Bluetongue Research Articles

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

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

Improved Efficiency of Bluetongue Viral Antigen Isolation for Successful Immunization

Bluetongue (BT) is a viral disease transmitted by Culicoides spp. The clinical presentation of BT varies widely among susceptible sheep, and in most cases results in severe illness and death in the infected animals. The mortality among susceptible sheep ranges from 2-30% but can occasionally be as high as 70%. Therefore, we investigated a new method to increase the purified BTV-antigen. BTV viral suspensions were purified using Freon-113 and ultracentrifugation through 40% sucrose. We obtained 94.5-95.8% purification of the BT-16 viral antigen. Sheep and cows were immunized with the isolated BTV antigen obtained from this method to confirm antibody specificity to BTV. The antibody activity measured by enzyme-linked immunosorbent assay (ELISA) from goat serum was 7.0log2 to 13.0log2 (on average 10.8±2.28log2) relative to that of sheep (P<0.05 to P<0.0001). We show here that this method can successfully purify BTV-16 antigen and could be used for large-scale production and other BTV serotypes.

First record of antibodies to the bluetongue virus in ewe (Ovis aries) in the state of Amazonas, Brazil

First record of antibodies to the bluetongue virus in ewe (Ovis aries) in the state of Amazonas, Brazil

Evaluation of oxidant and antioxidant profile in amniotic fluid of pregnant sheep experimentally infected with bluetongue virus serotype-1

A study was carried out to ascertain the oxidant and antioxidant profile in amniotic fluid (AF) of pregnant Muzaffarnagari ewes infected with bluetongue virus serotype-1. Twenty-seven pregnant ewes were divided into three groups as control and two treatment groups. BTV-1 was inoculated into two treatment groups during early and mid-gestation at 35 days and 60 days, respectively. Animals were sacrificed at periodical intervals. Amniotic fluid was collected after resection of uterus and evaluated for lipid peroxidation, superoxide dismutase (SOD) and total antioxidant capacity (TAC). Lipid peroxidation showed elevation in infected groups (1.234 and 1.240 |jmol/ml in early and mid-gestation infected group, respectively) as compared to control (1.107 jmol/ml), whereas SOD and TAC decreased in infected groups (SOD: 0.895 and 0.898 U/L; TAP: 0.445 and 0.451 mM/L in early and mid-gestation infected group, respectively) as compared to control (SOD: 1.280 U/L; TAP: 0.664 mM/L). Malondialdehyde (MDA) levels decreased gradually from early to mid-gestation stage and increased towards late gestation. Antioxidant enzyme SOD and TAC increased towards late gestation in control group. MDA levels also increased and SOD and TAC decreased towards last trimester in both infected groups. Among, thirty-two foetuses retrieved, two foetuses showed cerebellar malformation such as general cerebellar hypoplasia and Purkinje cell dysplasia towards the late gestation in infected groups. It was concluded that BTV-1 added oxidative stress to foetal growth in the womb and responsible for central nervous system malformation. Thus, amniotic fluid could be the valuable diagnostic sample to evaluate oxidative biomarkers in viral induced central nervous system malformations in foetuses.

Seroprevalence of Bluetongue Disease among Domestic Ruminants Raised in International Border Areas of Nepal

Seroprevalence of Bluetongue Disease among Domestic Ruminants Raised in International Border Areas of Nepal

Experimental bluetongue virus infection of Culicoides austropalpalis, collected from a farm environment in Victoria, Australia.

Following the emerging bluetongue virus transmission in European temperate regions, we question the vector competence of the abundant Culicoides austropalpalis Lee and Reye in South-East temperate Australia. Field collected Culicoides midges were membrane fed with a bluetongue virus serotype 1 (BTV-1). The average feeding rate was 50%. After 13 days, survival rate was 25% and virus RNA presence was checked by quantitative PCR targeting viral genome segment 10. Virus RNA was found in 7.4% of individually tested females with relative viral RNA load values lower than freshly fed females, indicating that viral replication was low or null. A second qPCR targeting viral genome segment 1 confirmed the presence of virus RNA in only four out of 29 previously positive specimens. After 10 days culture on Culicoides cells, none of these four confimed positive samples did show subsequent cytopathogenic effect on Vero cells or BTV antigen detection by ELISA. As control for this virus activity detection, 12 days after microinjection of BTV-1, Culex annulirostris mosquitoes showed, after culture on Kc cells, cytopathogenic effect on Vero cells, with ELISA-confirmed infection. Despite its abundance in farm environment of the temperate Australian regions, the results of this study make C. austropalpalis of unlikely epidemiological importance in the transmission of BTV in Australia.

Bluetongue and epizootic haemorrhagic disease in wildlife with emphasis on the South American scenario

Bluetongue and epizootic hemorrhagic disease affect domestic ruminants and cervids. However, other species may act as pathogen carriers in the transition of bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV). The wild species affected by these diseases manifest a variable range of clinical signs and lesions, and while some species appear to be extremely susceptible, showing high levels of mortality, some are resistant to these pathogens, acting as potential reservoirs of these orbiviruses. The purpose of the following review is to describe the clinical and pathological manifestations related to these diseases in wild species and to review studies performed on non-domestic species in South America, emphasizing the challenges of studying infectious diseases in free-living animals and the gaps in knowledge about bluetongue and epizootic haemorrhagic disease epidemiology. These gaps should be filled by more studies on the range of species affected and the transmission mechanisms, including in domestic species.

Antiviral Properties of Streptomyces tuirus DBZ39 Mediated Gold Nanoparticles Against Bluetongue virus.

<b>Background and Objective:</b> The proposed study involves the approach from the point of anti-viral activity of gold nanoparticles against the <i>Bluetongue virus</i>. Among viral diseases, Bluetongue is regarded as an economically scouring disease. Neither a vaccine nor an antiviral drug is available for the prevention or treatment of this disease. The antiviral activity of gold nanoparticles synthesized by a novel isolate of <i>Streptomyces tuirus</i> DBZ39 is the breakthrough of the study. <i>Streptomyces tuirus </i>DBZ39, a novel isolate obtained from alkaline soil was proved to be efficient actinomycetes, for the extracellular synthesis of gold nanoparticles. <b>Materials and Methods:</b> An upstream bioprocess was optimized and developed for the synthesis of controlled size gold nanoparticles with solitary mono dispersal pattern in aurum chloride solution. The characterization and confirmation of gold nanoparticles were illustrated by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Energy Dispersive X-ray Analysis (EDAX) and Fourier Transmission Infrared Radiation Analysis (FTIR). <b>Results:</b> Biomass size of 3 g, substrate concentration of 1 mM, pH of 8.5 and temperature of 45°C were observed as optimum conditions for the synthesis of 15-24 nm size gold nanoparticles. The <i>Bluetongue virus</i> (BTV) which belongs to the genus Orbivirus in the family Reoviridae with 26 serotypes is an etiological agent of infectious and non-contagious Bluetongue disease of main sheep and several other domestic animals. <b>Conclusion:</b> Gold nanoparticles for the 1st time, at a higher concentration of 1:64 dilutions revealed a very promising and novel antiviral property against the <i>Bluetongue virus</i>.

The Absence of Abdominal Pigmentation in Livestock Associated Culicoides following Artificial Blood Feeding and the Epidemiological Implication for Arbovirus Surveillance.

Culicoides midges (Diptera: Ceratopogonidae), the vectors of economically important arboviruses such as bluetongue virus and African horse sickness virus, are of global importance. In the absence of transovarial transmission, the parity rate of a Culicoides population provides imperative information regarding the risk of virus dispersal. Abdominal pigmentation, which develops after blood feeding and ovipositioning, is used as an indicator of parity in Culicoides. During oral susceptibility trials over the last three decades, a persistent proportion of blood engorged females did not develop pigment after incubation. The present study, combining a number of feeding trials and different artificial feeding methods, reports on this phenomenon, as observed in various South African and Italian Culicoides species and populations. The absence of pigmentation in artificial blood-fed females was found in at least 23 Culicoides species, including important vectors such as C. imicola, C. bolitinos, C. obsoletus, and C. scoticus. Viruses were repeatedly detected in these unpigmented females after incubation. Blood meal size seems to play a role and this phenomenon could be present in the field and requires consideration, especially regarding the detection of virus in apparent “nulliparous” females and the identification of overwintering mechanisms and seasonally free vector zones.

Bluetongue Disease in Small Ruminants in Palestine: A retrospective Study Between the period of 2005-2019

Small ruminant’s livestock industry provides a major source of livelihood for many people worldwide, particularly the rural poor in developing countries. Bluetongue (BT) is a reportable infectious non-contagious disease of the small ruminant that has considerable socioeconomic importance in livestock in Palestine. The objective of the current study was to investigate the epidemiological analysis of BT within small ruminants in Palestine over 14 years 2005 – 2019. The retrospective numbers of BT outbreaks, cases, and deaths from the World Organization for Animal Health were analyzed. The study indicated that BT is enzootic in Palestine, reported started from 2009. A total of 273 outbreaks include 1628 cases resulted in 351 deaths were reported. A total of 273 outbreaks, 1628 cases, and 351 deaths were recorded in small ruminants. The incidence rate average was 5.5%, and the average mortality rate was 1.6%. The case fatality rate ranged from 0.0- 39.29 % with an average of 20.2%. No vaccination practice was applied in Palestine. Temporal analysis obtained that BT is more endemic in the low-temperature season between September and December, with a higher peak in November, displaying `overwintering` of BTV in Palestine. At least five BTV serotypes BTV (2, 4, 5, 6 and 8) were continuously present in Palestine. Multiple serotypes were recorded in the same outbreak period increase the risk for re-assortment of individual BTV gene segments. Efficient data recording, enhance owner awareness, use of the vaccine and a systematic BT monitoring program are required to control and eradicate the disease.

Studies on Bluetongue virus (BTV) from Ruminant Collected Samples during 2016 in Egypt

Studies on Bluetongue virus (BTV) from Ruminant Collected Samples during 2016 in Egypt

Vaccination as a Strategy to Prevent Bluetongue Virus Vertical Transmission.

Bluetongue virus (BTV) produces an economically important disease in ruminants of compulsory notification to the OIE. BTV is typically transmitted by the bite of Culicoides spp., however, some BTV strains can be transmitted vertically, and this is associated with fetus malformations and abortions. The viral factors associated with the virus potency to cross the placental barrier are not well defined. The potency of vertical transmission is retained and sometimes even increased in live attenuated BTV vaccine strains. Because BTV possesses a segmented genome, the possibility of reassortment of vaccination strains with wild-type virus could even favor the transmission of this phenotype. In the present review, we will describe the non-vector-based BTV infection routes and discuss the experimental vaccination strategies that offer advantages over this drawback of some live attenuated BTV vaccines.

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

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

Antibodies to Bluetongue, Akabane and Schmallenberg viruses in native dromedary camels in Turkey

Sera from 86 Turkish native camels from seven provinces in Turkey were collected and tested for specific antibodies to Bluetongue virus (BTV), Akabane virus (AKAV) and Schmallenberg virus (SBV) using ELISA. The BTV, AKAV and SBV antibodies were found in 53.5%, 51.2% and 15.1%, respectively. Furthermore, the seropositivity for multiple infection was the highest for dual infection with AKAV and BTV (25.6%), followed by triple seropositivity (9.3%). These findings indicated that BTV, AKAV and SBV circulate in camels in Turkey at a relatively high rate, and that an active surveillance program is needed for the management and tracing the dynamics of these infections in the Turkish camel population.

Bluetongue Virus Infections in Cattle Herds of Manabí Province of Ecuador.

Bluetongue (BT) is a viral disease transmitted by Culicoides (Diptera: Ceratopogonidae) to domestic and wild ruminants. Infections in cattle are mainly subclinical, but severe necrotic and hemorrhagic illness and death may occur depending on the strain of the virus and other factors; cattle act as a reservoir for the virus. Although the Ecuadorian coast has climatic conditions that favor the presence of the vector, there are few serologic or virologic BTV studies available. Manabí is a coastal province in which livestock farming is mostly implemented in the northern part. We conducted two studies to assess, for the first time, the presence of active BTV infections in Manabí province. We collected 430 serum samples from 38 randomly selected farms between March and July 2019 to perform BTV competitive ELISA. In addition, six seropositive farms were selected to place eight sentinel BTV-naive calves. All these calves were blood sampled and the presence of BTV RNA and antibodies was tested for by RT-PCR and competitive ELISA, respectively, once a week for 6–8 weeks until seroconversion was evidenced. A high individual seroprevalence (99%) was obtained, and all investigated farms had BTV seropositive animals. All sentinel calves became BTV viremic and seroconverted. The first viremia appeared after 2–5 weeks from arrival at the farm; they seroconverted 1–3 weeks later. We demonstrate for the first time that there is a high level of BTV circulation north of Manabí, with active infections on these farms. Integrated control strategies such as hygienic measures on farms to reduce midge populations would be advisable for the owners as mitigation measures.

Updated checklist of Culicoides Latreille (Diptera: Ceratopogonidae) of Turkey with ten new records.

We investigated the Culicoides fauna in Turkey during the years 2016-2019 in the process of entomological surveillance for arboviral diseases. The entomological survey was conducted at 104 sampling stations in 51 provinces in Turkey during four consecutive years. There were approximately 450,000 specimens and 59 identified species collected during the surveillance. Ten species were newly recorded for Turkey: C. chiopterus, C. grisescens, C. paradoxalis, C. santonicus, C. poperinghensis, C. sergenti, C. tbilisicus, C. comosioculatus, C. haranti, and C. univittatus. Identification of C. chiopterus and C. grisescens was confirmed using species-specific PCR and DNA sequencing. With our recent findings, previous data were critically reviewed and updated, and the number of Culicoides species has been increased to 71 for Turkey. The presence of C. chiopterus has particular importance due to its potential vector status for bluetongue virus (BTV) and Schmallenberg virus (SBV). This study presents result of the first large-scale integrated faunistic survey on Culicoides species in Turkey.

Identification of the Genome Segments of Bluetongue Virus Type 26/Type 1 Reassortants Influencing Horizontal Transmission in a Mouse Model.

Bluetongue virus serotypes 1 to 24 are transmitted primarily by infected Culicoides midges, in which they also replicate. However, “atypical” BTV serotypes (BTV-25, -26, -27 and -28) have recently been identified that do not infect and replicate in adult Culicoides, or a Culicoides derived cell line (KC cells). These atypical viruses are transmitted horizontally by direct contact between infected and susceptible hosts (primarily small ruminants) causing only mild clinical signs, although the exact transmission mechanisms involved have yet to be determined. We used reverse genetics to generate a strain of BTV-1 (BTV-1 RGC7) which is less virulent, infecting IFNAR(−/−) mice without killing them. Reassortant viruses were also engineered, using the BTV-1 RGC7 genetic backbone, containing individual genome segments derived from BTV-26. These reassortant viruses were used to explore the genetic control of horizontal transmission (HT) in the IFNAR(−/−) mouse model. Previous studies showed that genome segments 1, 2 and 3 restrict infection of Culicoides cells, along with a minor role for segment 7. The current study demonstrates that genome segments 2, 5 and 10 of BTV-26 (coding for proteins VP2, NS1 and NS3/NS3a/NS5, respectively) are individually sufficient to promote HT.

Bluetongue virus capsid protein VP5 perforates membranes at low endosomal pH during viral entry.

Bluetongue virus (BTV) is a non-enveloped virus and causes substantial morbidity and mortality in ruminants such as sheep. Fashioning a receptor-binding protein (VP2) and a membrane penetration protein (VP5) on the surface, BTV releases its genome-containing core (VP3 and VP7) into the host cell cytosol after perforation of the endosomal membrane. Unlike enveloped ones, the entry mechanisms of non-enveloped viruses into host cells remain poorly understood. Here we applied single-particle cryo-electron microscopy, cryo-electron tomography and structure-guided functional assays to characterize intermediate states of BTV cell entry in endosomes. Four structures of BTV at the resolution range of 3.4-3.9 Å show the different stages of structural rearrangement of capsid proteins on exposure to low pH, including conformational changes of VP5, stepwise detachment of VP2 and a small shift of VP7. In detail, sensing of the low-pH condition by the VP5 anchor domain triggers three major VP5 actions: projecting the hidden dagger domain, converting a surface loop to a protonated β-hairpin that anchors VP5 to the core and stepwise refolding of the unfurling domains into a six-helix stalk. Cryo-electron tomography structures of BTV interacting with liposomes show a length decrease of the VP5 stalk from 19.5 to 15.5 nm after its insertion into the membrane. Our structures, functional assays and structure-guided mutagenesis experiments combined indicate that this stalk, along with dagger domain and the WHXL motif, creates a single pore through the endosomal membrane that enables the viral core to enter the cytosol. Our study unveils the detailed mechanisms of BTV membrane penetration and showcases general methods to study cell entry of other non-enveloped viruses.

Identification and Genetic Characterization of Viral Pathogens in Ruminant Gestation Abnormalities, Israel, 2015–2019

Infectious agents including viruses are important abortifacients and can cause fetal abnormalities in livestock animals. Here, samples that had been collected in Israel from aborted or malformed ruminant fetuses between 2015 and 2019 were investigated for the presence of the following viruses: the reoviruses bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV), the flaviviruses bovine viral diarrhea virus (BVDV) and border disease virus (BDV), the peribunyaviruses Shuni virus (SHUV) and Akabane virus (AKAV), bovine herpesvirus type 1 (BoHV-1) and bovine ephemeral fever virus (BEFV). Domestic (cattle, sheep, goat) and wild/zoo ruminants were included in the study. The presence of viral nucleic acid or antigen could be confirmed in 21.8 % of abnormal pregnancies (213 out of 976 investigated cases), with peribunyaviruses, reoviruses and pestiviruses being the most prevalent. At least four different BTV serotypes were involved in abnormal courses of pregnancy in Israel. The subtyping of pestiviruses revealed the presence of two BDV and several distinct BVDV type 1 strains. The peribunyaviruses AKAV and SHUV were identified annually throughout the study period, however, variation in the extent of virus circulation could be observed between the years. In 2018, AKAV even represented the most detected pathogen in cases of small domestic ruminant gestation abnormalities. In conclusion, it was shown that various viruses are involved in abnormal courses of pregnancy in ruminants in Israel.

Sialic Acid Binding Sites in VP2 of Bluetongue Virus and Their Use during Virus Entry.

ABSTRACTBluetongue virus (BTV), a member of the Orbivirus genus, is transmitted by biting midges (gnats, Culicoides sp.) and is one of the most widespread animal pathogens, causing serious outbreaks in domestic animals, particularly in sheep, with high economic impact. The non-enveloped BTV particle is a double-capsid structure of seven proteins and a genome of 10 double-stranded RNA segments. Although the outermost spike-like VP2 acts as the attachment protein during BTV entry, no specific host receptor has been identified for BTV. Recent high-resolution cryo-electron (cryoEM) structures and biological data have suggested that VP2 may interact with sialic acids (SAs). To confirm this, we have generated protein-based nanoparticles displaying multivalent VP2 and used them to probe glycan arrays. The data show that VP2 binds α2,3-linked SA with high affinity but also binds α2,6-linked SA. Further, Maackia amurensis lectin II (MAL II) and Sambucus nigra lectin (SNA), which specifically bind α2,3-linked and α2,6-linked SAs, respectively, inhibited BTV infection and virus growth in susceptible sheep cells while SNA alone inhibited virus growth in Culicoides-derived cells. A combination of hydrogen deuterium exchange mass spectrometry and site-directed mutagenesis allowed the identification of the specific SA binding residues of VP2. This study provides direct evidence that sialic acids act as key receptor for BTV and that the outer capsid protein VP2 specifically binds SA during BTV entry in both mammalian and insect cells.IMPORTANCE To date no receptor has been assigned for non-enveloped bluetongue virus. To determine if the outermost spike-like VP2 protein is responsible for host cell attachment via interaction with sialic acids, we first generated a protein-based VP2-nanoparticle, for the multivalent presentation of recombinant VP2 protein. Using nanoparticles displaying VP2 to probe a glycan array, we identified that VP2 binds both α2,3-linked and α2,6-linked sialic acids. Lectin inhibitors targeting both linkages of sialic acids showed strong inhibition to BTV infection and progeny virus production in mammalian cells; however the inhibition was only seen with the lectin targeting α2,6-linked sialic acid in insect vector cells. In addition, we identified the VP2 sialic acid binding sites in the exposed tip domain. Our data provides direct evidence that sialic acids act as key receptors for BTV attachment and entry in to both mammalian and insect cells.