Serological Cross-Reactions between Expressed VP2 Proteins from Different Bluetongue Virus Serotypes.

Bluetongue (BT) is a severe and economically important disease of ruminants that is widely distributed around the world, caused by the bluetongue virus (BTV). More than 28 different BTV serotypes have been identified in serum neutralisation tests (SNT), which, along with geographic variants (topotypes) within each serotype, reflect differences in BTV outer-capsid protein VP2. VP2 is the primary target for neutralising antibodies, although the basis for cross-reactions and serological variations between and within BTV serotypes is poorly understood. Recombinant BTV VP2 proteins (rVP2) were expressed in Nicotiana benthamiana, based on sequence data for isolates of thirteen BTV serotypes (primarily from Europe), including three 'novel' serotypes (BTV-25, -26 and -27) and alternative topotypes of four serotypes. Cross-reactions within and between these viruses were explored using rabbit anti-rVP2 sera and post BTV-infection sheep reference-antisera, in I-ELISA (with rVP2 target antigens) and SNT (with reference strains of BTV-1 to -24, -26 and -27). Strong reactions were generally detected with homologous rVP2 proteins or virus strains/serotypes. The sheep antisera were largely serotype-specific in SNT, but more cross-reactive by ELISA. Rabbit antisera were more cross-reactive in SNT, and showed widespread, high titre cross-reactions against homologous and heterologous rVP2 proteins in ELISA. Results were analysed and visualised by antigenic cartography, showing closer relationships in some, but not all cases, between VP2 topotypes within the same serotype, and between serotypes belonging to the same 'VP2 nucleotype'.

Serological survey of bluetongue virus in sheep from Minas Gerais / Inquérito sorológico do vírus da língua azul em ovinos de Minas Gerais

Bluetongue is an infectious, non-contagious disease that affects domestic and wild ruminants, caused by a virus from the Orbivirus genus, Reoviridae family, transmitted by arthropod vectors of the Culicoides genus. This paper aims to be the first serological survey of bluetongue in sheep from the Meso-regions of Campo das Vertentes and South and Southeast of Minas Gerais. Samples were collected from sheep from different properties. The serum samples were submitted to Agar Gel Immunodiffusion (AGID) and competitive Enzyme-Linked Immunosorbent Assay (cELISA). 303 serum samples were submitted to AGID and cELISA. In these samples, 164 (54.13%) were positive in the AGID technique, and 171 (56.44%) positive in the cELISA technique, with an almost perfect agreement between the techniques (kappa index = 0.887). In all visited properties, positive animals have been found in the herd. Animals acquired from properties of the studied mesoregions were more likely to be positive in IDGA and cELISA tests than animals acquired from properties in other regions of Brazil (p<0.001). These results suggest that bluetongue virus (BTV) is widespread in the mesoregions of Campo das Vertentes and South and Southeast of Minas Gerais.(AU)

A língua azul (LA) é uma doença infecciosa, não contagiosa, que acomete ruminantes domésticos e silvestres, causada por um vírus do gênero Orbivirus da família Reoviridae, transmitida por vetores artrópodes do gênero Culicoides. O presente estudo representa o primeiro trabalho a realizar um inquérito sorológico da língua azul em rebanhos ovinos nas Mesorregiões de Campo das Vertentes e Sul e Sudoeste de Minas Gerais. Foram coletadas amostras de soro de ovinos de diferentes propriedades. As amostras de soro foram submetidas aos testes de imunodifusão em gel de ágar (IDGA) e ensaio de imunoadsorção enzimática por competição (cELISA). Ao todo 303 amostras de soro foram submetidas ao IDGA e cELISA. Dessas amostras, 164 (54,13%) foram positivas na técnica de IDGA e 171 (56,44%) positivas na técnica de cELISA, havendo concordância quase perfeita entre as técnicas (índice kappa = 0,887). Em todas as propriedades visitadas, foram encontrados animais positivos no rebanho. Animais adquiridos de propriedades das Mesorregiões estudadas, tiveram mais chances de serem positivos nos testes de IDGA e cELISA do que animais adquiridos de propriedades de outras Regiões do Brasil (p<0,001). Esses resultados sugerem que o vírus da língua azul encontra-se disseminado em ovinos nas Mesorregiões de Campo das Vertentes e Sul e Sudoeste de Minas Gerais.(AU)

Evaluation of an IGM-specific ELISA for early detection of bluetongue virus infections in domestic ruminants sera.

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.

A competitive ELISA for detection of group specific antibody to bluetongue virus using anti-core antibody.

Bluetongue virus (BTV) is transmitted by biting midges, which infects domestic and wild ruminants. In present study, a competitive enzyme-linked immunosorbent assay (C-ELISA) for the detection of serogroup-specific antibodies against VP7 protein of BTV has been developed. The assay measures the competition between a group specific antibody against core protein of BTV and a test serum to an optimized concentration of BTV recombinant-VP7 (r-VP7) antigen. Serum samples (n = 895) collected from small and large ruminants were used to optimize the C-ELISA. Percent inhibition (PI) values were used for estimation of the cut-off value for the C-ELISA. On receiver operator characteristic (ROC) analysis, different cut-off values along with their diagnostic sensitivity (DSn) and diagnostic specificity (DSp) were obtained. Among these, >50% PI value was accepted as cut-off at which DSn and Dsp was achieved as 97.6% and 98.0% respectively, at >95% confidence interval. Results show the present C-ELISA assay described to be sensitive, specific and reliable and could be adopted for serological investigation of small and large ruminants.

Follicular dendritic cell disruption as a novel mechanism of virus-induced immunosuppression.

Arboviruses cause acute diseases that increasingly affect global health. We used bluetongue virus (BTV) and its natural sheep host to reveal a previously uncharacterized mechanism used by an arbovirus to manipulate host immunity. Our study shows that BTV, similarly to other antigens delivered through the skin, is transported rapidly via the lymph to the peripheral lymph nodes. Here, BTV infects and disrupts follicular dendritic cells, hindering B-cell division in germinal centers, which results in a delayed production of high affinity and virus neutralizing antibodies. Moreover, the humoral immune response to a second antigen is also hampered in BTV-infected animals. Thus, an arbovirus can evade the host antiviral response by inducing an acute immunosuppression. Although transient, this immunosuppression occurs at the critical early stages of infection when a delayed host humoral immune response likely affects virus systemic dissemination and the clinical outcome of disease.

Protective Efficacy in Sheep of Adenovirus-Vectored Vaccines against Bluetongue Virus Is Associated with Specific T Cell Responses.

Bluetongue virus (BTV) is an economically important Orbivirus of the Reoviridae family that causes a hemorrhagic disease in ruminants. Its control has been achieved by inactivated-vaccines that have proven to protect against homologous BTV challenge although unable to induce long-term immunity. Therefore, a more efficient control strategy needs to be developed. Recombinant adenovirus vectors are lead vaccine candidates for protection of several diseases, mainly because of their potency to induce potent T cell immunity. Here we report the induction of humoral and T-cell mediated responses able to protect animals against BTV challenge by recombinant replication-defective human adenovirus serotype 5 (Ad5) expressing either VP7, VP2 or NS3 BTV proteins. First we used the IFNAR(-/-) mouse model system to establish a proof of principle, and afterwards we assayed the protective efficacy in sheep, the natural host of BTV. Mice were completely protected against BTV challenge, developing humoral and BTV-specific CD8+- and CD4+-T cell responses by vaccination with the different rAd5. Sheep vaccinated with Ad5-BTV-VP2 and Ad5-BTV-VP7 or only with Ad5-BTV-VP7 and challenged with BTV showed mild disease symptoms and reduced viremia. This partial protection was achieved in the absence of neutralizing antibodies but strong BTV-specific CD8+ T cell responses in those sheep vaccinated with Ad5-BTV-VP7. These data indicate that rAd5 is a suitable vaccine vector to induce T cell immunity during BTV vaccination and provide new data regarding the relevance of T cell responses in protection during BTV infection.

Comparative analysis of cellular immune responses and cytokine levels in sheep experimentally infected with bluetongue virus serotype 1 and 8.

Protective immunity in sheep with bluetongue virus (BTV) infection as well as the role of BTV-induced cytokines during immune response remains unclear. Understanding the basis immunological mechanisms in sheep experimentally infected with serotypes 1 and 8 (BTV-1 and -8) was the aim of this study. A time-course study was carried out in order to evaluate cell-mediated immune response and serum concentrations of cytokines (IL-1ß, TNFα, IL-12, IFNγ, IL-4 and IL-10) with inflammatory and immunological functions. Depletion of T cell subsets (mainly CD4(+), γδ and CD25(+)) together with the absence of cytokines (IFNγ and IL-12) involved in the regulation of cell-mediated antiviral immunity at the first stage of the disease suggested that both BTV-1 and BTV-8 might impair host's capability against primary infections which would favor viral replication and spreading. However, cellular immune response and cytokines elicited an immune response in sheep that efficiently reduced viremia in the final stage of the experiment. Recovery of T cell subsets (CD4(+) and CD25(+)) together with a significant increase of CD8(+) T lymphocytes in both infected groups were observed in parallel with the decrease of viremia. Additionally, the recovery of CD4(+) T lymphocytes together with the significant increase of IL-4 serum levels at the final stage of the experiment might contribute to humoral immune response activation and neutralizing antibodies production against BTV previously described in the course of this experiment. These results suggested that both cellular and humoral immune response may contribute to protective immunity against BTV-1 and BTV-8 in sheep. The possible role played by IL-10 and CD25(+) cells in controlling inflammatory and immune response in the final stage of the experiment has also been suggested.

Expression of VP7, a Bluetongue virus group specific antigen by viral vectors: analysis of the induced immune responses and evaluation of protective potential in sheep.

Bluetongue virus (BTV) is an economically important Orbivirus transmitted by biting midges to domestic and wild ruminants. The need for new vaccines has been highlighted by the occurrence of repeated outbreaks caused by different BTV serotypes since 1998. The major group-reactive antigen of BTV, VP7, is conserved in the 26 serotypes described so far, and its role in the induction of protective immunity has been proposed. Viral-based vectors as antigen delivery systems display considerable promise as veterinary vaccine candidates. In this paper we have evaluated the capacity of the BTV-2 serotype VP7 core protein expressed by either a non-replicative canine adenovirus type 2 (Cav-VP7 R0) or a leporipoxvirus (SG33-VP7), to induce immune responses in sheep. Humoral responses were elicited against VP7 in almost all animals that received the recombinant vectors. Both Cav-VP7 R0 and SG33-VP7 stimulated an antigen-specific CD4+ response and Cav-VP7 R0 stimulated substantial proliferation of antigen-specific CD8+ lymphocytes. Encouraged by the results obtained with the Cav-VP7 R0 vaccine vector, immunized animals were challenged with either the homologous BTV-2 or the heterologous BTV-8 serotype and viral burden in plasma was followed by real-time RT-PCR. The immune responses triggered by Cav-VP7 R0 were insufficient to afford protective immunity against BTV infection, despite partial protection obtained against homologous challenge. This work underscores the need to further characterize the role of BTV proteins in cross-protective immunity.

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.

Type I interferon limits the capacity of bluetongue virus to infect hematopoietic precursors and dendritic cells in vitro and in vivo.

Hematopoietic stem cells (HSCs) give rise to progenitors with potential to produce multiple cell types, including dendritic cells (DCs). DCs are the principal antigen-presenting cells and represent the crucial link between innate and adaptive immune responses. Bluetongue virus (BTV), an economically important Orbivirus of the Reoviridae family, causes a hemorrhagic disease mainly in sheep and occasionally in other species of ruminants. BTV is transmitted between its mammalian hosts by certain species of biting midges (Culicoides spp.) and is a potent alpha interferon (IFN-α) inducer. In the present report, we show that BTV infects cells of hematopoietic origin but not HSCs in immunocompetent sheep. However, BTV infects HSCs in the absence of type I IFN (IFN-I) signaling in vitro and in vivo. Infection of HSCs in vitro results in cellular death by apoptosis. Furthermore, BTV infects bone marrow-derived DCs (BM-DCs), interfering with their development to mature DCs in the absence of type I IFN signaling. Costimulatory molecules CD80 and CD86 and costimulatory molecules CD40 and major histocompatibility complex class II (MHC-II) are affected by BTV infection, suggesting that BTV interferes with DC antigen-presenting capacity. In vivo, different DC populations are also affected during the course of infection, probably as a result of a direct effect of BTV replication in DCs and the production of infectious virus. These new findings suggest that BTV infection of HSCs and DCs can impair the immune response, leading to persistence or animal death, and that this relies on IFN-I.

A method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles.

Plant expression systems based on nonreplicating virus-based vectors can be used for the simultaneous expression of multiple genes within the same cell. They therefore have great potential for the production of heteromultimeric protein complexes. This work describes the efficient plant-based production and assembly of Bluetongue virus-like particles (VLPs), requiring the simultaneous expression of four distinct proteins in varying amounts. Such particles have the potential to serve as a safe and effective vaccine against Bluetongue virus (BTV), which causes high mortality rates in ruminants and thus has a severe effect on the livestock trade. Here, VLPs produced and assembled in Nicotiana benthamiana using the cowpea mosaic virus-based HyperTrans (CPMV-HT) and associated pEAQ plant transient expression vector system were shown to elicit a strong antibody response in sheep. Furthermore, they provided protective immunity against a challenge with a South African BTV-8 field isolate. The results show that transient expression can be used to produce immunologically relevant complex heteromultimeric structures in plants in a matter of days. The results have implications beyond the realm of veterinary vaccines and could be applied to the production of VLPs for human use or the coexpression of multiple enzymes for the manipulation of metabolic pathways.

Autoimmune/autoinflammatory syndrome induced by adjuvants (ASIA syndrome) in commercial sheep.

We describe a form of the autoimmune/autoinflammatory syndrome induced by adjuvants (ASIA syndrome) in commercial sheep, linked to the repetitive inoculation of aluminum-containing adjuvants through vaccination. The syndrome shows an acute phase that affects less than 0.5% of animals in a given herd, it appears 2-6 days after an adjuvant-containing inoculation and it is characterized by an acute neurological episode with low response to external stimuli and acute meningoencephalitis, most animals apparently recovering afterward. The chronic phase is seen in a higher proportion of flocks, it can follow the acute phase, and it is triggered by external stimuli, mostly low temperatures. The chronic phase begins with an excitatory phase, followed by weakness, extreme cachexia, tetraplegia and death. Gross lesions are related to a cachectic process with muscular atrophy, and microscopic lesions are mostly linked to a neurodegenerative process in both dorsal and ventral column of the gray matter of the spinal cord. Experimental reproduction of ovine ASIA in a small group of repeatedly vaccinated animals was successful. Detection of Al(III) in tissues indicated the presence of aluminum in the nervous tissue of experimental animals. The present report is the first description of a new sheep syndrome (ovine ASIA syndrome) linked to multiple, repetitive vaccination and that can have devastating consequences as it happened after the compulsory vaccination against bluetongue in 2008. The ovine ASIA syndrome can be used as a model of other similar diseases affecting both human and animals. A major research effort is needed in order to understand its complex pathogenesis.

Potential role of proinflammatory cytokines in the pathogenetic mechanisms of vascular lesions in goats naturally infected with bluetongue virus serotype 1.

In vitro studies have demonstrated that bluetongue virus (BTV)-induced vasoactive mediators could contribute to the endothelial cells dysfunction and increased vascular permeability responsible of lesions characteristic of bluetongue (BT) like oedema, haemorrhages and ischaemic necrosis in different tissues. However, few in vivo studies have been carried out to clarify the causes of these lesions. The aim of this study was to elucidate in vivo the pathogenetic mechanisms involved in the appearance of vascular lesions in different organs during BT. For this purpose, tissue samples from goats naturally infected with bluetongue virus serotype 1 (BTV-1) were taken for histopathological and immunohistochemical studies to determine the potential role of proinflammatory cytokines (tumour necrosis factor alpha, TNFα and interleukin one alpha, IL-1α) in the increased vascular permeability and their relationship with the presence of virus. Gross and histopathological examination revealed the presence of vascular damage leading to generalized oedema and haemorrhages. Immunohistochemical studies displayed that endothelial injury may have been due to the direct pathogenic effect of BTV infection on endothelial cells or may be a response to inflammatory mediators released by virus-infected endothelial cells and, possibly, other cell types such as monocytes/macrophages. These preliminary results of what appears to be the first in vivo study of tissue damage in small BT-infected ruminants suggest a direct link between the appearance of vascular changes and the presence of BTV-induced vasoactive cytokines.

Global gene expression analysis in skin biopsies of European red deer experimentally infected with bluetongue virus serotypes 1 and 8.

Bluetongue virus (BTV) is a double-stranded RNA virus transmitted by blood-feeding biting midges of the genus Culicoides to wild and domestic ruminants, causing high morbidity and variable mortality. The aim of this study was to characterize differential gene expression in skin biopsies of red deer (Cervus elaphus) hinds experimentally infected with BTV serotypes 1 and 8. Skin biopsies were collected from BTV-1 and BTV-8 experimentally infected and control hinds at 14 and 98 days post-infection (dpi). Global gene expression profile in response to BTV infection was characterized at 14 dpi using a bovine microarray together with real-time RT-PCR analysis of differentially expressed genes at 14 and 98 dpi. Eighteen genes were upregulated and three were downregulated in response to virus infection, with no significant differences between BTV-1 and BTV-8 infected hinds. Seven unique genes, six upregulated (ISG15, PSMB8, PSMB9, BOLA, C1qA, C4) and one downregulated (FOS) were over-represented after conditional test for biological process gene ontology, which affected five molecular pathways (RIG-1, proteasome, MHC-1, complement, TLR) implicated in host immune response. BTV infection had a minor and transient effect on gene expression in hinds, as shown by the very few genes that were differentially expressed in response to infection at 14 dpi, most of which had similar expression levels between infected and uninfected animals at 98 dpi. These results suggested that red deer could control BTV infection with little effect on host molecular pathways.

The double-stranded RNA bluetongue virus induces type I interferon in plasmacytoid dendritic cells via a MYD88-dependent TLR7/8-independent signaling pathway.

Dendritic cells (DCs), especially plasmacytoid DCs (pDCs), produce large amounts of alpha/beta interferon (IFN-α/ß) upon infection with DNA or RNA viruses, which has impacts on the physiopathology of the viral infections and on the quality of the adaptive immunity. However, little is known about the IFN-α/ß production by DCs during infections by double-stranded RNA (dsRNA) viruses. We present here novel information about the production of IFN-α/ß induced by bluetongue virus (BTV), a vector-borne dsRNA Orbivirus of ruminants, in sheep primary DCs. We found that BTV induced IFN-α/ß in skin lymph and in blood in vivo. Although BTV replicated in a substantial fraction of the conventional DCs (cDCs) and pDCs in vitro, only pDCs responded to BTV by producing a significant amount of IFN-α/ß. BTV replication in pDCs was not mandatory for IFN-α/ß production since it was still induced by UV-inactivated BTV (UV-BTV). Other inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-12p40, were also induced by UV-BTV in primary pDCs. The induction of IFN-α/ß required endo-/lysosomal acidification and maturation. However, despite being an RNA virus, UV-BTV did not signal through Toll-like receptor 7 (TLR7) for IFN-α/ß induction. In contrast, pathways involving the MyD88 adaptor and kinases dsRNA-activated protein kinase (PKR) and stress-activated protein kinase (SAPK)/Jun N-terminal protein kinase (JNK) were implicated. This work highlights the importance of pDCs for the production of innate immunity cytokines induced by a dsRNA virus, and it shows that a dsRNA virus can induce IFN-α/ß in pDCs via a novel TLR-independent and Myd88-dependent pathway. These findings have implications for the design of efficient vaccines against dsRNA viruses.

Myxomavirus as a vector for the immunisation of sheep: protection study against challenge with bluetongue virus.

Recombinant poxviruses are well suited for the development of new vaccine vectors. Our previous data supported the idea that Myxomavirus (MYXV) is efficient at priming antibody responses in sheep. To provide definitive evidence on the potential of MYXV for vaccination against infectious diseases in ruminants, we investigated the immune protection provided by recombinant MYXV against bluetongue, a devastating disease in sheep. To test this concept, sheep were injected twice with an MYXV expressing the immunodominant VP2 protein (SG33-VP2). The SG33-VP2 vector promoted the production of neutralising antibodies and partially protected sheep against disease after challenge with a highly virulent strain of serotype-8 bluetongue virus (BTV-8). In contrast, an MYXV expressing both VP2 and VP5 proteins (SG33-VP2/5) elicited very little protection. The expression levels of the VP2 and VP5 proteins suggested that, greater than the co-expression of the VP5 protein which was previously thought to favour anti-VP2 antibody response, the high expression of VP2 may be critical in the MYXV context to stimulate a protective response in sheep. This highlights the requirement for a careful examination of antigen expression before any conclusion can be drawn on the respective role of the protective antigens. As a proof of principle, our study shows that an MYXV vaccine vector is possible in ruminants.

Enhanced proinflammatory cytokine activity during experimental bluetongue virus-1 infection in Indian native sheep.

Bluetongue disease has been causing variable morbidity and mortality in sheep in India and other countries of the world. The experimental infection with Indian BTV-1 strain induced mild to sub-acute infection in native sheep. There were low induction of IL-12, IFN-γ and TNF-α cytokine mRNA expressions determined by real time RT-PCR in draining lymph nodes (DLN), spleen, and PBMCs during the initial stages of infection (8 days post inoculation, DPI) and higher around 15 DPI. The reduced pro-inflammatory cytokine (IFN-γ and TNF-α) responses during the initial stage of infection (8 DPI) was also accompanied by similarly decreased T-cell populations and overt clinical symptoms. Later up regulation of these cytokines and substantial increase in the proportion of CD8(+) T-cells occurred with reduction of clinical signs and disappearance of BTV-1 antigen from tissues as determined by immunohistochemistry and RT-PCR. Thus there is definite involvement of pro-inflammatory cytokines and CD8(+) T cell activity in disease induced by BTV-1 strain.

Bluetongue virus infection activates bovine monocyte-derived macrophages and pulmonary artery endothelial cells.

Bluetongue virus (BTV) is the cause of bluetongue (BT), an emerging, arthropod-transmitted disease of ungulates. The cellular tropism of BTV in ruminants includes macrophages, dendritic cells and endothelial cells (ECs), and fulminant infection is characterized by lesions consistent with those of so-called viral hemorrhagic fevers. Specifically, BT is characterized by vascular injury with hemorrhage, tissue infarction and widespread edema. To further investigate the pathogenesis of vascular injury in BT, we evaluated the responses of cultured bovine pulmonary artery EC (bPAEC) and monocyte-derived macrophages (bMDM) to BTV infection by measuring transcript levels of genes encoding molecules important in mediating EC activation and/or endothelial barrier dysregulation. The data confirm that BTV infection of bPAEC resulted in increased transcription of genes encoding chemokine ligand 2 (CCL2) and E-selectin, and BTV infection of bMDM resulted in increased transcription of genes encoding TNF-alpha, IL-1beta, IL-8, and inducible nitric oxide synthase (iNOS). The data from these in vitro studies provide further evidence that cytokines and other vasoactive substances produced in macrophages potentially contribute to vascular injury in BTV-infected ruminants, along with direct effects of the virus itself on ECs.

A rapid assay for detecting antibody against Bluetongue virus with a latex agglutination test using recombinant VP7 antigen.

A latex agglutination test (LAT) for detecting antibody against Bluetongue virus (BTV) in ruminants was developed using latex beads coupled with recombinant VP7 protein. Compared with competitive enzyme-linked immunosorbent assay (ELISA), the specificity and sensitivity of the LAT were 99.0% and 93.0%, respectively. There was excellent agreement between the results obtained by competitive ELISA and the LAT (kappa = 0.930). Because it is rapid and easy to use, the LAT could be used for BTV antibody detection, especially for screening many serum samples.

The role of endothelial cell-derived inflammatory and vasoactive mediators in the pathogenesis of bluetongue.

Bluetongue is an insect-transmitted disease of sheep and wild ruminants that is caused by bluetongue virus (BTV). Cattle are asymptomatic reservoir hosts of BTV. Infection of lung microvascular endothelial cells (ECs) is central to the pathogenesis of BTV infection of both sheep and cattle, but it is uncertain as to why sheep are highly susceptible to BTV-induced microvascular injury, whereas cattle are not. Thus, to better characterize the pathogenesis of bluetongue, the transcription of genes encoding a variety of vasoactive and inflammatory mediators was quantitated in primary ovine lung microvascular ECs (OLmVECs) exposed to BTV and/or inflammatory mediators. BTV infection of OLmVECs increased the transcription of genes encoding interleukin- (IL) 1 and IL-8, but less so IL-6, cyclooxygenase-2, and inducible nitric oxide synthase. In contrast, we previously have shown that transcription of genes encoding all of these same mediators is markedly increased in BTV-infected bovine lung microvascular ECs and that BTV-infected bovine ECs produce substantially greater quantities of prostacyclin than do sheep ECs. Thus, sheep and cattle were experimentally infected with BTV to further investigate the role of EC-derived vasoactive mediators in the pathogenesis of bluetongue. The ratio of thromboxane to prostacyclin increased during BTV infection of both sheep and cattle, but was significantly greater in sheep (P = 0.001). Increases in the ratio of thromboxane to prostacyclin, indicative of enhanced coagulation, coincided with the occurrence of clinical manifestations of bluetongue in BTV-infected sheep. The data suggest that inherent species-specific differences in the production and activities of EC-derived mediators contribute to the sensitivity of sheep to BTV-induced microvascular injury.