Emergence of Bluetongue Virus Serotype 3, the Netherlands, September 2023.

Since 1998, notifiable bluetongue virus (BTV) serotypes 1-4, 6, 8, 9, 11, and 16 have been reported in Europe. In August 2006, a bluetongue (BT) outbreak caused by BTV serotype 8 began in northwestern Europe. The Netherlands was declared BT-free in February 2012, and annual monitoring continued. On September 3, 2023, typical BT clinical manifestations in sheep were notified to the Netherlands Food and Product Safety Consumer Authority. On September 6, we confirmed BTV infection through laboratory diagnosis; notifications of clinical signs in cattle were also reported. We determined the virus was serotype 3 by whole-genome sequencing. Retrospective analysis did not reveal BTV circulation earlier than September. The virus source and introduction route into the Netherlands remains unknown. Continuous monitoring and molecular diagnostic testing of livestock will be needed to determine virus spread, and new prevention strategies will be required to prevent BTV circulation within the Netherlands and Europe.

Pathogenesis of wild-type- and vaccine-based recombinant peste des petits ruminants virus (PPRV) expressing EGFP in experimentally infected domestic goats.

Peste des petits ruminants virus (PPRV) is a highly contagious morbillivirus related to measles and canine distemper virus, mostly affecting small ruminants. The corresponding PPR disease has a high clinical impact in goats and is characterized by fever, oral and nasal erosions, diarrhoea and pneumonia. In addition, massive infection of lymphoid tissues causes lymphopaenia and immune suppression. This results in increased susceptibility to secondary bacterial infections, explaining the observed high mortality in some outbreaks. We studied the pathogenesis of PPR by experimental inoculation of Dutch domestic goats with a recombinant virulent PPRV strain modified to express EGFP and compared it to an EGFP-expressing vaccine strain of PPRV. After intratracheal inoculation with virulent PPRV, animals developed fever, viraemia and leucopaenia, and shed virus from the respiratory and gastro-intestinal tracts. Macroscopic evaluation of fluorescence at the peak of infection 7 days post-inoculation (dpi) showed prominent PPRV infection of the respiratory tract, lymphoid tissues, gastro-intestinal tract, mucosae and skin. Flow cytometry of PBMCs collected over time demonstrated a cell-associated viraemia mediated by infected lymphocytes. At 14 dpi, pathognomonic zebra stripes were detected in the mucosa of the large intestine. In contrast, vaccine strain-inoculated goats remained largely macroscopically fluorescence negative and did not present clinical signs. A low-level viraemia was detected by flow cytometry, but at necropsy no histological lesions were observed. Animals from both groups seroconverted as early as 7 dpi and sera efficiently neutralized virulent PPRV in vitro. Combined, this work presents a study of the pathogenesis of wild type- and vaccine-based PPRV in its natural host. This study shows the strength of recombinant EGFP-expressing viruses in fluorescence-guided pathogenesis studies.

"Frozen evolution" of an RNA virus suggests accidental release as a potential cause of arbovirus re-emergence.

The mechanisms underlying virus emergence are rarely well understood, making the appearance of outbreaks largely unpredictable. Bluetongue virus serotype 8 (BTV-8), an arthropod-borne virus of ruminants, emerged in livestock in northern Europe in 2006, spreading to most European countries by 2009 and causing losses of billions of euros. Although the outbreak was successfully controlled through vaccination by early 2010, puzzlingly, a closely related BTV-8 strain re-emerged in France in 2015, triggering a second outbreak that is still ongoing. The origin of this virus and the mechanisms underlying its re-emergence are unknown. Here, we performed phylogenetic analyses of 164 whole BTV-8 genomes sampled throughout the two outbreaks. We demonstrate consistent clock-like virus evolution during both epizootics but found negligible evolutionary change between them. We estimate that the ancestor of the second outbreak dates from the height of the first outbreak in 2008. This implies that the virus had not been replicating for multiple years prior to its re-emergence in 2015. Given the absence of any known natural mechanism that could explain BTV-8 persistence over this long period without replication, we hypothesise that the second outbreak could have been initiated by accidental exposure of livestock to frozen material contaminated with virus from approximately 2008. Our work highlights new targets for pathogen surveillance programmes in livestock and illustrates the power of genomic epidemiology to identify pathways of infectious disease emergence.

Virus-induced autophagic degradation of STAT2 as a mechanism for interferon signaling blockade.

The mammalian interferon (IFN) signaling pathway is a primary component of the innate antiviral response, and viral pathogens have evolved multiple mechanisms to antagonize this pathway and to facilitate infection. Bluetongue virus (BTV), an orbivirus of the Reoviridae family, is transmitted by midges to ruminants and causes a disease that produces important economic losses and restriction to animal trade and is of compulsory notification to the World Organization for Animal Health (OIE). Here, we show that BTV interferes with IFN-I and IFN-II responses in two ways, by blocking STAT1 phosphorylation and by degrading STAT2. BTV-NS3 protein, which is involved in virion egress, interacts with STAT2, and induces its degradation by an autophagy-dependent mechanism. This STAT2 degradative process requires the recruitment of an E3-Ub-ligase to NS3 as well as NS3 K63 polyubiquitination. Taken together, our study identifies a new mechanism by which a virus degrades STAT2 for IFN signaling blockade, highlighting the diversity of mechanisms employed by viruses to subvert the IFN response.

Novel Function of Bluetongue Virus NS3 Protein in Regulation of the MAPK/ERK Signaling Pathway.

Bluetongue virus (BTV) is an arbovirus transmitted by blood-feeding midges to a wide range of wild and domestic ruminants. In this report, we showed that BTV, through its nonstructural protein NS3 (BTV-NS3), is able to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, as assessed by phosphorylation levels of ERK1/2 and the translation initiation factor eukaryotic translation initiation factor 4E (eIF4E). By combining immunoprecipitation of BTV-NS3 and mass spectrometry analysis from both BTV-infected and NS3-transfected cells, we identified the serine/threonine-protein kinase B-Raf (BRAF), a crucial player in the MAPK/ERK pathway, as a new cellular interactor of BTV-NS3. BRAF silencing led to a significant decrease in the MAPK/ERK activation by BTV, supporting a model wherein BTV-NS3 interacts with BRAF to activate this signaling cascade. This positive regulation acts independently of the role of BTV-NS3 in counteracting the induction of the alpha/beta interferon response. Furthermore, the intrinsic ability of BTV-NS3 to bind BRAF and activate the MAPK/ERK pathway is conserved throughout multiple serotypes/strains but appears to be specific to BTV compared to other members of Orbivirus genus. Inhibition of MAPK/ERK pathway with U0126 reduced viral titers, suggesting that BTV manipulates this pathway for its own replication. Altogether, our data provide molecular mechanisms that unravel a new essential function of NS3 during BTV infection.IMPORTANCE Bluetongue virus (BTV) is responsible of the arthropod-borne disease bluetongue (BT) transmitted to ruminants by blood-feeding midges. In this report, we found that BTV, through its nonstructural protein NS3 (BTV-NS3), interacts with BRAF, a key component of the MAPK/ERK pathway. In response to growth factors, this pathway promotes cell survival and increases protein translation. We showed that BTV-NS3 enhances the MAPK/ERK pathway, and this activation is BRAF dependent. Treatment of MAPK/ERK pathway with the pharmacologic inhibitor U0126 impairs viral replication, suggesting that BTV manipulates this pathway for its own benefit. Our results illustrate, at the molecular level, how a single virulence factor has evolved to target a cellular function to increase its viral replication.

Structural Protein VP2 of African Horse Sickness Virus Is Not Essential for Virus Replication In Vitro.

The Reoviridae family consists of nonenveloped multilayered viruses with a double-stranded RNA genome consisting of 9 to 12 genome segments. The Orbivirus genus of the Reoviridae family contains African horse sickness virus (AHSV), bluetongue virus, and epizootic hemorrhagic disease virus, which cause notifiable diseases and are spread by biting Culicoides species. Here, we used reverse genetics for AHSV to study the role of outer capsid protein VP2, encoded by genome segment 2 (Seg-2). Expansion of a previously found deletion in Seg-2 indicates that structural protein VP2 of AHSV is not essential for virus replication in vitro In addition, in-frame replacement of RNA sequences in Seg-2 by that of green fluorescence protein (GFP) resulted in AHSV expressing GFP, which further confirmed that VP2 is not essential for virus replication. In contrast to virus replication without VP2 expression in mammalian cells, virus replication in insect cells was strongly reduced, and virus release from insect cells was completely abolished. Further, the other outer capsid protein, VP5, was not copurified with virions for virus mutants without VP2 expression. AHSV without VP5 expression, however, could not be recovered, indicating that outer capsid protein VP5 is essential for virus replication in vitro Our results demonstrate for the first time that a structural viral protein is not essential for orbivirus replication in vitro, which opens new possibilities for research on other members of the Reoviridae family. IMPORTANCE: Members of the Reoviridae family cause major health problems worldwide, ranging from lethal diarrhea caused by rotavirus in humans to economic losses in livestock production caused by different orbiviruses. The Orbivirus genus contains many virus species, of which bluetongue virus, epizootic hemorrhagic disease virus, and African horse sickness virus (AHSV) cause notifiable diseases according to the World Organization of Animal Health. Recently, it has been shown that nonstructural proteins NS3/NS3a and NS4 are not essential for virus replication in vitro, whereas it is generally assumed that structural proteins VP1 to -7 of these nonenveloped, architecturally complex virus particles are essential. Here we demonstrate for the first time that structural protein VP2 of AHSV is not essential for virus replication in vitro Our findings are very important for virologists working in the field of nonenveloped viruses, in particular reoviruses.

Replication-Deficient Particles: New Insights into the Next Generation of Bluetongue Virus Vaccines.

Bluetongue virus (BTV) is endemic in many parts of the world, often causing severe hemorrhagic disease in livestock. To date, at least 27 different serotypes have been recognized. Vaccination against all serotypes is necessary to protect susceptible animals and to prevent onward spread of the virus by insect vectors. In our previous studies, we generated replication-deficient (disabled infectious single-cycle [DISC]) virus strains for a number of serotypes and reported preliminary data on their protective efficacy in animals. In this report, to advance the DISC vaccines to the marketplace, we investigated different parameters of these DISC vaccines. First, we demonstrated the genetic stabilities of these vaccine strains and also the complementing cell line. Subsequently, the optimal storage conditions of vaccines, including additives, temperature, and desiccation, were determined and their protective efficacies in animals confirmed. Furthermore, to test if mixtures of different vaccine strains could be tolerated, we tested cocktails of DISC vaccines in combinations of three or six different serotypes in sheep and cattle, the two natural hosts of BTV. Groups of sheep vaccinated with a cocktail of six different vaccines were completely protected from challenge with individual virulent serotypes, both in early challenge and after 5 months of challenge without any clinical disease. There was no interference in protection between the different vaccines. Protection was also achieved in cattle with a mixture of three vaccine strains, albeit at a lesser level than in sheep. Our data support and validate the suitability of these virus strains as the next-generation vaccines for BTV. IMPORTANCE: Bluetongue (BT) is a debilitating and in many cases lethal disease that affects ruminants of economic importance. Classical vaccines that afford protection against bluetongue virus, the etiological agent, are not free from secondary and undesirable effects. A surge in new approaches to produce highly attenuated, safer vaccines was evident after the development of the BTV reverse-genetics system that allows the introduction of targeted mutations in the virus genome. We targeted an essential gene to develop disabled virus strains as vaccine candidates. The results presented in this report further substantiate our previous evidence and support the suitability of these virus strains as the next-generation BTV vaccines.

Current and next-generation bluetongue vaccines: Requirements, strategies, and prospects for different field situations.

Bluetongue virus (BTV) causes the hemorrhagic disease bluetongue (BT) in ruminants. The best way to control outbreaks is vaccination. Currently, conventionally modified-live and inactivated vaccines are commercially available, which have been successfully used to control BT, but nonetheless have their specific shortcomings. Therefore, there is a need for improved BT vaccines. The ideal BT vaccine is efficacious, safe, affordable, protective against multiple serotypes and enables the differentiation of infected from vaccinated animals. Different field situations require specific vaccine profiles. Single serotype outbreaks in former BT-free areas need rapid onset of protection against viremia of the respective serotype. In contrary, endemic multiple serotype situations require long-lasting protection against all circulating serotypes. The ideal BT vaccine for all field situations does not exist and balancing between vaccine properties is needed. Many new vaccines candidates, ranging from non-replicating subunits to replicating next-generation reverse genetics based vaccines, have been developed. Some have been tested extensively in large numbers of ruminants, whereas others were developed recently and have only been tested in vitro and in mice models. Most vaccine candidates are promising, but have their specific shortcomings and advantages. In this review, current and next-generation BT vaccines are discussed in the light of prerequisites for different field situations.

Balance of RNA sequence requirement and NS3/NS3a expression of segment 10 of orbiviruses.

Orbiviruses are insect-transmitted, non-enveloped viruses with a ten-segmented dsRNA genome of which the bluetongue virus (BTV) is the prototype. Viral non-structural protein NS3/NS3a is encoded by genome segment 10 (Seg-10), and is involved in different virus release mechanisms. This protein induces specific release via membrane disruptions and budding in both insect and mammalian cells, but also the cytopathogenic release that is only seen in mammalian cells. NS3/NS3a is not essential for virus replication in vitro with BTV Seg-10 containing RNA elements essential for virus replication, even if protein is not expressed. Recently, new BTV serotypes with distinct NS3/NS3a sequence and cell tropism have been identified. Multiple studies have hinted at the importance of Seg-10 in orbivirus replication, but the exact prerequisites are still unknown. Here, more insight is obtained with regard to the needs for orbivirus Seg-10 and the balance between protein expression and RNA elements. Multiple silent mutations in the BTV NS3a ORF destabilized Seg-10, resulting in deletions and sequences originating from other viral segments being inserted, indicating strong selection at the level of RNA during replication in mammalian cells in vitro. The NS3a ORFs of other orbiviruses were successfully exchanged in BTV1 Seg-10, resulting in viable chimeric viruses. NS3/NS3a proteins in these chimeric viruses were generally functional in mammalian cells, but not in insect cells. NS3/NS3a of the novel BTV serotypes 25 and 26 affected virus release from Culicoides cells, which might be one of the reasons for their distinct cell tropism.

VP2 Exchange and NS3/NS3a Deletion in African Horse Sickness Virus (AHSV) in Development of Disabled Infectious Single Animal Vaccine Candidates for AHSV.

UNLABELLED: African horse sickness virus (AHSV) is a virus species in the genus Orbivirus of the family Reoviridae. There are nine serotypes of AHSV showing different levels of cross neutralization. AHSV is transmitted by species of Culicoides biting midges and causes African horse sickness (AHS) in equids, with a mortality rate of up to 95% in naive horses. AHS has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climates appear to be competent vectors for the related bluetongue virus (BTV). To control AHS, live-attenuated vaccines (LAVs) are used in Africa. We used reverse genetics to generate "synthetic" reassortants of AHSV for all nine serotypes by exchange of genome segment 2 (Seg-2). This segment encodes VP2, which is the serotype-determining protein and the dominant target for neutralizing antibodies. Single Seg-2 AHSV reassortants showed similar cytopathogenic effects in mammalian cells but displayed different growth kinetics. Reverse genetics for AHSV was also used to study Seg-10 expressing NS3/NS3a proteins. We demonstrated that NS3/NS3a proteins are not essential for AHSV replication in vitro. NS3/NS3a of AHSV is, however, involved in the cytopathogenic effect in mammalian cells and is very important for virus release from cultured insect cells in particular. Similar to the concept of the bluetongue disabled infectious single animal (BT DISA) vaccine platform, an AHS DISA vaccine platform lacking NS3/NS3a expression was developed. Using exchange of genome segment 2 encoding VP2 protein (Seg-2[VP2]), we will be able to develop AHS DISA vaccine candidates for all current AHSV serotypes. IMPORTANCE: African horse sickness virus is transmitted by species of Culicoides biting midges and causes African horse sickness in equids, with a mortality rate of up to 95% in naive horses. African horse sickness has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climates are supposed to be competent vectors. By using reverse genetics, viruses of all nine serotypes were constructed by the exchange of Seg-2 expressing the serotype-determining VP2 protein. Furthermore, we demonstrated that the nonstructural protein NS3/NS3a is not essential for virus replication in vitro. However, the potential spread of the virus by biting midges is supposed to be blocked, since the in vitro release of the virus was strongly reduced due to this deletion. VP2 exchange and NS3/NS3a deletion in African horse sickness virus were combined in the concept of a disabled infectious single animal vaccine for all nine serotypes.

Turnover Rate of NS3 Proteins Modulates Bluetongue Virus Replication Kinetics in a Host-Specific Manner.

UNLABELLED: Bluetongue virus (BTV) is an arbovirus transmitted to livestock by midges of the Culicoides family and is the etiological agent of a hemorrhagic disease in sheep and other ruminants. In mammalian cells, BTV particles are released primarily by virus-induced cell lysis, while in insect cells they bud from the plasma membrane and establish a persistent infection. BTV possesses a ten-segmented double-stranded RNA genome, and NS3 proteins are encoded by segment 10 (Seg-10). The viral nonstructural protein 3 (NS3) plays a key role in mediating BTV egress as well as in impeding the in vitro synthesis of type I interferon in mammalian cells. In this study, we asked whether genetically distant NS3 proteins can alter BTV-host interactions. Using a reverse genetics approach, we showed that, depending on the NS3 considered, BTV replication kinetics varied in mammals but not in insects. In particular, one of the NS3 proteins analyzed harbored a proline at position 24 that leads to its rapid intracellular decay in ovine but not in Culicoides cells and to the attenuation of BTV virulence in a mouse model of disease. Overall, our data reveal that the genetic variability of Seg-10/NS3 differentially modulates BTV replication kinetics in a host-specific manner and highlight the role of the host-specific variation in NS3 protein turnover rate. IMPORTANCE: BTV is the causative agent of a severe disease transmitted between ruminants by biting midges of Culicoides species. NS3, encoded by Seg-10 of the BTV genome, fulfills key roles in BTV infection. As Seg-10 sequences from various BTV strains display genetic variability, we assessed the impact of different Seg-10 and NS3 proteins on BTV infection and host interactions. In this study, we revealed that various Seg-10/NS3 proteins alter BTV replication kinetics in mammals but not in insects. Notably, we found that NS3 protein turnover may vary in ovine but not in Culicoides cells due to a single amino acid residue that, most likely, leads to rapid and host-dependent protein degradation. Overall, this study highlights that genetically distant BTV Seg-10/NS3 influence BTV biological properties in a host-specific manner and increases our understanding of how NS3 proteins contribute to the outcome of BTV infection.

Requirements and comparative analysis of reverse genetics for bluetongue virus (BTV) and African horse sickness virus (AHSV).

BACKGROUND: Bluetongue virus (BTV) and African horse sickness virus (AHSV) are distinct arthropod borne virus species in the genus Orbivirus (Reoviridae family), causing the notifiable diseases Bluetongue and African horse sickness of ruminants and equids, respectively. Reverse genetics systems for these orbiviruses with their ten-segmented genome of double stranded RNA have been developed. Initially, two subsequent transfections of in vitro synthesized capped run-off RNA transcripts resulted in the recovery of BTV. Reverse genetics has been improved by transfection of expression plasmids followed by transfection of ten RNA transcripts. Recovery of AHSV was further improved by use of expression plasmids containing optimized open reading frames. RESULTS: Plasmids containing full length cDNA of the 10 genome segments for T7 promoter-driven production of full length run-off RNA transcripts and expression plasmids with optimized open reading frames (ORFs) were used. BTV and AHSV were rescued using reverse genetics. The requirement of each expression plasmid and capping of RNA transcripts for reverse genetics were studied and compared for BTV and AHSV. BTV was recovered by transfection of VP1 and NS2 expression plasmids followed by transfection of a set of ten capped RNAs. VP3 expression plasmid was also required if uncapped RNAs were transfected. Recovery of AHSV required transfection of VP1, VP3 and NS2 expression plasmids followed by transfection of capped RNA transcripts. Plasmid-driven expression of VP4, 6 and 7 was also needed when uncapped RNA transcripts were used. Irrespective of capping of RNA transcripts, NS1 expression plasmid was not needed for recovery, although NS1 protein is essential for virus propagation. Improvement of reverse genetics for AHSV was clearly demonstrated by rescue of several mutants and reassortants that were not rescued with previous methods. CONCLUSIONS: A limited number of expression plasmids is required for rescue of BTV or AHSV using reverse genetics, making the system much more versatile and generally applicable. Optimization of reverse genetics enlarge the possibilities to rescue virus mutants and reassortants, and will greatly benefit the control of these important diseases of livestock and companion animals.

Virus and host factors affecting the clinical outcome of bluetongue virus infection.

UNLABELLED: Bluetongue is a major infectious disease of ruminants caused by bluetongue virus (BTV), an arbovirus transmitted by Culicoides. Here, we assessed virus and host factors influencing the clinical outcome of BTV infection using a single experimental framework. We investigated how mammalian host species, breed, age, BTV serotypes, and strains within a serotype affect the clinical course of bluetongue. Results obtained indicate that in small ruminants, there is a marked difference in the susceptibility to clinical disease induced by BTV at the host species level but less so at the breed level. No major differences in virulence were found between divergent serotypes (BTV-8 and BTV-2). However, we observed striking differences in virulence between closely related strains of the same serotype collected toward the beginning and the end of the European BTV-8 outbreak. As observed previously, differences in disease severity were also observed when animals were infected with either blood from a BTV-infected animal or from the same virus isolated in cell culture. Interestingly, with the exception of two silent mutations, full viral genome sequencing showed identical consensus sequences of the virus before and after cell culture isolation. However, deep sequencing analysis revealed a marked decrease in the genetic diversity of the viral population after passaging in mammalian cells. In contrast, passaging in Culicoides cells increased the overall number of low-frequency variants compared to virus never passaged in cell culture. Thus, Culicoides might be a source of new viral variants, and viral population diversity can be another factor influencing BTV virulence. IMPORTANCE: Bluetongue is one of the major infectious diseases of ruminants. It is caused by an arbovirus known as bluetongue virus (BTV). The clinical outcome of BTV infection is extremely variable. We show that there are clear links between the severity of bluetongue and the mammalian host species infected, while at the breed level differences were less evident. No differences were observed in the virulence of two different BTV serotypes (BTV-8 and BTV-2). In contrast, we show that the European BTV-8 strain isolated at the beginning of the bluetongue outbreak in 2006 was more virulent than a strain isolated toward the end of the outbreak. In addition, we show that there is a link between the variability of the BTV population as a whole and virulence, and our data also suggest that Culicoides cells might function as an "incubator" of viral variants.

Bluetongue virus without NS3/NS3a expression is not virulent and protects against virulent bluetongue virus challenge.

Bluetongue is a disease in ruminants caused by the bluetongue virus (BTV), and is spread by Culicoides biting midges. Bluetongue outbreaks cause huge economic losses and death in sheep in several parts of the world. The most effective measure to control BTV is vaccination. However, both commercially available vaccines and recently developed vaccine candidates have several shortcomings. Therefore, we generated and tested next-generation vaccines for bluetongue based on the backbone of a laboratory-adapted strain of BTV-1, avirulent BTV-6 or virulent BTV-8. All vaccine candidates were serotyped with VP2 of BTV-8 and did not express NS3/NS3a non-structural proteins, due to induced deletions in the NS3/NS3a ORF. Sheep were vaccinated once with one of these vaccine candidates and were challenged with virulent BTV-8 3 weeks after vaccination. The NS3/NS3a knockout mutation caused complete avirulence for all three BTV backbones, including for virulent BTV-8, indicating that safety is associated with the NS3/NS3a knockout phenotype. Viraemia of vaccine virus was not detected using sensitive PCR diagnostics. Apparently, the vaccine viruses replicated only locally, which will minimize spread by the insect vector. In particular, the vaccine based on the BTV-6 backbone protected against disease and prevented viraemia of challenge virus, showing the efficacy of this vaccine candidate. The lack of NS3/NS3a expression potentially enables the differentiation of infected from vaccinated animals, which is important for monitoring virus spread in vaccinated livestock. The disabled infectious single-animal vaccine for bluetongue presented here is very promising and will be the subject of future studies.

Rapid generation of replication-deficient monovalent and multivalent vaccines for bluetongue virus: protection against virulent virus challenge in cattle and sheep.

Since 1998, 9 of the 26 serotypes of bluetongue virus (BTV) have spread throughout Europe, and serotype 8 has suddenly emerged in northern Europe, causing considerable economic losses, direct (mortality and morbidity) but also indirect, due to restriction in animal movements. Therefore, many new types of vaccines, particularly subunit vaccines, with improved safety and efficacy for a broad range of BTV serotypes are currently being developed by different laboratories. Here we exploited a reverse genetics-based replication-deficient BTV serotype 1 (BTV-1) (disabled infectious single cycle [DISC]) strain to generate a series of DISC vaccine strains. Cattle and sheep were vaccinated with these viruses either singly or in cocktail form as a multivalent vaccine candidate. All vaccinated animals were seroconverted and developed neutralizing antibody responses to their respective serotypes. After challenge with the virulent strains at 21 days postvaccination, vaccinated animals showed neither any clinical reaction nor viremia. Further, there was no interference with protection with a multivalent preparation of six distinct DISC viruses. These data indicate that a very-rapid-response vaccine could be developed based on which serotypes are circulating in the population at the time of an outbreak.

Bluetongue, Schmallenberg - what is next? Culicoides-borne viral diseases in the 21st Century.

In the past decade, two pathogens transmitted by Culicoides biting midges (Diptera: Ceratopogonidae), bluetongue virus and Schmallenberg virus, have caused serious economic losses to the European livestock industry, most notably affecting sheep and cattle. These outbreaks of arboviral disease have highlighted large knowledge gaps on the biology and ecology of indigenous Culicoides species. With these research gaps in mind, and as a means of assessing what potential disease outbreaks to expect in the future, an international workshop was held in May 2013 at Wageningen University, The Netherlands. It brought together research groups from Belgium, France, Germany, Spain, Switzerland, United Kingdom and The Netherlands, with diverse backgrounds in vector ecology, epidemiology, entomology, virology, animal health, modelling, and genetics. Here, we report on the key findings of this workshop.

Spatial Transmission Characteristics of the Bluetongue Virus Serotype 3 Epidemic in The Netherlands, 2023.

A devastating bluetongue (BT) epidemic caused by bluetongue virus serotype 3 (BTV-3) has spread throughout most of the Netherlands within two months since the first infection was officially confirmed in the beginning of September 2023. The epidemic comes with unusually strong suffering of infected cattle through severe lameness, often resulting in mortality or euthanisation for welfare reasons. In total, tens of thousands of sheep have died or had to be euthanised. By October 2023, more than 2200 locations with ruminant livestock were officially identified to be infected with BTV-3, and additionally, ruminants from 1300 locations were showing BTV-associated clinical symptoms (but not laboratory-confirmed BT). Here, we report on the spatial spread and dynamics of this BT epidemic. More specifically, we characterized the distance-dependent intensity of the between-holding transmission by estimating the spatial transmission kernel and by comparing it to transmission kernels estimated earlier for BTV-8 transmission in Northwestern Europe in 2006 and 2007. The 2023 BTV-3 kernel parameters are in line with those of the transmission kernel estimated previously for the between-holding spread of BTV-8 in Europe in 2007. The 2023 BTV-3 transmission kernel has a long-distance spatial range (across tens of kilometres), evidencing that in addition to short-distance dispersal of infected midges, other transmission routes such as livestock transports probably played an important role.

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.

Prevalence and predictors of vector-borne pathogens in Dutch roe deer.

BACKGROUND: The main objective of this study was to determine the prevalence of nine vector-borne pathogens or pathogen genera in roe deer (Capreolus capreolus) in the Netherlands, and to identify which host variables predict vector-borne pathogen presence in roe deer. The host variables examined were the four host factors 'age category', 'sex', 'nutritional condition' and 'health status', as well as 'roe deer density'. METHODS: From December 2009 to September 2010, blood samples of 461 roe deer were collected and analysed by polymerase chain reaction (PCR) for the presence of genetic material from Anaplasma phagocytophilum, Bartonella spp., Babesia spp., Borrelia burgdorferi sensu lato (s.l.), Borrelia miyamotoi, Neoehrlichia mikurensis, Rickettsia spp., and epizootic haemorrhagic disease virus (EHDV), and by commercial enzyme-linked immunosorbent assay (ELISA) for antibodies against bluetongue virus (BTV). The possible associations of host factors and density with pathogen prevalence and co-infection, and in the case of A. phagocytophilum with bacterial load, were assessed using generalized linear modelling. RESULTS AND CONCLUSION: Analysis revealed the following prevalence in roe deer: A. phagocytophilum 77.9%, Bartonella spp. 77.7%, Babesia spp. 17.4%, Rickettsia spp. 3.3%, B. burgdorferi sensu lato 0.2%. Various co-infections were found, of which A. phagocytophilum and Bartonella spp. (49.7% of infected roe deer) and A. phagocytophilum, Bartonella spp. and Babesia spp. (12.2% of infected roe deer) were the most common. Anaplasma phagocytophilum, Babesia spp., and co-infection prevalence were significantly higher in calves than in adult roe deer, whereas the prevalence of Bartonella spp. was lower in roe deer in good nutritional condition than in deer in poor nutritional condition. Local roe deer density was not associated with pathogen presence. The high prevalence of A. phagocytophilum, Bartonella spp., and Babesia spp. is evidence for the role of roe deer as reservoirs for these pathogens. Additionally, the results suggest a supportive role of roe deer in the life-cycle of Rickettsia spp. in the Netherlands.

Critical parameters of real time reverse transcription polymerase chain reaction (RT-PCR) diagnostics: Sensitivity and specificity for bluetongue virus.

A new variant of bluetongue virus serotype 3, BTV3 ITL 2018 (here named: BTV3), was included in serial dilutions in the BT Proficiency Test 2020. Although the OIE-recommended panBTV real time RT-PCR test targeting genome segment 10 (Seg-10) detected this variant, we showed that reverse transcription (RT) at 61 °C instead of 50 °C completely abolished detection. Another Seg-10 panBTV real time RT-PCR test detected BTV3, irrespective of the temperature of RT. In silico validation showed that each of the OIE-recommended PCR primers using IVI-primers contain single mismatches at the -3 position for BTV3. In contrast, WBVR-primers of a second test completely match to the BTV3 variant. Our results suggest that single mismatches caused false negative PCR results for BTV3 at high RT temperature. Indeed, correction of both IVI-primers for BTV3 led to positive results for BTV3 but negative results for all other samples of the BT Proficiency Test 2020. Apparently, variability of the -3 position is sufficient for discriminative PCR detection, although the single mismatch in the IVI-reverse primer was the most important for this phenomenon. Extensive in silico validation showed that targets of both Seg-10 panBTV RT-PCR tests are not completely conserved, and the detailed effect of single mismatches are hard to predict. Therefore, we recommend at least two panBTV RT-PCR tests to minimize the risk of false negatives. Preferably, their PCR targets should be located at completely different and highly conserved regions of the BTV genome to guarantee adequate detection of future BTV infections.