, epizootic hemorrhagic disease virus (EHDV), belongs to the Orbivirus genus and leads to significant regional disease outbreaks among livestock and wildlife in North America, Asia, Africa, and Oceania, causing significant morbidity and mortality. During the past decade, this viral disease has become a real threat for countries of the Mediterranean basin, with the recent occurrence of several important outbreaks in livestock. Moreover, the European Union registered the first cases of EHDV ever detected within its territory. Competent vectors involved in viral transmission, Culicoides midges, are expanding its distribution, conceivably due to global climate change. Therefore, livestock and wild ruminants around the globe are at risk for this serious disease. This review provides an overview of current knowledge about EHDV, including changes of distribution and virulence, an examination of different animal models of disease, and a discussion about potential treatments to control the disease.

Vaccinia Virus Strain MVA Expressing a Prefusion-Stabilized SARS-CoV-2 Spike Glycoprotein Induces Robust Protection and Prevents Brain Infection in Mouse and Hamster Models.

The COVID-19 pandemic has underscored the importance of swift responses and the necessity of dependable technologies for vaccine development. Our team previously developed a fast cloning system for the modified vaccinia virus Ankara (MVA) vaccine platform. In this study, we reported on the construction and preclinical testing of a recombinant MVA vaccine obtained using this system. We obtained recombinant MVA expressing the unmodified full-length SARS-CoV-2 spike (S) protein containing the D614G amino-acid substitution (MVA-Sdg) and a version expressing a modified S protein containing amino-acid substitutions designed to stabilize the protein a in a pre-fusion conformation (MVA-Spf). S protein expressed by MVA-Sdg was found to be expressed and was correctly processed and transported to the cell surface, where it efficiently produced cell-cell fusion. Version Spf, however, was not proteolytically processed, and despite being transported to the plasma membrane, it failed to induce cell-cell fusion. We assessed both vaccine candidates in prime-boost regimens in the susceptible transgenic K18-human angiotensin-converting enzyme 2 (K18-hACE2) in mice and in golden Syrian hamsters. Robust immunity and protection from disease was induced with either vaccine in both animal models. Remarkably, the MVA-Spf vaccine candidate produced higher levels of antibodies, a stronger T cell response, and a higher degree of protection from challenge. In addition, the level of SARS-CoV-2 in the brain of MVA-Spf inoculated mice was decreased to undetectable levels. Those results add to our current experience and range of vaccine vectors and technologies for developing a safe and effective COVID-19 vaccine.

Nanoparticle- and Microparticle-Based Vaccines against Orbiviruses of Veterinary Importance.

Bluetongue virus (BTV) and African horse sickness virus (AHSV) are widespread arboviruses that cause important economic losses in the livestock and equine industries, respectively. In addition to these, another arthropod-transmitted orbivirus known as epizootic hemorrhagic disease virus (EHDV) entails a major threat as there is a conducive landscape that nurtures its emergence in non-endemic countries. To date, only vaccinations with live attenuated or inactivated vaccines permit the control of these three viral diseases, although important drawbacks, e.g., low safety profile and effectiveness, and lack of DIVA (differentiation of infected from vaccinated animals) properties, constrain their usage as prophylactic measures. Moreover, a substantial number of serotypes of BTV, AHSV and EHDV have been described, with poor induction of cross-protective immune responses among serotypes. In the context of next-generation vaccine development, antigen delivery systems based on nano- or microparticles have gathered significant attention during the last few decades. A diversity of technologies, such as virus-like particles or self-assembled protein complexes, have been implemented for vaccine design against these viruses. In this work, we offer a comprehensive review of the nano- and microparticulated vaccine candidates against these three relevant orbiviruses. Additionally, we also review an innovative technology for antigen delivery based on the avian reovirus nonstructural protein muNS and we explore the prospective functionality of the nonstructural protein NS1 nanotubules as a BTV-based delivery platform.

Recombinant Modified Vaccinia Virus Ankara Development to Express VP2, NS1, and VP7 Proteins of Bluetongue Virus.

Modified vaccinia virus Ankara (MVA) is employed widely as an experimental vaccine vector for its abortive replication in mammalian cells and high expression level of foreign/heterologous genes. Recombinant MVAs (rMVAs) are used as platforms for protein production as well as vectors to generate vaccines against a wide range of infectious diseases and other pathologies. The portrait of the virus combines desirable elements such as high-level biological safety, the ability to activate appropriate innate immune mediators upon vaccination , and the capacity to deliver substantial amounts of heterologous antigens. rMVAs encoding proteins of Bluetongue virus (BTV), an orbivirus that infects domestic and wild ruminants through transmission by biting midges of the Culicoides species, are excellent vaccine candidates against this virus. In this chapter, we describe the methods for the generation of rMVAs encoding VP2, NS1, and VP7 proteins of BTV . The included protocols cover the cloning of VP2, NS1, and VP7 BTV-4 genes in a transfer plasmid, the construction of rMVAs, the titration of virus working stocks, and the protein expression analysis by immunofluorescence and radiolabeling of rMVA infected cells as well as virus purification procedure.

Identification of Amino Acid Residues Required for Inhibition of Host Gene Expression by Influenza Virus A/Viet Nam/1203/2004 H5N1 PA-X.

PA-X is a nonstructural protein of influenza A virus (IAV), which is encoded by the polymerase acidic (PA) N-terminal region that contains a C-terminal +1 frameshifted sequence. IAV PA-X protein modulates virus-induced host innate immune responses and viral pathogenicity via suppression of host gene expression or cellular shutoff, through cellular mRNA cleavage. Highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype naturally infect different avian species, they have an enormous economic impact in the poultry farming, and they also have zoonotic and pandemic potential, representing a risk to human public health. In the present study, we describe a novel bacterium-based approach to identify amino acid residues in the PA-X protein of the HPAIV A/Viet Nam/1203/2004 H5N1 that are important for its ability to inhibit host protein expression or cellular shutoff activity. Identified PA-X mutants displayed a reduced shutoff activity compared to that of the wild-type A/Viet Nam/1203/2004 H5N1 PA-X protein. Notably, this new bacterium-based screening allowed us to identify amino acid residues widely distributed over the entire N-terminal region of PA-X. Furthermore, we found that some of the residues affecting A/Viet Nam/1203/2004 H5N1 PA-X host shutoff activity also affect PA polymerase activity in a minigenome assay. This information could be used for the rational design of new and more effective compounds with antiviral activity against IAV. Moreover, our results demonstrate the feasibility of using this bacterium-based approach to identify amino acid residues important for the activity of viral proteins to inhibit host gene expression. IMPORTANCE Highly pathogenic avian influenza viruses continue to pose a huge threat to global animal and human health. Despite of the limited genome size of Influenza A virus (IAV), the virus encodes eight main viral structural proteins and multiple accessory nonstructural proteins, depending on the IAV type, subtype, or strain. One of the IAV accessory proteins, PA-X, is encoded by the polymerase acidic (PA) protein and is involved in pathogenicity through the modulation of IAV-induced host inflammatory and innate immune responses. However, the molecular mechanism(s) of IAV PA-X regulation of the host immune response is not well understood. Here, we used, for the first time, a bacterium-based approach for the identification of amino acids important for the ability of IAV PA-X to induce host shutoff activity and describe novel residues relevant for its ability to inhibit host gene expression, and their contribution in PA polymerase activity.

The Combined Expression of the Nonstructural Protein NS1 and the N-Terminal Half of NS2 (NS21-180) by ChAdOx1 and MVA Confers Protection against Clinical Disease in Sheep upon Bluetongue Virus Challenge.

Bluetongue, caused by bluetongue virus (BTV), is a widespread arthropod-borne disease of ruminants that entails a recurrent threat to the primary sector of developed and developing countries. In this work, we report modified vaccinia virus Ankara (MVA) and ChAdOx1-vectored vaccines designed to simultaneously express the immunogenic NS1 protein and/or NS2-Nt, the N-terminal half of protein NS2 (NS21-180). A single dose of MVA or ChAdOx1 expressing NS1-NS2-Nt improved the protection conferred by NS1 alone in IFNAR(-/-) mice. Moreover, mice immunized with ChAdOx1/MVA-NS1, ChAdOx1/MVA-NS2-Nt, or ChAdOx1/MVA-NS1-NS2-Nt developed strong cytotoxic CD8+ T-cell responses against NS1, NS2-Nt, or both proteins and were fully protected against a lethal infection with BTV serotypes 1, 4, and 8. Furthermore, although a single immunization with ChAdOx1-NS1-NS2-Nt partially protected sheep against BTV-4, the administration of a booster dose of MVA-NS1-NS2-Nt promoted a faster viral clearance, reduction of the period and level of viremia and also protected from the pathology produced by BTV infection. IMPORTANCE Current BTV vaccines are effective but they do not allow to distinguish between vaccinated and infected animals (DIVA strategy) and are serotype specific. In this work we have develop a DIVA multiserotype vaccination strategy based on adenoviral (ChAdOx1) and MVA vaccine vectors, the most widely used in current phase I and II clinical trials, and the conserved nonstructural BTV proteins NS1 and NS2. This immunization strategy solves the major drawbacks of the current marketed vaccines.

Natural Selection of H5N1 Avian Influenza A Viruses with Increased PA-X and NS1 Shutoff Activity.

Influenza A viruses (IAV) can infect a broad range of mammalian and avian species. However, the host innate immune system provides defenses that restrict IAV replication and infection. Likewise, IAV have evolved to develop efficient mechanisms to counteract host antiviral responses to efficiently replicate in their hosts. The IAV PA-X and NS1 non-structural proteins are key virulence factors that modulate innate immune responses and virus pathogenicity during infection. To study the determinants of IAV pathogenicity and their functional co-evolution, we evaluated amino acid differences in the PA-X and NS1 proteins of early (1996-1997) and more recent (since 2016) H5N1 IAV. H5N1 IAV have zoonotic and pandemic potential and represent an important challenge both in poultry farming and human health. The results indicate that amino acid changes occurred over time, affecting the ability of these two non-structural H5N1 IAV proteins to inhibit gene expression and affecting virus pathogenicity. These results highlight the importance to monitor the evolution of these two virulence factors of IAV, which could result in enhanced viral replication and virulence.

Viral Vector Vaccines against Bluetongue Virus.

Bluetongue virus (BTV), the prototype member of the genus Orbivirus (family Reoviridae), is the causative agent of an important livestock disease, bluetongue (BT), which is transmitted via biting midges of the genus Culicoides. To date, up to 29 serotypes of BTV have been described, which are classified as classical (BTV 1-24) or atypical (serotypes 25-27), and its distribution has been expanding since 1998, with important outbreaks in the Mediterranean Basin and devastating incursions in Northern and Western Europe. Classical vaccine approaches, such as live-attenuated and inactivated vaccines, have been used as prophylactic measures to control BT through the years. However, these vaccine approaches fail to address important matters like vaccine safety profile, effectiveness, induction of a cross-protective immune response among serotypes, and implementation of a DIVA (differentiation of infected from vaccinated animals) strategy. In this context, a wide range of recombinant vaccine prototypes against BTV, ranging from subunit vaccines to recombinant viral vector vaccines, have been investigated. This article offers a comprehensive outline of the live viral vectors used against BTV.

Recombinant Rift Valley fever viruses encoding bluetongue virus (BTV) antigens: Immunity and efficacy studies upon a BTV-4 challenge.

BACKGROUND: Many ruminant diseases of viral aetiology can be effectively prevented using appropriate vaccination measures. For diseases such as Rift Valley fever (RVF) the long inter-epizootic periods make routine vaccination programs unfeasible. Coupling RVF prophylaxis with seasonal vaccination programmes by means of multivalent vaccine platforms would help to reduce the risk of new RVF outbreaks. METHODOLOGY/PRINCIPAL FINDINGS: In this work we generated recombinant attenuated Rift Valley fever viruses (RVFVs) encoding in place of the virulence factor NSs either the VP2 capsid protein or a truncated form of the non-structural NS1 protein of bluetongue virus serotype 4 (BTV-4). The recombinant viruses were able to carry and express the heterologous BTV genes upon consecutive passages in cell cultures. In murine models, a single immunization was sufficient to protect mice upon RVFV challenge and to elicit a specific immune response against BTV-4 antigens that was fully protective after a BTV-4 boost. In sheep, a natural host for RVFV and BTV, both vaccines proved immunogenic although conferred only partial protection after a virulent BTV-4 reassortant Morocco strain challenge. CONCLUSIONS/SIGNIFICANCE: Though additional optimization will be needed to improve the efficacy data against BTV in sheep, our findings warrant further developments of attenuated RVFV as a dual vaccine platform carrying heterologous immune relevant antigens for ruminant diseases in RVF risk areas.

Inhibition of Orbivirus Replication by Aurintricarboxylic Acid.

Bluetongue virus (BTV) and African horse sickness virus (AHSV) are vector-borne viruses belonging to the Orbivirus genus, which are transmitted between hosts primarily by biting midges of the genus Culicoides. With recent BTV and AHSV outbreaks causing epidemics and important economy losses, there is a pressing need for efficacious drugs to treat and control the spread of these infections. The polyanionic aromatic compound aurintricarboxylic acid (ATA) has been shown to have a broad-spectrum antiviral activity. Here, we evaluated ATA as a potential antiviral compound against Orbivirus infections in both mammalian and insect cells. Notably, ATA was able to prevent the replication of BTV and AHSV in both cell types in a time- and concentration-dependent manner. In addition, we evaluated the effect of ATA in vivo using a mouse model of infection. ATA did not protect mice against a lethal challenge with BTV or AHSV, most probably due to the in vivo effect of ATA on immune system regulation. Overall, these results demonstrate that ATA has inhibitory activity against Orbivirus replication in vitro, but further in vivo analysis will be required before considering it as a potential therapy for future clinical evaluation.

A protective bivalent vaccine against Rift Valley fever and bluetongue.

Rift Valley fever (RVF) and bluetongue (BT) are two important ruminant diseases transmitted by arthropods. Both viruses have shown important geographic spread leading to endemicity of BT virus (BTV) in Africa and Europe. In this work, we report a dual vaccine that simultaneously induces protective immune responses against BTV and RVFV based on modified vaccinia Ankara virus (MVA) expressing BTV proteins VP2, NS1, or a truncated form of NS1 (NS1-Nt), and RVFV Gn and Gc glycoproteins. IFNAR(-/-) mice immunized with two doses of MVA-GnGc-VP2 developed a significant neutralizing antibody response against BTV-4 and RVFV. Furthermore, the homologous prime-boost immunization with MVA-GnGc-NS1 or MVA-GnGc-NS1-Nt triggered neutralizing antibodies against RVFV and NS1-specific cytotoxic CD8+ T cells in mice. Moreover, all mice immunized with MVA-GnGc-NS1 or MVA-GnGc-NS1-Nt remained healthy after lethal challenge with RVFV or BTV-4. The homologous prime-boost vaccination with MVA-GnGc-NS1, which was the best immunization strategy observed in mice, was assayed in sheep. Clinical signs and viremia were absent or highly reduced in vaccinated sheep after challenge with BTV-4 or RVFV. These results indicate that MVA-GnGc-NS1 vaccination elicits immune protection against RVFV and BTV in sheep.

Reverse genetics approaches: a novel strategy for African horse sickness virus vaccine design.

African horse sickness (AHS) is a devastating disease caused by African horse sickness virus (AHSV) and transmitted by arthropods between its equine hosts. AHSV is endemic in sub-Saharan Africa, where polyvalent live attenuated vaccine is in use even though it is associated with safety risks. This review article summarizes and compares new strategies to generate safe and effective AHSV vaccines based on protein, virus like particles, viral vectors and reverse genetics technology. Manipulating the AHSV genome to generate synthetic viruses by means of reverse genetic systems has led to the generation of potential safe vaccine candidates that are under investigation.

Heterologous Combination of ChAdOx1 and MVA Vectors Expressing Protein NS1 as Vaccination Strategy to Induce Durable and Cross-Protective CD8+ T Cell Immunity to Bluetongue Virus.

The sequence of non-structural protein NS1 of bluetongue virus (BTV), which contains immunodominant CD8+ T cell epitopes, is highly conserved among BTV serotypes, and has therefore become a major tool in the development of a universal BTV vaccine. In this work, we have engineered multiserotype BTV vaccine candidates based on recombinant chimpanzee adenovirus (ChAdOx1) and modified vaccinia virus Ankara (MVA) vectors expressing the NS1 protein of BTV-4 or its truncated form NS1-Nt. A single dose of ChAdOx1-NS1 or ChAdOx1-NS1-Nt induced a moderate CD8+ T cell response and protected IFNAR(-/-) mice against a lethal dose of BTV-4/MOR09, a reassortant strain between BTV-1 and BTV-4, although the animals showed low viremia after infection. Furthermore, IFNAR(-/-) mice immunized with a single dose of ChAdOx1-NS1 were protected after challenge with a lethal dose of BTV-8 in absence of viremia nor clinical signs. Additionally, the heterologous prime-boost ChAdOx1/MVA expressing NS1 or NS1-Nt elicited a robust NS1 specific CD8+ T cell response and protected the animals against BTV-4/MOR09 even 16 weeks after immunization, with undetectable levels of viremia at any time after challenge. Subsequently, the best immunization strategy based on ChAdOx1/MVA-NS1 was assayed in sheep. Non-immunized animals presented fever and viremia levels up to 104 PFU/mL after infection. In contrast, although viremia was detected in immunized sheep, the level of virus in blood was 100 times lower than in non-immunized animals in absence of clinical signs.

MVA Vectored Vaccines Encoding Rift Valley Fever Virus Glycoproteins Protect Mice against Lethal Challenge in the Absence of Neutralizing Antibody Responses.

In vitro neutralizing antibodies have been often correlated with protection against Rift Valley fever virus (RVFV) infection. We have reported previously that a single inoculation of sucrose-purified modified vaccinia Ankara (MVA) encoding RVFV glycoproteins (rMVAGnGc) was sufficient to induce a protective immune response in mice after a lethal RVFV challenge. Protection was related to the presence of glycoprotein specific CD8+ cells, with a low-level detection of in vitro neutralizing antibodies. In this work we extended those observations aimed to explore the role of humoral responses after MVA vaccination and to study the contribution of each glycoprotein antigen to the protective efficacy. Thus, we tested the efficacy and immune responses in BALB/c mice of recombinant MVA viruses expressing either glycoprotein Gn (rMVAGn) or Gc (rMVAGc). In the absence of serum neutralizing antibodies, our data strongly suggest that protection of vaccinated mice upon the RVFV challenge can be achieved by the activation of cellular responses mainly directed against Gc epitopes. The involvement of cellular immunity was stressed by the fact that protection of mice was strain dependent. Furthermore, our data suggest that the rMVA based single dose vaccination elicits suboptimal humoral immune responses against Gn antigen since disease in mice was exacerbated upon virus challenge in the presence of rMVAGnGc or rMVAGn immune serum. Thus, Gc-specific cellular immunity could be an important component in the protection after the challenge observed in BALB/c mice, contributing to the elimination of infected cells reducing morbidity and mortality and counteracting the deleterious effect of a subneutralizing antibody immune response.

Increasing the Safety Profile of the Master Donor Live Attenuated Influenza Vaccine.

Seasonal influenza epidemics remain one of the largest public health burdens nowadays. The best and most effective strategy to date in preventing influenza infection is a worldwide vaccination campaign. Currently, two vaccines are available to the public for the treatment of influenza infection, the chemically Inactivated Influenza Vaccine (IIV) and the Live Attenuated Influenza Vaccine (LAIV). However, the LAIV is not recommended for parts of the population, such as children under the age of two, immunocompromised individuals, the elderly, and pregnant adults. In order to improve the safety of the LAIV and make it available to more of the population, we sought to further attenuate the LAIV. In this study, we demonstrate that the influenza A virus (IAV) master donor virus (MDV) A/Ann Arbor/6/60 H2N2 LAIV can inhibit host gene expression using both the PA-X and NS1 proteins. Furthermore, we show that by removing PA-X, we can limit the replication of the MDV LAIV in a mouse model, while maintaining full protective efficacy. This work demonstrates a broadly applicable strategy of tuning the amount of host antiviral responses induced by the IAV MDV for the development of newer and safer LAIVs. Moreover, our results also demonstrate, for the first time, the feasibility of genetically manipulating the backbone of the IAV MDV to improve the efficacy of the current IAV LAIV.

Cross-protective immune responses against African horse sickness virus after vaccination with protein NS1 delivered by avian reovirus muNS microspheres and modified vaccinia virus Ankara.

African horse sickness virus (AHSV) is an insect-borne pathogen that causes acute disease in horses and other equids. In an effort to improve the safety of currently available vaccines and to acquire new knowledge about the determinants of AHSV immunogenicity, new generation vaccines are being developed. In this work we have generated and tested a novel immunization approach comprised of nonstructural protein 1 (NS1) of AHSV serotype 4 (AHSV-4) incorporated into avian reovirus muNS protein microspheres (MS-NS1) and/or expressed using recombinant modified vaccinia virus Ankara vector (MVA-NS1). The protection conferred against AHSV by a homologous MS-NS1 or heterologous MS-NS1 and MVA-NS1 prime/boost was evaluated in IFNAR (-/-) mice. Our results indicate that immunization based on MS-NS1 and MVA-NS1 afforded complete protection against the infection with homologous AHSV-4. Moreover, priming with MS-NS1 and boost vaccination with MVA-NS1 (MS-MVA-NS1) triggered NS1 specific cytotoxic CD8 + T cells and prevented AHSV disease in IFNAR (-/-) mice after challenge with heterologous serotype AHSV-9. Cross-protective immune responses are highly important since AHS can be caused by nine different serotypes, which means that a universal polyvalent vaccination would need to induce protective immunity against all serotypes.

Modeling Arboviral Infection in Mice Lacking the Interferon Alpha/Beta Receptor.

Arboviruses are arthropod-borne viruses that exhibit worldwide distribution and are a constant threat, not only for public health but also for wildlife, domestic animals, and even plants. To study disease pathogenesis and to develop efficient and safe therapies, the use of an appropriate animal model is a critical concern. Adult mice with gene knockouts of the interferon α/ß (IFN-α/ß) receptor (IFNAR(-/-)) have been described as a model of arbovirus infections. Studies with the natural hosts of these viruses are limited by financial and ethical issues, and in some cases, the need to have facilities with a biosafety level 3 with sufficient space to accommodate large animals. Moreover, the number of animals in the experiments must provide results with statistical significance. Recent advances in animal models in the last decade among other gaps in knowledge have contributed to the better understanding of arbovirus infections. A tremendous advantage of the IFNAR(-/-) mouse model is the availability of a wide variety of reagents that can be used to study many aspects of the immune response to the virus. Although extrapolation of findings in mice to natural hosts must be done with care due to differences in the biology between mouse and humans, experimental infections of IFNAR(-/-) mice with several studied arboviruses closely mimics hallmarks of these viruses in their natural host. Therefore, IFNAR(-/-) mice are a good model to facilitate studies on arbovirus transmission, pathogenesis, virulence, and the protective efficacy of new vaccines. In this review article, the most important arboviruses that have been studied using the IFNAR(-/-) mouse model will be reviewed.