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.

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.

Mosquito Salivary Proteins and Arbovirus Infection: From Viral Enhancers to Potential Targets for Vaccines.

Arthropod-borne viruses present important public health challenges worldwide. Viruses such as DENV, ZIKV, and WNV are of current concern due to an increasing incidence and an expanding geographic range, generating explosive outbreaks even in non-endemic areas. The clinical signs associated with infection from these arboviruses are often inapparent, mild, or nonspecific, but occasionally develop into serious complications marked by rapid onset, tremors, paralysis, hemorrhagic fever, neurological alterations, or death. They are predominately transmitted to humans through mosquito bite, during which saliva is inoculated into the skin to facilitate blood feeding. A new approach to prevent arboviral diseases has been proposed by the observation that arthropod saliva facilitates transmission of pathogens. Viruses released within mosquito saliva may more easily initiate host invasion by taking advantage of the host's innate and adaptive immune responses to saliva. This provides a rationale for creating vaccines against mosquito salivary proteins, especially because of the lack of licensed vaccines against most of these viruses. This review aims to provide an overview of the effects on the host immune response by the mosquito salivary proteins and how these phenomena alter the infection outcome for different arboviruses, recent attempts to generate mosquito salivary-based vaccines against flavivirus including DENV, ZIKV, and WNV, and the potential benefits and pitfalls that this strategy involves.

Current Advances in Zika Vaccine Development.

Zika virus (ZIKV), an emerging arthropod-borne flavivirus, was first isolated in Uganda in 1947 from monkeys and first detected in humans in Nigeria in 1952; it has been associated with a dramatic burden worldwide. Since then, interventions to reduce the burden of ZIKV infection have been mainly restricted to mosquito control, which in the end proved to be insufficient by itself. Hence, the situation prompted scientists to increase research on antivirals and vaccines against the virus. These efforts are still ongoing as the pathogenesis and immune evasion mechanisms of ZIKV have not yet been fully elucidated. Understanding the viral disease mechanism will provide a better landscape to develop prophylactic and therapeutic strategies against ZIKV. Currently, no specific vaccines or drugs have been approved for ZIKV. However, some are undergoing clinical trials. Notably, different platforms have been evaluated for the design of vaccines, including DNA, mRNA, viral vectors, virus-like particles (VLPs), inactivated virus, live attenuated virus, peptide and protein-based vaccines, passive immunizations by using monoclonal antibodies (MAbs), and vaccines that target vector-derived antigens. These vaccines have been shown to induce specific humoral and cellular immune responses and reduce viremia and viral RNA titers, both in vitro and in vivo. This review provides a comprehensive summary of current advancements in the development of vaccines against Zika virus.

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.

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.

Aedes aegypti anti-salivary proteins IgG levels in a cohort of DENV-like symptoms subjects from a dengue-endemic region in Colombia.

Dengue fever, caused by the dengue virus (DENV), is currently a threat to about half of the world's population. DENV is mainly transmitted to the vertebrate host through the bite of a female Aedes mosquito while taking a blood meal. During this process, salivary proteins are introduced into the host skin and blood to facilitate blood acquisition. These salivary proteins modulate both local (skin) and systemic immune responses. Several salivary proteins have been identified as immunogenic inducing the production of antibodies with some of those proteins also displaying immunomodulatory properties enhancing arboviral infections. IgG antibody responses against salivary gland extracts of a diverse number of mosquitoes, as well as antibody responses against the Ae. aegypti peptide, Nterm-34 kDa, have been suggested as biomarkers of human exposure to mosquito bites while antibodies against AgBR1 and NeSt1 proteins have been investigated for their potential protective effect against Zika virus (ZIKV) and West Nile virus infections. Thus, we were interested in evaluating whether IgG antibodies against AgBR1, NeSt1, Nterm-34 kDa peptide, and SGE were associated with DENV infections and clinical characteristics. For this, we tested samples from volunteers living in a dengue fever endemic area in Colombia in 2019 for the presence of IgG antibodies against those salivary proteins and peptides using an ELISA test. Results from this pilot study suggest an involvement of antibody responses against salivary proteins in dengue disease progression.

The Lyme disease agent co-opts adiponectin receptor-mediated signaling in its arthropod vector.

Adiponectin-mediated pathways contribute to mammalian homeostasis; however, little is known about adiponectin and adiponectin receptor signaling in arthropods. In this study, we demonstrate that Ixodes scapularis ticks have an adiponectin receptor-like protein (ISARL) but lack adiponectin, suggesting activation by alternative pathways. ISARL expression is significantly upregulated in the tick gut after Borrelia burgdorferi infection, suggesting that ISARL signaling may be co-opted by the Lyme disease agent. Consistent with this, RNA interference (RNAi)-mediated silencing of ISARL significantly reduced the B. burgdorferi burden in the tick. RNA-seq-based transcriptomics and RNAi assays demonstrate that ISARL-mediated phospholipid metabolism by phosphatidylserine synthase I is associated with B. burgdorferi survival. Furthermore, the tick complement C1q-like protein 3 interacts with ISARL, and B. burgdorferi facilitates this process. This study identifies a new tick metabolic pathway that is connected to the life cycle of the Lyme disease spirochete.

Many countries around the world are seeing an increase in the number of patients diagnosed with Lyme disease, with often serious joint, heart, and neurologic complications. This illness is caused by species of 'spirochete' bacteria that live and multiply inside black-legged ticks, and get injected into mammals upon a bite. Ticks are not simply 'syringes' however, and a complex relationship is established between spirochetes and their host. This is particularly true since Lyme disease-causing bacteria such as Borrelia burgdorferi rely on ticks to obtain energy and nutrients. Tang, Cao et al. delved into these complex interactions by focusing on the molecular cascades (or pathways) involving adiponectin, a hormone essential for regulating sugar levels and processing fats. Analyses of gene and protein databases highlighted that ticks carry a receptor-like protein for adiponectin but not the hormone itself, suggesting that an alternative pathway is at play. This may involve B. burgdorferi, which gets its fats and sugars from its host. And indeed, experiments showed that ticks produced more of the adiponectin receptor-like protein when they carried B. burgdorferi; conversely, silencing the receptor reduced the number of surviving spirochetes inside the tick. Further exploration showed that the receptor mediates molecular cascades that help to process fat molecules; these are associated with spirochete survival. In addition, the receptor-like protein was activated by C1QL3, a 'complement 1q domain-contained' molecule which might be part of the tick energy-making or immune systems. Larger quantities of C1QL3 were found in ticks upon B. burgdorferi infection, suggesting that the spirochete facilitates an interaction that boosts activity of the adiponectin receptor-like protein. Overall, the work by Tang and Cao et al. revealed a new pathway which B. burgdorferi takes advantage of to infect their host and multiply. Targeting this molecular cascade could help to interfere with the life cycle of the spirochete, as well as fight Lyme disease and other insect-borne conditions.

Aedes aegypti SNAP and a calcium transporter ATPase influence dengue virus dissemination.

Dengue virus (DENV) is a flavivirus that causes marked human morbidity and mortality worldwide, and is transmitted to humans by Aedes aegypti mosquitoes. Habitat expansion of Aedes, mainly due to climate change and increasing overlap between urban and wild habitats, places nearly half of the world's population at risk for DENV infection. After a bloodmeal from a DENV-infected host, the virus enters the mosquito midgut. Next, the virus migrates to, and replicates in, other tissues, like salivary glands. Successful viral transmission occurs when the infected mosquito takes another blood meal on a susceptible host and DENV is released from the salivary gland via saliva into the skin. During viral dissemination in the mosquito and transmission to a new mammalian host, DENV interacts with a variety of vector proteins, which are uniquely important during each phase of the viral cycle. Our study focuses on the interaction between DENV particles and protein components in the A. aegypti vector. We performed a mass spectrometry assay where we identified a set of A. aegypti salivary gland proteins which potentially interact with the DENV virion. Using dsRNA to silence gene expression, we analyzed the role of these proteins in viral infectivity. Two of these candidates, a synaptosomal-associated protein (AeSNAP) and a calcium transporter ATPase (ATPase) appear to play a role in viral replication both in vitro and in vivo, observing a ubiquitous expression of these proteins in the mosquito. These findings suggest that AeSNAP plays a protective role during DENV infection of mosquitoes and that ATPase protein is required for DENV during amplification within the vector.

AgBR1 and NeSt1 antisera protect mice from Aedes aegypti-borne Zika infection.

Zika virus(ZIKV) is primarily spread by Aedes. aegyptimosquitoes. Infection with ZIKV can result in diverse clinical symptoms in humans, ranging from mild to severe. Previously, we demonstrated that passive immunization against A. aegypti AgBR1 or NeSt1 antiserum, two mosquito saliva proteins that are transmitted with the virus, conferred partial protection against ZIKV in mice. Each individual antiserum altered the early host response in the skin and reduced viremia. Here, we show that passive immunization with a combination of AgBR1- and NeSt1-specific antibodies enhanced survival and reduced the viral burden in blood, thereby protecting mice from mosquito-borne ZIKV infection. This finding suggests that targeting a combination of mosquito saliva proteins, with AgBR1 and NeSt1 as model antigens, may be used as a vaccine strategy to help prevent mosquito-borne ZIKV infection.

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.

An Ixodes scapularis Protein Disulfide Isomerase Contributes to Borrelia burgdorferi Colonization of the Vector.

Borrelia burgdorferi causes Lyme disease, the most common tick-transmitted illness in North America. When Ixodes scapularis feed on an infected vertebrate host, spirochetes enter the tick gut along with the bloodmeal and colonize the vector. Here, we show that a secreted tick protein, I. scapularisprotein disulfide isomerase A3 (IsPDIA3), enhances B. burgdorferi colonization of the tick gut. I. scapularis ticks in which ispdiA3 has been knocked down using RNA interference have decreased spirochete colonization of the tick gut after engorging on B. burgdorferi-infected mice. Moreover, administration of IsPDIA3 antiserum to B. burgdorferi-infected mice reduced the ability of spirochetes to colonize the tick when feeding on these animals. We show that IsPDIA3 modulates inflammatory responses at the tick bite site, potentially facilitating spirochete survival at the vector-host interface as it exits the vertebrate host to enter the tick gut. These data provide functional insights into the complex interactions between B. burgdorferi and its arthropod vector and suggest additional targets to interfere with the spirochete life cycle.

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.

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.

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.

Vaccination with Aedes aegypti AgBR1 Delays Lethal Mosquito-Borne Zika Virus Infection in Mice.

Zika Virus (ZIKV) is transmitted primarily by Aedes aegypti mosquitoes, resulting in asymptomatic infection, or acute illness with a fever and headache, or neurological complications, such as Guillain-Barre syndrome or fetal microcephaly. Previously, we determined that AgBR1, a mosquito salivary protein, induces inflammatory responses at the bite site, and that passive immunization with AgBR1 antiserum influences mosquito-transmitted ZIKV infection. Here, we show that the active immunization of mice with AgBR1 adjuvanted with aluminum hydroxide delays lethal mosquito-borne ZIKV infection, suggesting that AgBR1 may be used as part of a vaccine to combat ZIKV.

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.