Exploring type I interferon pathway: virulent vs. attenuated strain of African swine fever virus revealing a novel function carried by MGF505-4R.

African swine fever virus represents a significant reemerging threat to livestock populations, as its incidence and geographic distribution have surged over the past decade in Europe, Asia, and Caribbean, resulting in substantial socio-economic burdens and adverse effects on animal health and welfare. In a previous report, we described the protective properties of our newly thermo-attenuated strain (ASFV-989) in pigs against an experimental infection of its parental Georgia 2007/1 virulent strain. In this new study, our objective was to characterize the molecular mechanisms underlying the attenuation of ASFV-989. We first compared the activation of type I interferon pathway in response to ASFV-989 and Georgia 2007/1 infections, employing both in vivo and in vitro models. Expression of IFN-α was significantly increased in porcine alveolar macrophages infected with ASFV-989 while pigs infected with Georgia 2007/1 showed higher IFN-α than those infected by ASFV-989. We also used a medium-throughput transcriptomic approach to study the expression of viral genes by both strains, and identified several patterns of gene expression. Subsequently, we investigated whether proteins encoded by the eight genes deleted in ASFV-989 contribute to the modulation of the type I interferon signaling pathway. Using different strategies, we showed that MGF505-4R interfered with the induction of IFN-α/ß pathway, likely through interaction with TRAF3. Altogether, our data reveal key differences between ASFV-989 and Georgia 2007/1 in their ability to control IFN-α/ß signaling and provide molecular mechanisms underlying the role of MGF505-4R as a virulence factor.

Detection, Characterization and Sequencing of BTV Serotypes Circulating in Cuba in 2022.

In Cuba, despite a high sero-prevalence of bluetongue virus (BTV), circulating serotypes remain unknown. The aim of this study was to identify circulating BTV serotypes in farms throughout the western region of Cuba. Blood samples were collected from 200 young cattle and sheep between May and July 2022 for virological analyses (PCR, viral isolation and virus neutralization) and genome sequencing. The results confirmed viral circulation, with viro-prevalence of 25% for BTV. The virus was isolated from 18 blood samples and twelve BTV serotypes were identified by sequencing RT-PCR products targeting the segment 2 of the BTV genome (BTV-1, 2, 3, 6, 10, 12, 13, 17, 18, 19, 22 and 24). Finally, the full genome sequences of 17 Cuban BTV isolates were recovered using a Sequence Independent Single Primer Amplification (SISPA) approach combined to MinION Oxford Nanopore sequencing technology. All together, these results highlight the co-circulation of a wide diversity of BTV serotypes in a quite restricted area and emphasize the need for entomological and livestock surveillance, particularly in light of recent changes in the global distribution and nature of BTV infections.

Experimental infection of cattle, sheep, and goats with the newly emerged epizootic hemorrhagic disease virus serotype 8.

Epizootic hemorrhagic disease virus serotype 8 (EHDV-8) emerged in Europe for the first time in late 2022. In this study, we investigated the kinetics of EHDV-8 infection in cattle, sheep, and goats.  Following experimental infection with EHDV-8, four out of five calves displayed fever, while another calf exhibited ulcerative and crusty lesions of the muzzle. RNAemia peaked at day 7 post infection in all calves and remained relatively stable till the end of the study, at 78 days post infection. Infectious virus was isolated up to 21 days post infection in one calf. As far as small ruminants are concerned, one sheep experienced fever and two out of five had consistent RNAemia that lasted until the end of the study. Remarkably, infectious virus was evidenced at day 7 post infection in one sheep. In goats, no RNA was observed. All infected animals seroconverted, and a neutralizing immune response was observed in all species, with calves exhibiting a more robust response than sheep and goats. Our study provides insights into the kinetics of EHDV-8 infection and the host immune responses. We also highlight that sheep may also play a role in EHDV-8 epidemiology. Altogether, the data gathered in this study could have important implications for disease control and prevention strategies, providing crucial information to policy makers to mitigate the impact of this viral disease on livestock.

Correction: Sarry et al. Host-Specific Interplay between Foot-and-Mouth Disease Virus 3D Polymerase and the Type-I Interferon Pathway. Viruses 2023, 15, 666.

In the original publication [...].

Activation of Inflammasome during Bluetongue Virus Infection.

Bluetongue virus (BTV), a double-stranded RNA virus belonging to the Sedoreoviridae family, provokes an economically important disease in ruminants. In this study, we show that the production of activated caspase-1 and interleukin 1 beta (IL-1ß) is induced in BTV-infected cells. This response seems to require virus replication since a UV-inactivated virus is unable to activate this pathway. In NLRP3-/- cells, BTV could not trigger further IL-1ß synthesis, indicating that it occurs through NLRP3 inflammasome activation. Interestingly, we observed differential activation levels in bovine endothelial cells depending on the tissue origin. In particular, inflammasome activation was stronger in umbilical cord cells, suggesting that these cells are more prone to induce the inflammasome upon BTV infection. Finally, the strength of the inflammasome activation also depends on the BTV strain, which points to the importance of viral origin in inflammasome modulation. This work reports the crucial role of BTV in the activation of the NLRP3 inflammasome and further shows that this activation relies on BTV replication, strains, and cell types, thus providing new insights into BTV pathogenesis.

Circulation of Bluetongue Virus Serotypes 1, 4, 8, 10 and 16 and Epizootic Hemorrhagic Disease Virus in the Sultanate of Oman in 2020-2021.

The circulation of Bluetongue (BT) and Epizootic Hemorrhagic Disease (EHD) in the Middle East has already been reported following serological analyses carried out since the 1980s, mostly on wild ruminants. Thus, an EHD virus (EHDV) strain was isolated in Bahrain in 1983 (serotype 6), and more recently, BT virus (BTV) serotypes 1, 4, 8 and 16 have been isolated in Oman. To our knowledge, no genomic sequence of these different BTV strains have been published. These same BTV or EHDV serotypes have circulated and, for some of them, are still circulating in the Mediterranean basin and/or in Europe. In this study, we used samples from domestic ruminant herds collected in Oman in 2020 and 2021 for suspected foot-and-mouth disease (FMD) to investigate the presence of BTV and EHDV in these herds. Sera and whole blood from goats, sheep and cattle were tested for the presence of viral genomes (by PCR) and antibodies (by ELISA). We were able to confirm the presence of 5 BTV serotypes (1, 4, 8, 10 and 16) and the circulation of EHDV in this territory in 2020 and 2021. The isolation of a BTV-8 strain allowed us to sequence its entire genome and to compare it with another BTV-8 strain isolated in Mayotte and with homologous BTV sequences available on GenBank.

Emergence of epizootic hemorrhagic disease in Europe.

Epizootic hemorrhagic disease (EHD) is a non-contagious arthropod-borne disease transmitted by blood-sucking midges of the genus Culicoides. It affects domestic and wild ruminants, mainly white-tailed deer and cattle. At the end of October and in November 2022, outbreaks of EHD were confirmed in several cattle farms in Sardinia and Sicily. This is the first detection of EHD in Europe. The loss of free status and the lack of effective prophylactic measures could have significant economic consequences for infected countries.

[Emergence of epizootic hemorrhagic disease in Europe]. / Émergence de la maladie hémorragique épizootique en Europe.

Epizootic hemorrhagic disease (EHD) is a non-contagious arthropod-borne disease transmitted by blood-sucking midges of the genus Culicoides. It affects domestic and wild ruminants, mainly white-tailed deer and cattle. At the end of October and in November 2022, outbreaks of EHD were confirmed in several cattle farms in Sardinia and Sicily. This is the first detection of EHD in Europe. The loss of free status and the lack of effective prophylactic measures could have significant economic consequences for infected countries.

Host-Specific Interplay between Foot-and-Mouth Disease Virus 3D Polymerase and the Type-I Interferon Pathway.

Foot-and-mouth disease (FMD) is a highly contagious viral disease affecting cloven-hoofed animals. One of the issues related to this disease is the persistence of its causative agent, foot-and-mouth disease virus (FMDV). While the mechanisms of FMDV persistence remain unclear, there are clues that it may be related to protein-protein interactions (PPI) between viral proteins and cellular proteins involved in the interferon (IFN) response. Since FMDV persistence has been described in cattle, sheep and goats but not in swine, we screened PPI involving FMDV proteins and sixteen major type-I IFN pathway proteins from these four species by nanoluciferase-2-hybrid complementation assay, in order to identify new PPI and determine their host specificity. As the results concerning the 3Dpol were the most interesting in view of the limited data concerning its role in immune escape, we decided to focus particularly on this protein. The identified PPI were confirmed by GST pull-down. We identified PPI between 3Dpol and seven IFN pathway proteins, namely, IKKα, IKKε, IRF3, IRF7, NEMO, MDA5 and MAVS. These PPI are conserved among the four studied species, with the exception of the one between 3Dpol and MAVS, which was only found with the swine protein. We also showed, using luciferase reporter assays, that 3Dpol could inhibit the induction phase of the IFN pathway. These results demonstrate, for the first time, a putative role for 3Dpol in FMDV innate immune escape.

Different viral genes modulate virulence in model mammal hosts and Culex pipiens vector competence in Mediterranean basin lineage 1 West Nile virus strains.

West Nile virus (WNV) is a single-stranded positive-sense RNA virus (+ssRNA) belonging to the genus Orthoflavivirus. Its enzootic cycle involves mosquito vectors, mainly Culex, and wild birds as reservoir hosts, while mammals, such as humans and equids, are incidental dead-end hosts. It was first discovered in 1934 in Uganda, and since 1999 has been responsible for frequent outbreaks in humans, horses and wild birds, mostly in America and in Europe. Virus spread, as well as outbreak severity, can be influenced by many ecological factors, such as reservoir host availability, biodiversity, movements and competence, mosquito abundance, distribution and vector competence, by environmental factors such as temperature, land use and precipitation, as well as by virus genetic factors influencing virulence or transmission. Former studies have investigated WNV factors of virulence, but few have compared viral genetic determinants of pathogenicity in different host species, and even fewer have considered the genetic drivers of virus invasiveness and excretion in Culex vector. In this study, we characterized WNV genetic factors implicated in the difference in virulence observed in two lineage 1 WNV strains from the Mediterranean Basin, the first isolated during a significant outbreak reported in Israel in 1998, and the second from a milder outbreak in Italy in 2008. We used an innovative and powerful reverse genetic tool, e.g., ISA (infectious subgenomic amplicons) to generate chimeras between Israel 1998 and Italy 2008 strains, focusing on non-structural (NS) proteins and the 3'UTR non-coding region. We analyzed the replication of these chimeras and their progenitors in mammals, in BALB/cByJ mice, and vector competence in Culex (Cx.) pipiens mosquitoes. Results obtained in BALB/cByJ mice suggest a role of the NS2B/NS3/NS4B/NS5 genomic region in viral attenuation in mammals, while NS4B/NS5/3'UTR regions are important in Cx. pipiens infection and possibly in vector competence.

Serological Responses in Cattle following Booster Vaccination against Serotypes 4 and 8 Bluetongue Virus with Two Bivalent Commercial Inactivated Vaccines.

Since the outbreak of bluetongue in Northern Europe in 2006, numerous outbreaks involving several serotypes have been observed. Since 2008, compulsory or voluntary vaccination campaigns with inactivated vaccines have been carried out to eradicate these serotypes. In France, serotypes 8 and 4 have been enzootic since 2017, and currently, the majority of vaccinations take place in the context of animal movements, to comply with the regulations of the importing countries. Several vaccine manufacturers have developed inactivated vaccines against serotypes 4 and 8 (mono or bivalent). In this study, we investigated and compared the serological responses to a booster vaccination with two different bivalent inactivated vaccines (BTVPUR suspension injectable® 4 + 8, Boehringer Ingelheim or SYVAZUL ® BTV 4 + 8, Biové) following a primary vaccination with BTVPUR® 4 + 8 in the previous year. The results show that using an alternative vaccine for booster vaccination is at least as effective as using the homologous vaccine. Indeed, the antibody response against BTV-8 is higher in the case of a heterologous vaccination and identical for BTV-4. This information could allow more flexibility in the choice of vaccines used for booster vaccination, particularly in cases where homologous vaccines are in short supply or unavailable.

African horse sickness: an ancient disease for a current threat / La peste équine : une maladie ancienne pour une menace actuelle

African horse sickness (AHS) is a major arthropod-borne disease that causes significant losses in horses in sub-Saharan Africa. It is caused by the African horse sickness virus (AHSV), which is transmitted during a blood meal by Culicoides biting midges. The distribution of historical African culicoid vectors increases due to global warming. In addition, recent (Thailand, 2020) and earlier (Iberian Peninsula, 1965-66/1987-90) AHS outbreaks outside Africa demonstrate the adaptation of the virus to endogenous species in AHS-free regions, similar to what has been observed for bluetongue disease in recent decades. Therefore, many regions are considered at risk of introduction of AHS which could have important economic consequences for the equine industry. Overall, this prone the European Union to launch research programs to get better diagnostic and prophylactic tools.

La peste équine est une arbovirose majeure qui entraîne des pertes importantes chez les chevaux en Afrique subsaharienne. Elle est provoquée par le virus de la peste équine (African horse sickness virus, AHSV) dont la transmission s'effectue au cours d'un repas sanguin par des petits moucherons hématophages appartenant au genre Culicoides. En outre, les espèces vectrices historiques de culicoïdes présentes en Afrique voient leur aire de répartition s'étendre en lien avec le réchauffement climatique à l'échelle mondiale. Par ailleurs, des épisodes épizootiques récents (Thaïlande, 2020) ou un peu plus anciens (péninsule ibérique, 1965-66/1987-90) en dehors du continent africain soulignent la capacité d'adaptation du virus à des espèces vectrices autochtones, à l'instar de ce qui a été observé pour la fièvre catarrhale ovine ces dernières décennies. Ces facteurs laissent craindre à tout moment une introduction de la peste équine dans des régions indemnes. L'urgence est donc donnée actuellement par l'Union européenne pour se doter de meilleurs outils diagnostiques et prophylactiques afin de prévenir des conséquences économiques brutales pour l'industrie équine.

Modulation of the innate immune response by African swine fever virus / Modulation de la réponse immunitaire innée par le virus de la peste porcine africaine

African swine fever (ASF) is a highly pathogenic disease causing haemorrhagic fever in domestic and wild swine. It is responsible for numerous epizootics, particularly in Europe and Asia, causing major economic losses for the pig industry. African Swine Fever virus (ASFV) is the etiological agent responsible for this disease. It is a very large double-stranded DNA virus, encoding for over 150 proteins. Various studies have shown that there is a close relationship between the ability of some viral proteins to inhibit the type I interferon (IFNI) response and the attenuation and virulence processes of ASFV. This review describes the mechanisms of inhibition of the IFN-I response by ASFV proteins, which provide a molecular explanation of how ASFV escapes the innate immune response.

La peste porcine africaine (PPA) est une maladie hautement pathogène causant une fièvre hémorragique chez les suidés domestiques et sauvages. Elle est responsable de nombreuses épizooties notamment en Europe et en Asie, causant de grandes pertes économiques pour la filière porcine. Le virus de la peste porcine africaine (ASFV) est l'agent étiologique responsable de cette maladie. C'est un virus avec un génome à ADN double brin de grande taille, codant pour plus de 150 protéines. Différents travaux ont montré qu'il existe une étroite relation entre la capacité de certaines protéines virales à inhiber la réponse interféron de type I (IFN-I) et les processus d'atténuation et de virulence pour l'ASFV. Cette revue décrit les mécanismes d'inhibition de la réponse IFN-I par les protéines d'ASFV permettant d'expliquer sur le plan moléculaire l'échappement à la réponse immunitaire innée.

Foot-and-Mouth Disease Virus: Molecular Interplays with IFN Response and the Importance of the Model.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals with a significant socioeconomic impact. One of the issues related to this disease is the ability of its etiological agent, foot-and-mouth disease virus (FMDV), to persist in the organism of its hosts via underlying mechanisms that remain to be elucidated. The establishment of a virus-host equilibrium via protein-protein interactions could contribute to explaining these phenomena. FMDV has indeed developed numerous strategies to evade the immune response, especially the type I interferon response. Viral proteins target this innate antiviral response at different levels, ranging from blocking the detection of viral RNAs to inhibiting the expression of ISGs. The large diversity of impacts of these interactions must be considered in the light of the in vitro models that have been used to demonstrate them, some being sometimes far from biological systems. In this review, we have therefore listed the interactions between FMDV and the interferon response as exhaustively as possible, focusing on both their biological effect and the study models used.

Comparative Virus-Host Protein Interactions of the Bluetongue Virus NS4 Virulence Factor.

Bluetongue virus (BTV) is the etiologic agent of a non-contagious arthropod-borne disease transmitted to wild and domestic ruminants. BTV induces a large panel of clinical manifestations ranging from asymptomatic infection to lethal hemorrhagic fever. Despite the fact that BTV has been studied extensively, we still have little understanding of the molecular determinants of BTV virulence. In our report, we have performed a comparative yeast two-hybrid (Y2H) screening approach to search direct cellular targets of the NS4 virulence factor encoded by two different serotypes of BTV: BTV8 and BTV27. This led to identifying Wilms' tumor 1-associated protein (WTAP) as a new interactor of the BTV-NS4. In contrast to BTV8, 1, 4 and 25, NS4 proteins from BTV27 and BTV30 are unable to interact with WTAP. This interaction with WTAP is carried by a peptide of 34 amino acids (NS422-55) within its putative coil-coiled structure. Most importantly, we showed that binding to WTAP is restored with a chimeric protein where BTV27-NS4 is substituted by BTV8-NS4 in the region encompassing residue 22 to 55. We also demonstrated that WTAP silencing reduces viral titers and the expression of viral proteins, suggesting that BTV-NS4 targets a cellular function of WTAP to increase its viral replication.

Development and Validation of an ELISA for the Detection of Bluetongue Virus Serotype 4-Specific Antibodies.

In this article, we describe the development and evaluation of a double antigen sandwich enzyme-linked immunosorbent assay (ELISA) able to detect serotype 4-specific antibodies from BTV-4 infected or vaccinated animals using a recombinant BTV-4 VP2 protein. The coding sequence of VP2 was inserted into a pVote plasmid by recombination in the Gateway® cloning system. Vaccinia virus (VacV) was used as a vector for the expression of the recombinant VP2. After production in BSR cells, recombinant VP2 was purified by immunoprecipitation using a FLAG tag and then used both as the coated ELISA antigen and as the HRP-tagged conjugate. The performance of the ELISA was evaluated with 1186 samples collected from BTV negative, infected or vaccinated animals. The specificity and sensitivity of the BTV-4 ELISA were above the expected standards for the detection of anti-BTV-4 VP2 antibodies in animals reared in Europe or in the Mediterranean basin. Cross-reactions were observed with reference sera for serotypes 10 and 20, and to a lesser extent with serotypes 12, 17 and 24, due to their genetic proximity to serotype 4. Nevertheless, these serotypes have never been detected in Europe and the Mediterranean area. This ELISA, which requires only the production of a recombinant protein, can be used to detect BTV serotype 4-specific antibodies and is therefore an attractive alternative diagnostic method to serum neutralization.