In vivo and in vitro propagation and transmission of Toggenburg orbivirus

The Toggenburg orbivirus (TOV), a recently discovered virus related to bluetongue virus (BTV), has been identified in goats in Switzerland, Italy and Germany. Isolation of TOV in vitro has not yet been achieved and the transmission mechanisms are still unknown. In the experimental infection of pregnant goats described here, TOV could not be detected in secretion/excretion samples or fetal blood. Material from the goat experiment was used as inoculum for propagating the virus in vitro. To enhance the infectivity of TOV several modified protocols, e.g. pretreatment of the virus with trypsin, polyethylene glycol-mediated infection and lipofection were applied. Isolation of TOV, attempts to infect Culicoides nubeculosus by feeding TOV-positive blood and intracerebral inoculation of newborn mice were unsuccessful. The results of these studies suggest that TOV requires specific but different factors than other BTVs for infection and replication outside of its natural caprine host.     

Dynamics of viral spread in bluetongue virus infected calves

The kinetics of viremia and sites of viral replication in bluetongue virus (BTV) infected calves were characterized by virus isolation, serology and immunofluorescence staining procedures. In addition, the role of the regional lymph node and lymphatics draining inoculated skin in the pathogenesis of BTV infection was determined by analyzing efferent lymph collected from indwelling cannulas. Viremia persisted for 35 to 42 days after inoculation (DAI) and virus co-circulated with neutralizing antibodies for 23 to 26 days. Virus was first isolated from peripheral blood mononuclear (PBM) cells at 3 DAI, after stimulation of PBM cells with interleukin 2 and mitogen. BTV was frequently isolated from erythrocytes, platelets and stimulated PBM cells but never from granulocytes and rarely from plasma during viremia. Virus was consistently isolated from erythrocytes late in the course of veremia. Interruption of efferent lymph flow by cannulation delayed the onset of viremia to 7 DAI. BTV was infrequently isolated from lymph cells, and few fluorescence positive cells were observed after lymph and PBM cells were labelled with a BTV-specific monoclonal antibody. Virus was isolated from spleen by 4 DAI and most tissues by 6 DAI, whereas virus was isolated from bone marrow only at 10 DAI. Virus was not isolated from any tissue after termination of viremia. It is concluded that primary viral replication occurred in the local lymph node and BTV then was transported in low titer to secondary sites of replication via infected lymph and PBM cells. We speculate that virus replication in spleen resulted in release of virus into the circulation and non-selective infection of blood cells which disseminated BTV to other tissues. Virus association with erythrocytes likely was responsible for prolonged viremia, although infected erythrocytes eventually were cleared from the circulation and persistent BTV infection of calves did not occur.     

4种重要虫媒病的核酸液相芯片高通量检测方法的建立

为建立可检测鹿流行性出血热病毒(EHDV)、阿卡斑病毒(AKV)、蓝舌病病毒(BTV)和水泡性口炎病毒(VSV)的液相芯片快速检测技术,用DNAStar软件对GenBank中BTV的VP7基因、EHDV的VP7基因、AKV的N基因和VSV的NP基因序列进行序列分析,设计针对这些基因的特异性探针并标记生物素,分别与不同编号的荧光编码微球偶联后再与这些病毒相应基因的PCR产物杂交反应,用液相芯片检测仪(Liquichip 200)检测荧光信号建立了以上4种虫媒病的快速液相芯片检测方法。检测结果显示,该方法具有较好的特异性,偶联特异性探针的微球只与相应的病毒基因的PCR产物反应,而不与其他虫媒病病毒反应;检测灵敏度达到50~100个TCID50。本研究建立了可以同时检测鹿流行性出血热病毒、阿卡斑病毒、蓝舌病病毒和水泡性口炎病毒的快速高通量液相芯片技术,为其他类似病毒的快速高通量检测提供了借鉴和经验。     

Prevalence of bluetongue virus antibodies and associated risk factors among cattle in East Darfur State,Western Sudan

Background

Bluetongue virus (BTV) is an insect-transmitted virus, which causes bluetongue disease (BT) in sheep and a fatal hemorrhagic infection in North American white-tailed deer. However, in cattle the disease is typically asymptomatic and no overt clinical signs of disease appear to be associated with BTV infection. Serological evidence and isolation of different BTV serotypes have been reported in Sudan, however, no information is currently available in regard to previous exposure of Sudanese livestock to BTV infection in East Darfur State, Sudan.

Aims

To determine the prevalence of BTV antibodies and to identify the potential risk factors associated with BTV infection among cattle in East Darfur State, Sudan.

Methods

A total of 224 blood samples were collected randomly from five localities in East Darfur State, Sudan. The serum samples were screened for detection of BTV-specific immunoglobulin G (IgG) antibodies using a competitive enzyme-linked immunosorbent assay (c-ELISA).

Results

Serological evidence of BTV infection was observed in 150 out of 224 animals accounting for a 67% prevalence rate among cattle in East Darfur State. Older cattle (>2 years of age) were six times more likely to be infected with BTV (OR = 6.62, CI = 2.87-15.26, p-value = 0.01). Regarding animal source (contact with other herds) as a risk factor, it was shown that cattle purchased from market or introduced from other herds were 3 times at higher risk of being infected with BTV (OR = 3.87, CI = 1.07-13.87, p value = 0.03). Exposure of cattle to the insect vector increased the risk of contracting BTV infection by six times compared to non-exposed cattle (OR = 6.44, CI = 1.53-27.08, p value = 0.01).

Conclusion

The present study indicated that age, animal source and the intensity of the insect vector are influential risk factors for BTV infection in cattle in the Darfur region. Surveillance for BTV infection should be extended to include other susceptible ruminants and to study the distribution of the insect vectors to better predict and respond to a possible BTV outbreak in the State of East Darfur, Sudan.     

Detection of bluetongue virus RNA by in situ hybridization: comparison with virus isolation and antigen detection.

An in situ nucleic acid hybridization (ISH) technique was developed to detect bluetongue virus (BTV) RNA in cell culture. The sensitivity of the ISH technique was compared with virus isolation (VI) and antigen detection, using an indirect fluorescent-antibody (IFA) or an enzyme immunocytoassay (EICA) technique, for detection of 5 BTV serotypes indigenous to the United States. The VI was the most sensitive technique, detecting BTV early after infection of the cells. The IFA and EICA were of similar sensitivity; BTV antigen could be detected shortly after demonstration of virus by isolation. The sensitivity of ISH for detection of BTV-17 was equivalent to that of antigen detection. The ISH was not as sensitive as VI or antigen detection when assaying for the other BTV serotypes.     

Involvement of the skin during bluetongue virus infection and replication in the ruminant host

ABSTRACT: Bluetongue virus (BTV) is a double stranded (ds) RNA virus (genus Orbivirus; family Reoviridae), which is considered capable of infecting all species of domestic and wild ruminants, although clinical signs are seen mostly in sheep. BTV is arthropod-borne ("arbovirus") and able to productively infect and replicate in many different cell types of both insects and mammalian hosts. Although the organ and cellular tropism of BTV in ruminants has been the subject of several studies, many aspects of its pathogenesis are still poorly understood, partly because of inherent problems in distinguishing between "virus replication" and "virus presence". BTV replication and organ tropism were studied in a wide range of infected sheep tissues, by immuno-fluorescence-labeling of non-structural or structural proteins (NS2 or VP7 and core proteins, respectively) using confocal microscopy to distinguish between virus presence and replication. These results are compared to gross and microscopic pathological findings in selected organs from infected sheep. Replication was demonstrated in two major cell types: vascular endothelial cells, and agranular leukocytes which morphologically resemble lymphocytes, monocytes/ macrophages and/or dendritic cells. Two organs (the skin and tonsils) were shown to support relatively high levels of BTV replication, although they have not previously been proposed as important replication sites during BTV infection. The high level of BTV replication in the skin is thought to be of major significance for the pathogenesis and transmission of BTV (via biting insects) and a refinement of our current model of BTV pathogenesis is discussed.     

Ovine and murine T cell epitopes from the non-structural protein 1 (NS1) of bluetongue virus serotype 8 (BTV-8) are shared among viral serotypes

Bluetongue virus (BTV) is a non-enveloped dsRNA virus that causes a haemorrhagic disease mainly in sheep. It is an economically important Orbivirus of the Reoviridae family. In order to estimate the importance of T cell responses during BTV infection, it is essential to identify the epitopes targeted by the immune system. In the present work, we selected potential T cell epitopes (3 MHC-class II-binding and 8 MHC-class I binding peptides) for the C57BL/6 mouse strain from the BTV-8 non-structural protein NS1, using H2^b-binding predictive algorithms. Peptide binding assays confirmed all MHC-class I predicted peptides bound MHC-class I molecules. The immunogenicity of these 11 predicted peptides was then determined using splenocytes from BTV-8-inoculated C57BL/6 mice. Four MHC-class I binding peptides elicited specific IFN-γ production and generated cytotoxic T lymphocytes (CTL) in BTV-8 infected mice. CTL specific for 2 of these peptides were also able to recognise target cells infected with different BTV serotypes. Similarly, using a combination of IFN-γ ELISPOT, intracellular cytokine staining and proliferation assays, two MHC-class II peptides were identified as CD4⁺ T cell epitopes in BTV-8 infected mice. Importantly, two peptides were also consistently immunogenic in sheep infected with BTV-8 using IFN-γ ELISPOT assays. Both of these peptides stimulated CD4⁺ T cells that cross-reacted with other BTV serotypes. The characterisation of these T cell epitopes can help develop vaccines protecting against a broad spectrum of BTV serotypes and differentiate infected from vaccinated animals.     

Temporal development of bluetongue virus protein-specific antibody in sheep following natural infection

Humoral immune responses of sheep to natural bluetongue virus (BTV) infection were studied on a temporal basis. The temporal development of viral protein-specific IgG was determined by western immunoblotting; virus neutralization and agar gel immunodiffusion (AGID) were conducted for comparative purposes. Prior to the emergence of the arthropod vector and the associated transmission of BTV, virus-neutralizing antibody was absent from all sentinel sheep; 3 sheep had pre-existing AGID antibody and all sheep had IgG, specific for 4 viral proteins, as determined by immunoblotting. Following emergence of the BTV vector, 9 of 11 sheep became infected, as determined by virus isolation, with BTV. All sheep developed virus-neutralizing and AGID antibody. However, only those sheep with a demonstrable viremia experienced an increase in viral protein-specific antibody. Development of viral protein-specific IgG varied with the individual animal and no obvious correlation between a specific response and protective immunity or viral clearance was noted. From a diagnostic viewpoint, the immunoblotting procedure was superior in identifying past exposure to BTV, as compared with neutralization and AGID. In addition, the application of immunoblotting to paired serum samples appeared to be a sensitive indicator of viremia.     

Comparative studies on the growth of Australian bluetongue virus serotypes in continuous cell lines and embryonated chicken eggs

The replication of cell culture passaged Australian bluetongue virus (BTV) isolates, serotypes 20 (CSIRO19) and 1 (CSIRO156), and an untyped BTV (CSIRO154) was assessed in eight continuous cell lines (one derived from baby hamster kidney cells, BHK-21; three derived from monkey kidney cells, Vero, LLC-MK2 and CV-1P; a foetal ovine lung and a mouse fibroblast cell line, CSL503 and L929, respectively, a Super-Vero-Porcine stable cell line, SVP; and a mosquito cell line, Aedes albopictus cells) and in 11-day-old embryonated chicken eggs (ECE) at different multiplicities of infection. All three viruses replicated in the cell lines tested, maximum extracellular virus yields being attained from BHK-21 cells at high multiplicities of infection (approximately 10 PFU per cell). Also BHK-21 cells produced much higher yields of virus than the other cell lines tested when low multiplicities of infection were used (approximately 10(-4) PFU per cell). All BTV serotypes multiplied in Singh's Aedes albopictus cells with no cytopathogenic effects over the 4 day period tested. The viruses also replicated in 11-day-old ECE; however, the sensitivity of ECE for growth of the Australian serotypes was not as high as has been reported for BTV isolates in other countries. In all cell culture systems and in ECE, BTV1 and BTV20 replicated more efficiently than did CSIRO154 virus.     

Evidence of mixed infection of peste des petits ruminants virus and bluetongue virus in a flock of goats as confirmed by detection of antigen, antibody and nucleic acid of both the viruses

A mixed infection with peste des petits ruminants virus (PPRV) and bluetongue virus (BTV) occurred in goats which exhibited symptoms characteristic of PPR. A number of samples were collected from ailing or dead goats for labrotory diagnosis. Antibody to BTV and PPRV was detected in sera samples by competitive ELISA. No PPRV antigen was detected in tissue samples like lung and spleen, however, presence of PPRV antigen in some sera samples was confirmed by sandwich ELISA. All the blood samples collected from the ailing animals were found positive for BTV antigen by a sandwich ELISA. BTV- and PPRV nucleic acids were amplified from the pooled blood and tissue samples respectively by RT-PCR assays. The identity of the amplicons was confirmed by cloning and sequencing. All these tests confirm that the goats were infected with PPRV and BTV simultaneously. Isolation of viruses from the clinical samples is underway.     

Evaluation of the efficacy of commercial vaccines against bluetongue virus serotypes 1 and 8 in experimentally infected red deer (Cervus elaphus)

Red deer (Cervus elaphus) is a widespread and abundant species susceptible to bluetongue virus (BTV) infection. Inclusion of red deer vaccination among BTV control measures should be considered. Four out of twelve BTV antibody negative deer were vaccinated against serotype 1 (BTV-1), and four against serotype 8 (BTV-8). The remaining four deer acted as unvaccinated controls. Forty-two days after vaccination (dpv), all deer were inoculated with a low cell passage of the corresponding BTV strains. Serological and virological responses were analyzed from vaccination until 28 days after inoculation (dpi). The vaccinated deer reached statistically significant (P<0.05) higher specific antibody levels than the non vaccinated deer from 34 (BTV-8) and 42 (BTV-1) dpv, maintaining stable neutralizing antibodies until 28 dpi. The non vaccinated deer remained seronegative until challenge, showing neutralizing antibodies from 7 dpi. BTV RNA was detected in the blood of the non vaccinated deer from 2 to 28 dpi, whereas no BTV RNA was found in the vaccinated deer. BTV was isolated from the blood of non vaccinated deer from 7 to 28 dpi (BTV-1) and from 9 to 11 dpi (BTV-8). BTV RNA could be identified by RT-PCR at 28 dpi in spleen and lymph nodes, but BTV could not be isolated from these samples. BT-compatible clinical signs were inapparent and no gross lesions were found at necropsy. The results obtained in the present study confirm that monovalent BTV-1 and BTV-8 vaccines are safe and effective to prevent BTV infection in red deer. This finding indicates that vaccination programs on farmed or translocated red deer could be a useful tool to control BTV.     

感染16型蓝舌病病毒后绵羊部分细胞因子消长特点研究

本试验旨在探明绵羊感染16型蓝舌病病毒(Bluetongue virus type 16,BTV16)后细胞因子IFN-γ、IL-2、IL-4和IL-10的消长特点。用实时荧光定量PCR检测方法对感染BTV16后3只绵羊上述4种因子mRNA进行检测,同时设立阴性对照绵羊,并以0 d mRNA为基准,计算mRNA的相对表达量,同时检测病毒抗体效价、测量绵羊体温。结果显示,接种BTV16的3只绵羊均不同程度产生抗体和体温症状,4种细胞因子的mRNA在接种病毒2~4 d内均出现显著上升,其中IFN-γ峰值在2.58~27.84倍之间,IL-2峰值在5.24~17.19倍之间,IL-4峰值在2.16~3.43倍之间,IL-10峰值在15.78~48.77倍之间,个体上升幅度存在显著差异,4种细胞因子均在高水平持续6 d左右后逐渐下降。对照绵羊上述参数在正常范围内波动。本研究阐明了接种BTV16后绵羊细胞因子IFN-γ、IL-2、IL-4、IL-10在转录水平上的消长特点,为进一步深入开展BTV感染特征、宿主机体免疫机制研究提供参考。     

The impact of naturally-occurring,trans-placental bluetongue virus serotype-8 infection on reproductive performance in sheep

Infection with bluetongue virus serotype (BTV)-8 occurred in ruminants in 2006 in Central-Western Europe. The trans-placental passage of this virus has been demonstrated in naturally- and experimentally-infected cattle and in experimentally-infected sheep. Trans-placental transmission is potentially important in the ‘over-wintering’ of this virus and its subsequent impact on reproductive performance. This epidemiological study was carried out on a sheep flock in Belgium that had experienced a severe outbreak of BTV-8 infection, and where the seroprevalence had increased from 1.3% to 88% between January and November 2007. In total, 476 lambs and 26 aborted fetuses from 300 ewes, lambing at four distinct time periods, were investigated between November 2007 and May 2008.The following evidence suggested that BTV-8 infection occurred in utero: (1) positive PCR results from splenic tissue from aborted fetuses (n = 4); (2) fetal malformations suggestive of BTV infection (n = 10); (3) positive PCR results from red blood cells in-lambs (n = 7), and (4) the presence of antibody at birth in viable lambs prior to the intake of colostrum (n = 9). The evidence provided by this investigation strongly suggests that trans-placental BTV-8 infection occurs in naturally-infected sheep and the impact of infection on the reproductive performance of such a naïve flock was considerable, with up to 25% of ewes aborting and with flock fertility reduced by 50%. The contribution of in utero-infected lambs to the over-wintering of BTV appears limited.     

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.     

Bluetongue virus serotype 26: Infection kinetics,pathogenesis and possible contact transmission in goats

The aim of this study was to assess the pathogenicity and infection kinetics of Bluetongue virus serotype 26 (BTV-26) in goats. Out of a group of six goats housed in insect free accommodation, five were experimentally infected with BTV-26 and one was kept uninfected as an in-contact control. Samples taken throughout the study were used to determine the kinetics of infection using a pan specific BTV real time RT-PCR assay and a group specific ELISA. The five infected goats did not show clinical signs of BTV, however high levels of viral RNA were detected and virus was isolated from the blood of all 5 goats. Antibodies against BTV were first detected between 7 and 11 dpi in all 5 experimentally infected goats. Interestingly at 21 dpi viral RNA was detected in, and virus was isolated from, the blood of the in-contact control goat, which also seroconverted. These results suggest that BTV-26 replicates to high levels in goats, causing no obvious clinical disease, suggesting that goats may be the natural host for this virus. Preliminary evidence also indicates that BTV-26 may be spread by contact transmission between goats, however a more detailed study is required in order to confirm this observation.     

蓝舌病病原分子生物学及免疫学研究进展

蓝舌病病毒通过吸血昆虫(库蠓)在易感反刍动物之间叮咬进行传播。在家畜中,蓝舌病易发于某些品种的羊,具有典型症状,呈地方性流行;牛感染蓝舌病通常不表现出临床症状。作者分析和总结了近年蓝舌病疫情发生和传播可能的潜在路线,病毒分子生物学研究概况,致病机理及宿主对蓝舌病病毒的免疫反应,并对蓝舌病疫苗的研究进展作了介绍,建议要加强对该病的深入研究,防患于未然。