Hsp90 Chaperones Bluetongue Virus Proteins and Prevents Proteasomal Degradation.

The molecular chaperone machinery is important for the maintenance of protein homeostasis within the cells. The principle activities of the chaperone machinery are to facilitate protein folding and organize conformationally dynamic client proteins. Prominent among the members of the chaperone family are heat shock protein 70 (Hsp70) and 90 (Hsp90). Like cellular proteins, viral proteins depend upon molecular chaperones to mediate their stabilization and folding. Bluetongue virus (BTV), which is a model system for the Reoviridae family, is a nonenveloped arbovirus that causes hemorrhagic disease in ruminants. This constitutes a significant burden upon animals of commercial significance, such as sheep and cattle. Here, for the first time, we examined the role of chaperone proteins in the viral lifecycle of BTV. Using a combination of molecular, biochemical, and microscopic techniques, we examined the function of Hsp90 and its relevance to BTV replication. We demonstrate that Hsp70, the chaperone that is commonly usurped by viral proteins, does not influence virus replication, while Hsp90 activity is important for virus replication by stabilizing BTV proteins and preventing their degradation via the ubiquitin-proteasome pathway. To our knowledge this is the first report showing the involvement of Hsp90 as a modulator of BTV infection.IMPORTANCE Protein chaperones are instrumental for maintaining protein homeostasis, enabling correct protein folding and organization; prominent members include heat shock proteins 70 and 90. Virus infections place a large burden on this homeostasis. Identifying and understanding the underlying mechanisms that facilitate Bluetongue virus replication and spread through the usurpation of host factors is of primary importance for the development of intervention strategies. Our data identify and show that heat shock protein 90, but not heat shock protein 70, stabilizes bluetongue virus proteins, safeguarding them from proteasomal degradation.

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.

A low-passage insect-cell isolate of bluetongue virus uses a macropinocytosis-like entry pathway to infect natural target cells derived from the bovine host.

Bluetongue virus (BTV) causes an economically important disease in domestic and wildlife ruminants and is transmitted by Culicoides biting midges. In ruminants, BTV has a wide cell tropism that includes endothelial cells of vascular and lymphatic vessels as important cell targets for virus replication, and several cell types of the immune system including monocytes, macrophages and dendritic cells. Thus, cell-entry represents a particular challenge for BTV as it infects many different cell types in widely diverse vertebrate and invertebrate hosts. Improved understanding of BTV cell-entry could lead to novel antiviral approaches that can block virus transmission from cell to cell between its invertebrate and vertebrate hosts. Here, we have investigated BTV cell-entry using endothelial cells derived from the natural bovine host (BFA cells) and purified whole virus particles of a low-passage, insect-cell isolate of a virulent strain of BTV-1. Our results show that the main entry pathway for infection of BFA cells is dependent on actin and dynamin, and shares certain characteristics with macropinocytosis. The ability to use a macropinocytosis-like entry route could explain the diverse cell tropism of BTV and contribute to the efficiency of transmission between vertebrate and invertebrate hosts.

Phosphoproteomic Analysis Reveals the Importance of Kinase Regulation During Orbivirus Infection.

Bluetongue virus (BTV) causes infections in wild and domesticated ruminants with high morbidity and mortality and is responsible for significant economic losses in both developing and developed countries. BTV serves as a model for the study of other members of the Orbivirus genus. Previously, the importance of casein kinase 2 for BTV replication was demonstrated. To identify intracellular signaling pathways and novel host-cell kinases involved during BTV infection, the phosphoproteome of BTV infected cells was analyzed. Over 1000 phosphosites were identified using mass spectrometry, which were then used to determine the corresponding kinases involved during BTV infection. This analysis yielded protein kinase A (PKA) as a novel kinase activated during BTV infection. Subsequently, the importance of PKA for BTV infection was validated using a PKA inhibitor and activator. Our data confirmed that PKA was essential for efficient viral growth. Further, we showed that PKA is also required for infection of equid cells by African horse sickness virus, another member of the Orbivirus genus. Thus, despite their preference in specific host species, orbiviruses may utilize the same host signaling pathways during their replication.

Proteomic analysis of sheep primary testicular cells infected with bluetongue virus.

Bluetongue virus (BTV) causes a non-contagious, arthropod-transmitted disease in wild and domestic ruminants, such as sheep. In this study, we used iTRAQ labeling coupled with LC-MS/MS for quantitative identification of differentially expressed proteins in BTV-infected sheep testicular (ST) cells. Relative quantitative data were obtained for 4455 proteins in BTV- and mock-infected ST cells, among which 101 and 479 proteins were differentially expressed at 24 and 48 h post-infection, respectively, indicating further proteomic changes during the later stages of infection. Ten corresponding genes of differentially expressed proteins were validated via real-time RT-PCR. Expression levels of three representative proteins, eIF4a1, STAT1 and HSP27, were further confirmed via western blot analysis. Bioinformatics analysis disclosed that the differentially expressed proteins are primarily involved in biological processes related to innate immune response, signal transduction, nucleocytoplasmic transport, transcription and apoptosis. Several upregulated proteins were associated with the RIG-I-like receptor signaling pathway and endocytosis. To our knowledge, this study represents the first attempt to investigate proteome-wide dysregulation in BTV-infected cells with the aid of quantitative proteomics. Our collective results not only enhance understanding of the host response to BTV infection but also highlight multiple potential targets for the development of antiviral agents.

Dual modulation of type I interferon response by bluetongue virus.

UNLABELLED: Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus that causes an economically important disease in ruminants. BTV infection is a strong inducer of type I interferon (IFN-I) in multiple cell types. It has been shown recently that BTV and, more specifically, the nonstructural protein NS3 of BTV are able to modulate the IFN-I synthesis pathway. However, nothing is known about the ability of BTV to counteract IFN-I signaling. Here, we investigated the effect of BTV on the IFN-I response pathway and, more particularly, the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription protein (STAT) signaling pathway. We found that BTV infection triggered the expression of IFN-stimulated genes (ISGs) in A549 cells. However, when BTV-infected cells were stimulated with external IFN-I, we showed that activation of the IFN-stimulated response element (ISRE) promoter and expression of ISGs were inhibited. We found that this inhibition involved two different mechanisms that were dependent on the time of infection. After overnight infection, BTV blocked specifically the phosphorylation and nuclear translocation of STAT1. This inhibition correlated with the redistribution of STAT1 in regions adjacent to the nucleus. At a later time point of infection, BTV was found to interfere with the activation of other key components of the JAK/STAT pathway and to induce the downregulation of JAK1 and TYK2 protein expression. Overall, our study indicates for the first time that BTV is able to interfere with the JAK/STAT pathway to modulate the IFN-I response. IMPORTANCE: Bluetongue virus (BTV) causes a severe disease in ruminants and has an important impact on the livestock economy in areas of endemicity such as Africa. The emergence of strains, such as serotype 8 in Europe in 2006, can lead to important economic losses due to commercial restrictions and prophylactic measures. It has been known for many years that BTV is a strong inducer of type I interferon (IFN-I) in vitro and in vivo in multiple cell types. However, the ability of BTV to interact with the IFN-I system remains unclear. Here, we report that BTV is able to modulate the IFN-I response by interfering with the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription protein (STAT) signaling pathway. These findings contribute to knowledge of how BTV infection interferes with the host's innate immune response and becomes pathogenic. This will also be important for the design of efficacious vaccine candidates.

Identification and characterization of a novel non-structural protein of bluetongue virus.

Bluetongue virus (BTV) is the causative agent of a major disease of livestock (bluetongue). For over two decades, it has been widely accepted that the 10 segments of the dsRNA genome of BTV encode for 7 structural and 3 non-structural proteins. The non-structural proteins (NS1, NS2, NS3/NS3a) play different key roles during the viral replication cycle. In this study we show that BTV expresses a fourth non-structural protein (that we designated NS4) encoded by an open reading frame in segment 9 overlapping the open reading frame encoding VP6. NS4 is 77-79 amino acid residues in length and highly conserved among several BTV serotypes/strains. NS4 was expressed early post-infection and localized in the nucleoli of BTV infected cells. By reverse genetics, we showed that NS4 is dispensable for BTV replication in vitro, both in mammalian and insect cells, and does not affect viral virulence in murine models of bluetongue infection. Interestingly, NS4 conferred a replication advantage to BTV-8, but not to BTV-1, in cells in an interferon (IFN)-induced antiviral state. However, the BTV-1 NS4 conferred a replication advantage both to a BTV-8 reassortant containing the entire segment 9 of BTV-1 and to a BTV-8 mutant with the NS4 identical to the homologous BTV-1 protein. Collectively, this study suggests that NS4 plays an important role in virus-host interaction and is one of the mechanisms played, at least by BTV-8, to counteract the antiviral response of the host. In addition, the distinct nucleolar localization of NS4, being expressed by a virus that replicates exclusively in the cytoplasm, offers new avenues to investigate the multiple roles played by the nucleolus in the biology of the cell.

The Timing and Magnitude of the Type I Interferon Response Are Correlated with Disease Tolerance in Arbovirus Infection.

Infected hosts possess two alternative strategies to protect themselves against the negative impact of virus infections: resistance, used to abrogate virus replication, and disease tolerance, used to avoid tissue damage without controlling viral burden. The principles governing pathogen resistance are well understood, while less is known about those involved in disease tolerance. Here, we studied bluetongue virus (BTV), the cause of bluetongue disease of ruminants, as a model system to investigate the mechanisms of virus-host interactions correlating with disease tolerance. BTV induces clinical disease mainly in sheep, while cattle are considered reservoirs of infection, rarely exhibiting clinical symptoms despite sustained viremia. Using primary cells from multiple donors, we show that BTV consistently reaches higher titers in ovine cells than cells from cattle. The variable replication kinetics of BTV in sheep and cow cells were mostly abolished by abrogating the cell type I interferon (IFN) response. We identified restriction factors blocking BTV replication, but both the sheep and cow orthologues of these antiviral genes possess anti-BTV properties. Importantly, we demonstrate that BTV induces a faster host cell protein synthesis shutoff in primary sheep cells than cow cells, which results in an earlier downregulation of antiviral proteins. Moreover, by using RNA sequencing (RNA-seq), we also show a more pronounced expression of interferon-stimulated genes (ISGs) in BTV-infected cow cells than sheep cells. Our data provide a new perspective on how the type I IFN response in reservoir species can have overall positive effects on both virus and host evolution. IMPORTANCE The host immune response usually aims to inhibit virus replication in order to avoid cell damage and disease. In some cases, however, the infected host avoids the deleterious effects of infection despite high levels of viral replication. This strategy is known as disease tolerance, and it is used by animal reservoirs of some zoonotic viruses. Here, using a virus of ruminants (bluetongue virus [BTV]) as an experimental system, we dissected virus-host interactions in cells collected from species that are susceptible (sheep) or tolerant (cow) to disease. We show that (i) virus modulation of the host antiviral type I interferon (IFN) responses, (ii) viral replication kinetics, and (iii) virus-induced cell damage differ in tolerant and susceptible BTV-infected cells. Understanding the complex virus-host interactions in disease tolerance can allow us to disentangle the critical balance between protective and damaging host immune responses.

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

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

The VP3 Protein of Bluetongue Virus Associates with the MAVS Complex and Interferes with the RIG-I-Signaling Pathway.

Bluetongue virus (BTV), an arbovirus transmitted by Culicoides biting midges, is a major concern of wild and domestic ruminants. While BTV induces type I interferon (alpha/beta interferon [IFN-α/ß]) production in infected cells, several reports have described evasion strategies elaborated by this virus to dampen this intrinsic, innate response. In the present study, we suggest that BTV VP3 is a new viral antagonist of the IFN-ß synthesis. Indeed, using split luciferase and coprecipitation assays, we report an interaction between VP3 and both the mitochondrial adapter protein MAVS and the IRF3-kinase IKKε. Overall, this study describes a putative role for the BTV structural protein VP3 in the control of the antiviral response.

Initial sequencing and tissue distribution of Toll-like receptor 3 mRNA in white-tailed deer (Odocoileus virginianus).

Toll-like receptor (TLR) 3 recognizes double-stranded RNA (dsRNA) and activates a signal transduction pathway that results in the release of a variety of chemokines and cytokines and apoptotic activity. Variability in TLR3 expression may play an important role in disease susceptibility of white-tailed deer (WTD; Odocoileus virginianus) to bluetongue and epizootic hemorrhagic disease viruses, which are dsRNA viruses. Because little is known about TLR3 in WTD, our objective was to sequence WTD TLR3 mRNA and to determine baseline levels of tissue expression. A 209-base pair sequence of TLR3 mRNA was obtained from WTD peripheral blood mononuclear cells. Dot blots confirmed that the sequence obtained was part of total WTD mRNA. Variable expression or ligand binding of TLR3 may contribute to observed susceptibility differences between populations of WTD, so the level of TLR3 in small intestine, skin, spleen, heart, cecum, rumen, lymph node, lung, kidney, and liver from WTD fawns (n=2) was analyzed using real-time reverse transcriptase-polymerase chain reaction. Tissue expression of TLR3 mRNA relative to the housekeeping gene beta-actin was highest in spleen, heart, skin, and lung.

Attenuation of Bluetongue Virus (BTV) in an in ovo Model Is Related to the Changes of Viral Genetic Diversity of Cell-Culture Passaged BTV.

The embryonated chicken egg (ECE) is routinely used for the laboratory isolation and adaptation of Bluetongue virus (BTV) in vitro. However, its utility as an alternate animal model has not been fully explored. In this paper, we evaluated the pathogenesis of BTV in ovo using a pathogenic isolate of South African BTV serotype 3 (BTV-3) derived from the blood of an infected sheep. Endothelio- and neurotropism of BTV-3 were observed by immunohistochemistry of non-structural protein 1 (NS1), NS3, NS3/3a, and viral protein 7 (VP7) antigens. In comparing the pathogenicity of BTV from infectious sheep blood with cell-culture-passaged BTV, including virus propagated through a Culicoides-derived cell line (KC) or ECE, we found virus attenuation in ECE following cell-culture passage. Genomic analysis of the consensus sequences of segments (Seg)-2, -5, -6, -7, -8, -9, and -10 identified several nucleotide and amino-acid mutations among the cell-culture-propagated BTV-3. Deep sequencing analysis revealed changes in BTV-3 genetic diversity in various genome segments, notably a reduction of Seg-7 diversity following passage in cell culture. Using this novel approach to investigate BTV pathogenicity in ovo, our findings support the notion that pathogenic BTV becomes attenuated in cell culture and that this change is associated with virus quasispecies evolution.

Functional mapping of bluetongue virus proteins and their interactions with host proteins during virus replication.

Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus which is transmitted by blood-feeding gnats to wild and domestic ruminants, causing high morbidity and often high mortality. Partly due to this BTV has been in the forefront of molecular studies for last three decades and now represents one of the best understood viruses at the molecular and structural levels. BTV, like the other members of the Reoviridae family is a complex non-enveloped virus with seven structural proteins and a RNA genome consisting of 10 dsRNA segments of different sizes. In virus infected cells, three other virus encoded nonstructural proteins are synthesized. Significant recent advances have been made in understanding the structure-function relationships of BTV proteins and their interactions during virus assembly. By combining structural and molecular data it has been possible to make progress on the fundamental mechanisms used by the virus to invade, replicate in, and escape from, susceptible host cells. Data obtained from studies over a number of years have defined the key players in BTV entry, replication, assembly and egress. Specifically, it has been possible to determine the complex nature of the virion through three dimensional structure reconstructions; atomic structure of proteins and the internal capsid; the definition of the virus encoded enzymes required for RNA replication; the ordered assembly of the capsid shell and the protein sequestration required for it; and the role of three NS proteins in virus replication, assembly and release. Overall, this review demonstrates that the integration of structural, biochemical and molecular data is necessary to fully understand the assembly and replication of this complex RNA virus.

MicroRNA expression profiling of primary sheep testicular cells in response to bluetongue virus infection.

Bluetongue virus (BTV) is a member of the genus Orbivirus within the family Reoviridae and causes a non-contagious, insect-transmitted disease in domestic and wild ruminants, mainly in sheep and occasionally in cattle and some species of deer. Virus infection can trigger the changes of the cellular microRNA (miRNA) expression profile, which play important post-transcriptional regulatory roles in gene expression and can greatly influence viral replication and pathogenesis. Here, we employed deep sequencing technology to determine which cellular miRNAs were differentially expressed in primary sheep testicular (ST) cells infected with BTV. A total of 25 known miRNAs and 240 novel miRNA candidates that were differentially expressed in BTV-infected and uninfected ST cells were identified, and 251 and 8428 predicted target genes were annotated, respectively. Nine differentially expressed miRNAs and their mRNA targets were validated by quantitative reverse transcription-polymerase chain reaction. Targets prediction and functional analysis of these regulated miRNAs revealed significant enrichment for several signaling pathways including MAPK, PI3K-Akt, endocytosis, Hippo, NF-kB, viral carcinogenesis, FoxO, and JAK-STAT signaling pathways. This study provides a valuable basis for further investigation on the roles of miRNAs in BTV replication and pathogenesis.

Nucleotide sequence analysis of VP7 gene of Indian isolates of bluetongue virus vis-à-vis other serotypes from different parts of the world.

Bluetongue virus (BTV), a member of genus Orbivirus, a family Reoviridae, is a non-enveloped with double shelled structure and ten segmented double stranded (ds) RNA genome. The RNA segment S7 encodes an inner capsid serogroup specific viral protein VP7. To amplify coding region of VP7 gene of BTV, new primers, forward primer (18-38 bp) and reverse primer (1156-1136 bp), were designed using VP7 gene sequences available in GenBank. This primer pair successfully amplified cell culture adapted Indian isolates of BTV belonging to two different serotypes 1 and 18. The coding sequences of two Indian isolates of BTV (BTV-1H and BTV-18B) were cloned into pPCR Script-Amp SK (+) plasmid vector and transformed into XL10-Gold Kan ultracompetent E. coli cells. The positive clones selected by blue-white screening and colony touch PCR were sequenced. The sequence analysis revealed that there was 93-97% nucleotide sequence identity in VP7 gene of three different Indian serotypes of BTV. The VP7 gene sequences of Indian isolates have comparatively less sequence homology (< 80%) with American (US), and French isolates compared to South African (SA), Australian (AUS) and Chinese (PRC) isolates. In silico restriction enzyme profile analysis of VP7 gene sequences revealed that Indian isolates of BTV-1 can be differentiated from other BTV-1 isolates reported from SA, AUS and PRC using TaqI. Similarly the Indian isolates of BTV belonging to three different serotypes can be differentiated using EcoRI, Hae III and TaqI restriction enzymes.

Pathological Characterization Of IFNAR(-/-) Mice Infected With Bluetongue Virus Serotype 4.

Bluetongue virus (BTV) replicates in lymphoid tissues where infected mononuclear leukocytes secrete proinflammatory and vasoactive mediators that can contribute to bluetongue (BT) pathogenesis. Using the well-characterized IFNAR(-/-) mice animal model, we have now studied the histopathology and dynamics of leukocyte populations in different target tissues (spleen, thymus, and lung) during BTV-4 infection by histological and immunohistochemical techniques. The spleen and thymus of BTV-4 infected mice showed severe lymphoid depletion on H&E stained sections. This finding was confirmed by IHC, showing moderate decreased immunopositivity against CD3 in the thymus, and scarce immunoreactivity against CD3 and CD79 in the rest of the white pulp in the spleen, together with an increase in MAC387 immunostaining. BTV-4 infection also induced the expression of active caspase-3 in the spleen, where apoptotic debris was observed by H&E. A dramatic increase in iNOS immunoreactivity associated to necrotic areas of the white pulp was observed, being less noticeable in the thymus and the lung. The induction of pro-inflammatory cytokines in tissues where BTV replicates was evaluated by measuring transcript levels by RT-qPCR. BTV-4 infection led to enhance transcription of IFN-γ, TNF, IL-6, IL-12-p40, and IL-1ß mRNA in the thymus, spleen and lung, correlating with the level of virus replication in these tissues. Disease progression and pathogenesis in IFNAR(-/-) mice closely mimics hallmarks of bluetongue disease in ruminants. IFNAR(-/-) mice are a good choice to facilitate a faster advance in the field of orbiviruses.

Bluetongue virus-induced activation of primary bovine lung microvascular endothelial cells.

Bluetongue is an insect-transmitted viral disease of sheep and some species of wild ruminants. Infection of lung microvascular endothelial cells (ECs) is central to the pathogenesis of bluetongue virus (BTV) infection of ruminants, but it is uncertain as to why cattle are resistant to BTV-induced microvascular injury and bluetongue disease. Thus, in order to better understand the pathogenesis of BTV infection of cattle, mRNAs encoding a variety of inflammatory mediators were quantitated by real-time polymerase chain reaction in primary bovine lung microvascular ECs (BLmVECs) exposed to BTV and/or EC-derived mediators. BTV infection of BLmVECs significantly increased the transcription of genes encoding interleukin-1 (IL-1), IL-6, IL-8, cyclooxygenase-2, and inducible nitric oxide synthase. Treatment of BLmVECs with EC-lysates that contained BTV as well as cytokines increased both the incidence of apoptosis and expression of cellular adhesion molecules, as compared to infection of BLmVECs with BTV alone. Thus, BTV infection caused activation of BLmVECs with production of inflammatory mediators that alter the mechanism of cell death of BLmVECs and exert potentially potent effects on blood coagulation. The activities of BTV-induced-EC-derived inflammatory mediators likely contribute to the resistance of cattle to BTV-induced microvascular injury and bluetongue disease.

Bluetongue virus infection: activation of the MAP kinase-dependent pathway is required for apoptosis.

Bluetongue virus (BTV) is a double-stranded RNA virus that induces apoptosis both in mammalian cell cultures and in target tissues. Based on information that members of the mitogen-activated protein kinase family (MAPKs) are mediators of apoptosis, we have examined in detail the MAPK-dependent apoptosis in BTV infection. Previously, we have shown that apoptosis in BTV infection requires the participation of mitochondrial apoptotic pathways. In addition, we demonstrated that NF-κB is activated and that its inhibition substantially reduces cellular apoptosis. For the first time, here we demonstrated the activation of MAPKs after BTV infection. Moreover, by pre-treatment with MAPK inhibitors, c-Jun N-terminal kinases (JNKs) and p38 MAPK, but not extracellular signal-related kinase (ERK), significantly decreased the induction of apoptosis. JNK and p38 activation regulated the cytochrome c released from mitochondria and caspase 3 activation. These results strengthen the understanding of BTV infection and contribute to our previous data confirming that BTV infection induces robust apoptosis in mammalian cells and is likely to play a primary role in BTV pathophysiology.

Inhibin and reproduction. First communication: changes to inhibin level in Barki ewes after artificial infection with bluetongue virus.

Bluetongue virus type 16, isolated from sheep in Egypt, was injected to 4 normally cycling Barki ewes and caused high levels of inhibin. This was assayed by a biological method, using suppression of the luteinising hormone (LH) of castrated rats. Albumin (fraction 2) was injected to normally cycling ewes. The sera of injected ewes were investigated 1 day after injection and weekly up to the 4th one injection. There was a gradual decrease of LH (4.8 +/- 0.52 I.U./ml serum) until the minimum level (1.17 +/- 0.25 I.U./ml serum) was reached, in comparison to the control serum LH which was 5.26 +/- 0.52 I.U./ml serum during the dioestrous phase.

Histamine and eicosanoid levels in plasma and peripheral blood leucocyte cultures during experimental infection of cattle with bluetongue virus serotype 11.

Three calves were sensitized with three doses of inactivated BTV-11 UC8 strain and then experimentally infected with the homologous virus. In addition, four BTV-seronegative heifers were also experimentally infected with BTV-11. Granulocyte rich fractions of peripheral blood leucocyte (PBL-GRF) cultures from BTV-sensitized/infected calves and from control unexposed cattle were exposed in vitro with BTV-11. Histamine, leukotriene (LT) C4 and prostaglandin (PG) D2 were assayed in supernatant fluids. Plasma histamine levels increased in BTV-infected heifers from 10.1 +/- 2 ng/ml at Day 0 to 23.1 +/- 6.6 ng/ml at Day 12 following virus exposure. In addition, in this experimental group the concentration of PGF2 alpha (mean 551.97 +/- 243.54 pg/ml) increased significantly (P < or = 0.05) compared with control cattle (mean 467.3 +/- 73.9 pg/ml). Bluetongue virus induced histamine and LTC4 release after in vitro infection of PBL-GRF. Release of LTC4 was significantly (P < or = 0.05) higher in PBL-GRF cultures from sensitized and control animals than in unexposed PBL-GRF cultures. In contrast to these results, PGD2 was not released after BTV infection of PBL-GRF in vitro. The histamine release caused by BTV was virus-specific and mainly mediated by an immunological reaction, since the release was significantly reduced by removal of cell surface immunoglobulins.