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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-a was significantly increased in porcine alveolar macrophages infected with ASFV-989 while pigs infectedwith Georgia 2007/1 showed higher IFN-a 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/β 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/β signaling and provide molecular mechanisms underlying the role of MGF505-4R as a virulence factor. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Copyright of Summa, Animali da Reddito is the property of Point Veterinaire Italie s.r.l. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

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. [ABSTRACT FROM AUTHOR]

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-1β) 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-1β 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Résumé: 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. 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. [ABSTRACT FROM AUTHOR]

Résumé: 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. 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. [ABSTRACT FROM AUTHOR]

Bluetongue is an infectious, noncontagious, arthropodborne viral disease of domestic and wild ruminants (1). Twenty-six distinct bluetongue virus (BTV) serotypes have been identified (2). The first detection of bluetongue virus [...]

To the Editor: In 2011, Schmallenberg virus (SBV) emerged in Europe (1); the virus spread into France in January 2012 (2). During January March 2012, a total of >1,000 cases [...]

Abstract Since its introduction into northern Europe in 2006, bluetongue has become a major threat to animal health. While the efficacy of commercial vaccines has been clearly demonstrated in livestock, little is known regarding the effect of maternal immunity on vaccinal efficacy. Here, we have investigated the duration and amplitude of colostral antibody-induced immunity in calves born to dams vaccinated against bluetongue virus serotype 8 (BTV-8) and the extent of colostral antibody-induced interference of vaccination in these calves. Twenty-two calf-cow pairs were included in this survey. The median age at which calves became seronegative for BTV was 84 and 112 days as assayed by seroneutralisation test (SNT) and VP7 BTV competitive ELISA (cELISA), respectively. At the mean age of 118 days, 13/22 calves were immunized with inactivated BTV-8 vaccine. In most calves vaccination elicited a weak immune response, with seroconversion in only 3/13 calves. The amplitude of the humoral response to vaccination was inversely proportional to the maternal antibody level prior to vaccination. Thus, the lack of response was attributed to the persistence of virus-specific colostral antibodies that interfered with the induction of the immune response. These data suggest that the recommended age for vaccination of calves born to vaccinated dams needs to be adjusted in order to optimize vaccinal efficacy.

This document is a correction notice for an article titled "Host-Specific Interplay between Foot-and-Mouth Disease Virus 3D Polymerase and the Type-I Interferon Pathway" published in the journal Viruses. The correction addresses an error in Figure 4 of the original publication, where the graphs were switched with those in Figure S1. The corrected version of Figure 4 has been provided, and the authors state that this correction does not affect the scientific conclusions of the study. The original publication has been updated accordingly. [Extracted from the article]

Bluetongue (BT) and epizootic haemorrhagic disease (EHD) are vector‐borne viral diseases affecting domestic and wild ruminants. Both are notifiable under OIE rules. BT and EHD viruses (BTV and EHDV) are closely related Orbiviruses with structural, antigenic and molecular similarities. Both viruses can produce analogous clinical signs in susceptible animals. Serological tests are commonly used for BT and EHD diagnosis and surveillance. Competitive ELISA (c‐ELISA) is the most widely used serological test for the specific detection of BTV or EHDV viral protein 7 (VP7) antibodies (Abs). The specificity and sensitivity of the BTV c‐ELISA kits available on the market are recognized for the detection of BTV Abs. Concerning EHD, a single commercial EHDV c‐ELISA kit (ELISA A kit) commonly used for diagnosis in Europe and Africa was available between 2011 and 2018 but is now no longer on the market. In this study, we evaluated a new commercial c‐ELISA to detect ruminant EHDV VP7 Abs in 2,199 serum samples from cattle, sheep, goats, wild deer and zoo animals. The results showed that this ELISA kit is specific and can detect the presence of IgG anti‐EHDV VP7 with a very good diagnostic specificity and a satisfactory sensitivity in domestic ruminants, zoo animals and wild deer. Therefore, the evaluated c‐ELISA can detect the introduction of EHDV into an area where BTV‐seropositive domestic animals are present. The performance of this kit is similar to that of the c‐ELISA A kit and can thus be used for diagnosis. [ABSTRACT FROM AUTHOR]

Bluetongue virus serotype 8 (BTV‐8) caused an epizootic in Europe in 2006/09. Transplacental transmission of BTV‐8 was demonstrated leading to abortions, congenital malformations or nervous clinical signs in newborn calves. BTV‐8 re‐emerged in France in 2015. Although the re‐emergent strain is nearly genetically identical to the one that had circulated in 2006/2009, it has caused very few clinical cases. However, from mid‐December 2018 to April 2019, cases of calves with congenital malformations or displaying nervous clinical signs occurred in some departments (French administrative unit) in mainland France. Blood samples from these animals were sent to local laboratories, and the positive ones were confirmed at the French Bluetongue reference laboratory (BT‐NRL). Out of 580 samples found positive at the local laboratories, 544 were confirmed as RT‐PCR BTV‐8 positive. The 36 samples found positive in the local laboratories and negative in the BT‐NRL were all at the limit of RT‐PCR detection. Hundred eighty‐eight of the confirmed samples were also tested for the presence of Schmallenberg virus (SBV) and bovine virus diarrhoea virus (BVDV) infection: 4 were found positive for BVDV and none for SBV. The main clinical signs recorded for 244 calves, for which a reporting form was completed by veterinarians, included nervous clinical signs (81%), amaurosis (72%) and decrease/ no suckling reflex (40%). Hydranencephaly and microphthalmia were reported in 19 calves out of 27 in which a necropsy was practiced after death or euthanasia. These results indicate that the re‐emergent strain of BTV‐8 can cross the transplacental barrier and cause congenital malformations or nervous clinical signs in calves. [ABSTRACT FROM AUTHOR]

Competitive‐ELISA (c‐ELISA) is the most widely used serological test for the detection of Bluetongue virus (BTV) viral protein 7 (VP7) antibodies (Ab). However, these BTV c‐ELISAs cannot to distinguish between IgG and IgM. IgM Ab are generated shortly after the primary immune response against an infectious agent, indicating a recent infection or exposure to antigens, such as after vaccination. Because the BTV genome or anti‐VP7 Ab can be detected in ruminant blood months after infection, BTV diagnostic tools cannot discriminate between recent and old infections. In this study, we evaluated an IgM‐capture ELISA prototype to detect ruminant anti‐BTV VP7 IgM on 1,650 serum samples from cattle, sheep, or goats. Animals were BTV‐naive, infected, or/and vaccinated with BTV‐1, ‐2, ‐4, ‐8, ‐9, ‐16, or ‐27, and we also included 30 sera from cattle infected with the Epizootic haemorrhagic disease virus (EHDV) serotype 6. Results demonstrated that this ELISA kit is specific and can detect the presence of IgM with satisfactory diagnostic specificity and sensitivity from 1 to 5 weeks after BTV infection in domestic ruminants (for goats and cattle; for sheep, at least up to 24 days). The peak of anti‐VP7 IgM was reached when the level of infectious viruses and BTV RNA in blood were the highest. The possibility of detecting BTV‐RNA in IgM‐positive sera allows the amplification and sequencing of the partial RNA segment 2 (encoding the serotype specific to VP2) to determine the causative BTV serotype/strain. Therefore, BTV IgM ELISA can detect the introduction of BTV (or EHDV) in an area with BTV‐seropositive domestic animals regardless of their serological BTV status. This approach may also be of particular interest for retrospective epidemiological studies on frozen serum samples. [ABSTRACT FROM AUTHOR]

Copyright of Virologie is the property of John Libbey Eurotext Ltd. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

To the Editor: After unexpected emergence of bluetongue virus serotype 8 in northern Europe in 2006 (7), another arbovirus, Schmallenberg virus (SBV), which is transmitted by Culicoides spp. biting midges, [...]

Abstract: In November 2017, a 15‐day‐old calf located in France (Haute‐Savoie department) was found positive for bluetongue virus (BTV) RNA by RT‐PCR. Laboratory investigations allowed the isolation and identification of the serotype: BTV‐4. The analysis of the full viral genome showed that all the 10 genome segments were closely related to BTV‐4 strains involved in a large BT outbreak in the Balkan Peninsula, in Italy since 2014 and in Corsica since the end of October 2016. These results together with epidemiological data suggest that BTV‐4 has been introduced to mainland France from Corsica or Italy where BTV‐4 outbreaks have been reported in summer and autumn 2016. This is the first report of the introduction of BTV‐4 in mainland France. [ABSTRACT FROM AUTHOR]

Since the unexpected emergence of BTV-8 in Northern Europe and the incursion of BTV-8 and 1 in France in 2006-2007, molecular diagnosis has considerably evolved. Several real-time RT- PCR (rt RT- PCR) methods have been developed and published, and are currently being used in many countries across Europe for BTV detection and typing. In France, the national reference laboratory ( NRL) for orbiviruses develops and validates 'ready-to-use' kits with private companies for viral RNA detection. The regional laboratories network that was set up to deal with a heavy demand for analyses has used these available kits. From 2007, ring tests were organized to monitor the performance of the French laboratories. This study presents the results of 63 regional laboratories in the ring trial organized in 2016. Blood samples were sent to the laboratories. Participants were asked to use the rt RT- PCR methods in place in their laboratory, for detection of all BTV serotypes and specifically BTV-8. The French regional laboratories are able to detect and genotype BTV in affected animals. Despite the use of several methods (i.e. RNA extraction and different commercial rt RT- PCRs), the network is homogeneous. The ring trial demonstrated that the French regional veterinary laboratories have reliable and robust BTV diagnostic tools for BTV genome detection. [ABSTRACT FROM AUTHOR]

Background: After viral infection and the stimulation of some pattern-recognition receptors, TANK-binding kinase I (TBK1) is activated by K63-linked polyubiquitination followed by trans-autophosphorylation. While the activated TBK1 induces type I interferon production by phosphorylating the transcription factor IRF3, the precise molecular mechanisms underlying TBK1 activation remain unclear. Results: We report here the localization of the ubiquitinated and phosphorylated active form of TBK1 to the Golgi apparatus after the stimulation of RIG-I-like receptors (RLRs) or Toll-like receptor-3 (TLR3), due to TBK1 K63-linked ubiquitination on lysine residues 30 and 401. The ubiquitin-binding protein optineurin (OPTN) recruits ubiquitinated TBK1 to the Golgi apparatus, leading to the formation of complexes in which TBK1 is activated by trans-autophosphorylation. Indeed, OPTN deficiency in various cell lines and primary cells impairs TBK1 targeting to the Golgi apparatus and its activation following RLR or TLR3 stimulation. Interestingly, the Bluetongue virus NS3 protein binds OPTN at the Golgi apparatus, neutralizing its activity and thereby decreasing TBK1 activation and downstream signaling. Conclusions: Our results highlight an unexpected role of the Golgi apparatus in innate immunity as a key subcellular gateway for TBK1 activation after RNA virus infection. [ABSTRACT FROM AUTHOR]

Epizootic haemorrhagic disease virus (EHDV) may cause severe clinical episodes in some species of deer and sometimes in cattle. Laboratory diagnosis provides a basis for the design and timely implementation of disease control measures. There are seven distinct EHDV serotypes, VP2 coding segment 2 being the target for serotype specificity. This paper reports the development and validation of eight duplex real-time RT-PCR assays to simultaneously amplify the EHDV target (S9 for the pan-EHDV real-time RT-PCR assay and S2 for the serotyping assays) and endogenous control gene Beta-actin. Analytical and diagnostic sensitivity and specificity, inter- and intra-assay variation and efficiency were evaluated for each assay. All were shown to be highly specific and sensitive. [ABSTRACT FROM AUTHOR]

A newly developed Enzym Like Immuno Sorbant Assay (ELISA) based on the recombinant nucleocapsid protein (N) of Schmallenberg virus (SBV) was evaluated and validated for the detection of SBV-specific IgG antibodies in ruminant sera by three European Reference Laboratories. Validation data sets derived from sheep, goat and bovine sera collected in France and Germany (n = 1515) in 2011 and 2012 were categorized according to the results of a virus neutralization test (VNT) or an indirect immuno-flurorescence assay (IFA). The specificity was evaluated with 1364 sera from sheep, goat and bovine collected in France and Belgium before 2009. Overall agreement between VNT and ELISA was 98.9% and 98.3% between VNT and IFA, indicating a very good concordance between the different techniques. Although cross-reactions with other Orthobunyavirus from the Simbu serogroup viruses might occur, it is a highly sensitive, specific and robust ELISA-test validated to detect anti-SBV antibodies. This test can be applied for SBV sero-diagnostics and disease-surveillance studies in ruminant species in Europe. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Rotavirus mRNAs are capped but not polyadenylated, and viral proteins are translated by the cellular translation machinery. This is accomplished through the action of the viral nonstructural protein NSP3, which specifically binds the 3′ consensus sequence of viral mRNAs and interacts with the eukaryotic translation initiation factor eIF4G I. To further our understanding of the role of NSP3 in rotavirus replication, we looked for other cellular proteins capable of interacting with this viral protein. Using the yeast two-hybrid assay, we identified a novel cellular protein-binding partner for rotavirus NSP3. This 110-kDa cellular protein, named RoXaN (rotavirus X protein associated with NSP3), contains a minimum of three regions predicted to be involved in protein-protein or nucleic acid-protein interactions. A tetratricopeptide repeat region, a protein-protein interaction domain most often found in multiprotein complexes, is present in the amino-terminal region. In the carboxy terminus, at least five zinc finger motifs are observed, further suggesting the capacity of RoXaN to bind other proteins or nucleic acids. Between these two regions exists a paxilfin leucine-aspartate repeat (LD) motif which is involved in protein-protein interactions. RoXaN is capable of interacting with NSP3 in vivo and during rotavirus infection. Domains of interaction were mapped and correspond to the dimerization domain of NSP3 (amino acids 163 to 237) and the LD domain of RoXaN (amino acids 244 to 341). The interaction between NSP3 and RoXaN does not impair the interaction between NSP3 and eIF4G I, and a ternary complex made of NSP3, RoXaN, and eIF4G I can be detected in rotavirus-infected cells, implicating RoXaN in translation regulation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]