Evidence for Transmission of Bluetongue Virus Serotype 26 through Direct Contact

The aim of this study was to assess the mechanisms of transmission of bluetongue virus serotype 26 (BTV-26) in goats. A previous study, which investigated the pathogenicity and infection kinetics of BTV-26 in goats, unexpectedly revealed that one control goat may have been infected through a direct contact transmission route. To investigate the transmission mechanisms of BTV-26 in more detail an experimental infection study was carried out in which three goats were infected with BTV-26, three goats were kept uninfected, but were housed in direct contact with the infected goats, and an additional four goats were kept in indirect contact separated from infected goats by metal gates. This barrier allowed the goats to have occasional face-to-face contact in the same airspace, but feeding, watering, sampling and environmental cleaning was carried out separately. The three experimentally infected goats did not show clinical signs of BTV, however high levels of viral RNA were detected and virus was isolated from their blood. At 21 dpi viral RNA was detected in, and virus was isolated from the blood of the three direct contact goats, which also seroconverted. The four indirect barrier contact goats remained uninfected throughout the duration of the experiment. In order to assess replication in a laboratory model species of Culicoides biting midge, more than 300 Culicoides sonorensis were fed a BTV-26 spiked blood meal and incubated for 7 days. The dissemination of BTV-26 in individual C. sonorensis was inferred from the quantity of virus RNA and indicated that none of the insects processed at day 7 possessed transmissible infections. This study shows that BTV-26 is easily transmitted through direct contact transmission between goats, and the strain does not seem to replicate in C. sonorensis midges using standard incubation conditions

Measurement of the Infection and Dissemination of Bluetongue Virus in Culicoides Biting Midges Using a Semi-Quantitative RT-PCR Assay and Isolation of Infectious Virus

Culicoides biting midges (Diptera: Ceratopogonidae) are the biological vectors of globally significant arboviruses of livestock including bluetongue virus (BTV), African horse sickness virus (AHSV) and the recently emerging Schmallenberg virus (SBV). From 2006–2009 outbreaks of BTV in northern Europe inflicted major disruption and economic losses to farmers and several attempts were made to implicate Palaearctic Culicoides species as vectors. Results from these studies were difficult to interpret as they used semi-quantitative RT-PCR (sqPCR) assays as the major diagnostic tool, a technique that had not been validated for use in this role. In this study we validate the use of these assays by carrying out time-series detection of BTV RNA in two colony species of Culicoides and compare the results with the more traditional isolation of infectious BTV on cell culture

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

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

Development and evaluation of real time RT-PCR assays for detection and typing of Bluetongue virus

Bluetongue virus is the type species of the genus Orbivirus, family Reoviridae. Bluetongue viruses (BTV) are transmitted between their vertebrate hosts primarily by biting midges (Culicoides spp.) in which they also replicate. Consequently BTV distribution is dependent on the activity, geographic distribution, and seasonal abundance of Culicoides spp. The virus can also be transmitted vertically in vertebrate hosts, and some strains/serotypes can be transmitted horizontally in the absence of insect vectors. The BTV genome is composed of ten linear segments of double-stranded (ds) RNA, numbered in order of decreasing size (Seg-1 to Seg-10). Genome segment 2 (Seg-2) encodes outer-capsid protein VP2, the most variable BTV protein and the primary target for neutralising antibodies. Consequently VP2 (and Seg-2) determine the identity of the twenty seven serotypes and two additional putative BTV serotypes that have been recognised so far. Current BTV vaccines are serotype specific and typing of outbreak strains is required in order to deploy appropriate vaccines. We report development and evaluation of multiple ‘TaqMan’ fluorescence-probe based quantitative real-time type-specific RT-PCR assays targeting Seg-2 of the 27+1 BTV types. The assays were evaluated using orbivirus isolates from the ‘Orbivirus Reference Collection’ (ORC) held at The Pirbright Institute. The assays are BTV-type specific and can be used for rapid, sensitive and reliable detection / identification (typing) of BTV RNA from samples of infected blood, tissues, homogenised Culicoides, or tissue culture supernatants. None of the assays amplified cDNAs from closely related but heterologous orbiviruses, or from uninfected host animals or cell cultures

Interaction between Orbiviruses and their <em>Culicoides</em> Vectors

Resumen en inglès, ver archivo PD

Evidence for Transmission of Bluetongue Virus Serotype 26 through Direct Contact

The aim of this study was to assess the mechanisms of transmission of bluetongue virus serotype 26 (BTV-26) in goats. A previous study, which investigated the pathogenicity and infection kinetics of BTV-26 in goats, unexpectedly revealed that one control goat may have been infected through a direct contact transmission route. To investigate the transmission mechanisms of BTV-26 in more detail an experimental infection study was carried out in which three goats were infected with BTV-26, three goats were kept uninfected, but were housed in direct contact with the infected goats, and an additional four goats were kept in indirect contact separated from infected goats by metal gates. This barrier allowed the goats to have occasional face-to-face contact in the same airspace, but feeding, watering, sampling and environmental cleaning was carried out separately. The three experimentally infected goats did not show clinical signs of BTV, however high levels of viral RNA were detected and virus was isolated from their blood. At 21 dpi viral RNA was detected in, and virus was isolated from the blood of the three direct contact goats, which also seroconverted. The four indirect barrier contact goats remained uninfected throughout the duration of the experiment. In order to assess replication in a laboratory model species of Culicoides biting midge, more than 300 Culicoides sonorensis were fed a BTV-26 spiked blood meal and incubated for 7 days. The dissemination of BTV-26 in individual C. sonorensis was inferred from the quantity of virus RNA and indicated that none of the insects processed at day 7 possessed transmissible infections. This study shows that BTV-26 is easily transmitted through direct contact transmission between goats, and the strain does not seem to replicate in C. sonorensis midges using standard incubation conditions

Clinical disease in sheep caused by bluetongue virus serotype 8, and prevention by an inactivated vaccine.

The ability to reduce clinical signs, induce neutralizing antibodies, and perhaps most importantly, to prevent or reduce viraemia (and therefore virus-transmission), represent primary criteria for assessment of bluetongue virus (BTV) vaccine efficacy. Identification of BTV challenge-strains that reliably induce viraemia and clinical signs comparable to those in naturally infected animals, is therefore important for vaccine evaluation. Texel cross-breed and Dorset Poll sheep vaccinated with inactivated BTV-8 vaccine ('Bovilis(®) BTV8' from MSD Animal Health), were challenged with low-passage BTV-8 (Northern European strain) grown in either insect (Culicoides) or mammalian cell-cultures. The severity of clinical signs was recorded (using a modified numerical scoring-system, which is described) along with viraemia and serum neutralizing (SN) antibody levels. Low level SN-antibodies were detected at the time of challenge (three weeks after vaccination). All unvaccinated control animals became infected after challenge, developing high SN-antibody titres by 21 days post challenge (dpc). Vaccinees showed faster increases in SN-antibody titres ('booster' response), with significantly higher titres at 6 dpc than unvaccinated controls. Although only limited clinical-signs could be attributed to BTV in younger animals infected with the mammalian-cell-culture derived virus, both BTV-8 challenge preparations induced severe clinical signs comparable to 'bluetongue' observed during natural outbreaks in older unvaccinated animals. Challenge with BTV-8 grown in Culicoides cell-cultures seemed to induce greater severity of clinical-scores and 'post-mortem lesions' than the mammalian-derived BTV-8 strain. Vaccination reduced clinical signs, fever, and viraemia equally well after challenge with either virus preparation

Clinical disease in sheep caused by bluetongue virus serotype 8, and prevention by an inactivated vaccine.

The ability to reduce clinical signs, induce neutralizing antibodies, and perhaps most importantly, to prevent or reduce viraemia (and therefore virus-transmission), represent primary criteria for assessment of bluetongue virus (BTV) vaccine efficacy. Identification of BTV challenge-strains that reliably induce viraemia and clinical signs comparable to those in naturally infected animals, is therefore important for vaccine evaluation. Texel cross-breed and Dorset Poll sheep vaccinated with inactivated BTV-8 vaccine ('Bovilis(®) BTV8' from MSD Animal Health), were challenged with low-passage BTV-8 (Northern European strain) grown in either insect (Culicoides) or mammalian cell-cultures. The severity of clinical signs was recorded (using a modified numerical scoring-system, which is described) along with viraemia and serum neutralizing (SN) antibody levels. Low level SN-antibodies were detected at the time of challenge (three weeks after vaccination). All unvaccinated control animals became infected after challenge, developing high SN-antibody titres by 21 days post challenge (dpc). Vaccinees showed faster increases in SN-antibody titres ('booster' response), with significantly higher titres at 6 dpc than unvaccinated controls. Although only limited clinical-signs could be attributed to BTV in younger animals infected with the mammalian-cell-culture derived virus, both BTV-8 challenge preparations induced severe clinical signs comparable to 'bluetongue' observed during natural outbreaks in older unvaccinated animals. Challenge with BTV-8 grown in Culicoides cell-cultures seemed to induce greater severity of clinical-scores and 'post-mortem lesions' than the mammalian-derived BTV-8 strain. Vaccination reduced clinical signs, fever, and viraemia equally well after challenge with either virus preparation