Epizootic Haemorrhagic Disease Of Deer Virus Research Articles

Bluetongue in white-tailed deer (Odocoileus virginianus) in Northeastern Mexico

Abstract Bluetongue (BT) and epizootic hemorrhagic disease of deer (EHD) are two distinct viral hemorrhagic diseases of domestic and wild ruminants caused by members of the family Reoviridae and transmitted by Culicoides midges. These conditions have been recognized in Canada and the United States for many years, but not in Mexico. Although in Mexico there is serologic evidence of EHD virus (EHDV) and bluetongue virus (BTV) in domestic and wild ruminants, to our knowledge, there have never been reports of clinical illness or fatalities attributed to either of these viruses. Two free ranging white-tailed deer (Odocoileus virginianus) in two licensed hunting ranches in Northern Mexico near the Texas border died unexpectedly. Postmortem and microscopic examinations revealed hemorrhagic lesions compatible with viral hemorrhagic disease (Reoviridae: Orbivirus). Tissues from one animal tested positive by RT-PCR for BTV but negative for EHDV. To our knowledge, this is the first time in Mexico where deer dying with hemorrhagic lesions consistent with Bluetongue tested positive for BTV by PCR. Key words: hemorrhagic disease, white-tailed deer, Orbivirus, Mexico.

PCR detection of African horse sickness virus serogroup based on genome segment three sequence analysis

A nested reverse transcriptase (RT) polymerase chain reaction (RT-PCR), for rapid detection of African horse sickness virus (AHSV) double-stranded ribonucleic acid (dsRNA) in cell culture and tissue samples, was developed and evaluated. Using an outer pair of primers (P1 and P2), selected from genome segment three of AHSV serotype 6 (AHSV-6), the RT-PCR-based assay resulted in amplification of a 890 base pair (bp) primary PCR product. RNAs from the nine vaccine strains of AHSV, and a number of AHSV field isolates including the Central African isolates of AHSV-9 and AHSV-6, propagated in cell cultures, were detected by this assay. A second pair of nested primers (P3 and P4) was used to produce a 240-bp PCR product. The RT-PCR described below detected as little as 0.1 fg of AHSV RNA, which is equivalent to six viral particles. The nested amplification confirmed the integrity of the primary PCR product and increased the sensitivity of the PCR assay by at least 1000-fold. Application of this RT-PCR assay to clinical samples resulted in direct detection of AHSV dsRNA from blood and a variety of tissue samples collected from equines infected experimentally and naturally. The specificity studies indicated that the primary or the nested PCR products were not amplified from, closely related orbiviruses including, bluetongue virus (BTV) prototypes serotypes 1, 2, 4, 10, 16 and 17; epizootic hemorrhagic disease of deer virus (EHDV) prototypes serotypes 1 and 2; EHDV-318, Sudanese isolates of palyam serogroup of orbiviruses; total nucleic acid extracts from uninfected Vero cells; or unfractionated blood from horses and donkeys that were AHSV-seronegative and virus isolation negative. The RT-PCR provides a valuable tool for study of the epidemiology of AHSV and can be recommended for rapid diagnosis during an outbreak of the disease among susceptible equines.

Diagnostic des orbivirus dans un pays d'endémicité

Diagnostic des orbivirus dans un pays d'endémicité

Bluetongue virus in the French Island of Reunion

This paper records the results of a bluetongue virus (BTV) serological survey and reports the first isolation of BTV on the French Island of Reunion. In January 2003, the French Island of Reunion, located off the coast of Madagascar, reported an outbreak of disease in cattle that resembled clinical bluetongue (BT) in sheep. The suspected causal agent was isolated and identified as epizootic haemorrhagic disease of deer virus (EHDV). However, because of the similarity in the clinical signs to those of BT, a retrospective survey against BTV was carried out using sera collected in 2002. Results revealed the presence of antibody in all sera tested indicating that BTV has been resident on the Island since 2002, and probably earlier. Although up to July 2003 no clinical BT had ever been reported in sheep, BTV viral RNA was amplified by RT-PCR from a single sheep blood collected in February that year, which strongly suggested that BTV was currently circulating on the Island. Following a second outbreak of disease in August 2003, this time involving a flock of Merino sheep, infectious BTV was finally isolated, and identified by both traditional and molecular techniques as serotype 3. The nucleotide and amino-acid sequences of the RT-PCR products amplified for BTV segments 7 and 10 from the sheep blood collected in February and August from different areas of the Island, were sufficiently diverse as to suggest that they were of different origins and/or different BTV serotypes.

Effect of temperature on the transmission of orbiviruses by the biting midge, Culicoides sonorensis.

The influence of temperature on the likelihood of Culicoides sonorensis Wirth & Jones (Diptera: Ceratopogonidae) transmitting African horse sickness virus (AHSV) serotypes 4 and 6, bluetongue virus (BTV) serotypes 10 and 16 and epizootic haemorrhagic disease of deer virus (EHDV) serotype 1 was investigated. Extrinsic incubation periods (EIP), vector competence and vector survival were determined at 15, 20, 25 and 30 degrees C. The effect of humidity on vector survival was also investigated by maintaining adult C. sonorensis at 40, 75 and 85% r.h. at each temperature. Higher temperatures were associated with a shorter EIP for all virus serotypes except AHSV6, to which C. sonorensis was orally refractory, increased vector competence for AHSV4 and EHDV1, but not for BTV10 or BTV16, and a reduction in vector survival. Humidity interacted with temperature in influencing vector survival, such that at low temperatures, lower humidity (40 and 75% r.h.) was detrimental for survival (up to 18% reduction in longevity), whereas at high temperatures, high humidity (85% r.h.) was detrimental (up to 36% reduction in longevity). In general, the transmission potential of C. sonorensis for AHSV4, EHDV1, BTV10 and BTV16 was greater at higher temperatures, because although vector survival was reduced, this was more than compensated for by the accompanying decrease in duration of the EIP.

Development of an enzyme-linked immunosorbent assay for the detection of antibody to epizootic hemorrhagic disease of deer virus.

An enzyme-linked immunosorbent assay has been developed to detect antibodies to epizootic hemorrhagic disease of deer virus (EHDV). The assay incorporates a monoclonal antibody to EHDV serotype 2 (EHDV-2) that demonstrates specificity for the viral structural protein, VP7. The assay was evaluated with sequential sera collected from cattle experimentally infected with EHDV serotype 1 (EHDV-1) and EHDV-2, as well as the four serotypes of bluetongue virus (BTV), BTV-10, BTV-11, BTV-13, and BTV-17, that currently circulate in the US. A competitive and a blocking format as well as the use of antigen produced from both EHDV-1- and EHDV-2-infected cells were evaluated. The assay was able to detect specific antibody as early as 7 days after infection and could differentiate animals experimentally infected with EHDV from those experimentally infected with BTV. The diagnostic potential of this assay was demonstrated with field-collected serum samples from cattle, deer, and buffalo.

Antigen-capture enzyme-linked immunosorbent assay using monoclonal antibody for detection of bluetongue virus antigen An

antigen-capture enzyme-linked immunosorbent assay (ELISA) using a specific anti-bluetongue virus group was applied to detect bluetongue viral antigen . The test was specific for bluetongue viruses and did not detect the closely related epizootic haemorrhagic disease of deer viruses (EHDV) and other orbiviruses . It was easy to perform and could be established in laboratories which have simple facilities . The antigen-capture ELISA technique is an alternative method to agar gel immunodiffusion and immuno-dot blotting tests to detect bluetongue antigen in infected tissues, Vero cells, Aedes albopictus cells and BHK-21 cell cultures . Keywords : ELISA, monoclonal antibody, antigen detection, BT

Monoclonal antibody based competitive ELISA for the detection of antibodies against epizootic haemorrhagic disease of deer virus.

A monoclonal antibody based competitive enzyme-linked immunosorbent assay (MC-ELISA) for the detection of antibodies against epizootic haemorrhagic disease of deer viruses (EHDV) is described. Test sera were competed with a monoclonal antibody against the VP7 protein of EHDV serotype 1. The assay was capable of detecting antibodies to all serotypes of EHDV but unlike the agar gel immunodiffusion (AGID) test, gave no cross-reactions with antibodies against bluetongue, Palyam or Tilligery viruses. The MC-ELISA was more sensitive than a polyclonal based ELISA reported previously (Thevasagayam et al., 1995b) and would be ideal for epidemiological surveys since it is suitable for the examination of antisera from all animal species without the need for individual anti-species enzyme conjugates.

Detection and differentiation of epizootic haemorrhagic disease of deer and bluetongue viruses by serogroup-specific sandwich ELISA.

A serogroup specific sandwich ELISA was developed for the detection of epizootic haemorrhagic disease of deer viruses (EHDV) in infected insects and tissue culture preparations. Polyclonal rabbit antiserum against purified EHDV core particles was used to capture viral antigen and specific binding detected using guinea pig antisera against EHDV core particles followed by anti-guinea pig immunoglobulin enzyme-labelled conjugate. The assay is EHDV specific and detects all 8 serotypes. No cross-reactions were found with related viruses such as bluetongue (BTV), Palyam, Tilligery or African horse sickness virus (AHSV). A similar serogroup specific sandwich ELISA was also developed for BTV. The assays showed a similar sensitivity in detecting the respective EHDV or BTV antigens in a pool of 500 midges where only 2 were infected. These assays allow a simple and rapid means of detecting and differentiating members of these closely related serogroups. The sensitivity of the tests will allow more extensive studies on vector competence and virus/vector distribution.

Competitive ELISA for the detection of antibodies against epizootic haemorrhagic disease of deer virus.

A competitive enzyme-linked immunosorbent assay is described for the detection of antibodies against epizootic haemorrhagic disease of deer viruses (EHDV). Test antisera were tested against a guinea-pig antiserum raised against EHDV core particles. The assay detected antibodies to all serotypes of EHDV, but unlike the agar gel immunodiffusion (AGID) test, gave no cross-reactions with antibodies against bluetongue, Palyam and Tilligery viruses. The C-ELISA would be ideal for use in epidemiological surveys since it is suitable for the examination of antisera from all susceptible species without the need for individual species-specific enzyme conjugates.

Identification of epizootic haemorrhagic disease of deer virus serotypes using a fluorescence inhibition test

A fluorescence inhibition test (FIT) is described for serotyping rapidly isolates of epizootic haemorrhagic disease of deer virus (EHDV). The test used a serogroup-reactive monoclonal antibody in a immunofluorescence procedure to detect virus which resisted neutralisation by antisera to any of the eight known EHDV serotypes. The EHDV FIT provided an accurate serotype identification procedure for all eight reference serotypes and, in comparison with the plaque inhibition assay, abbreviated the serotyping process by three to four days.

A competitive ELISA for the detection of anti-tubule antibodies using a monoclonal antibody against bluetongue virus non-structural protein NSI

A monoclonal antibody directed against the largest bluetongue virus (BTV) non-structural protein (NS1) was used in a competitive ELISA to detect antibodies to tubules (composed of NS1) in serum samples. Anti-tubule antibodies were detected at approximately 10 days post-infection in BTV infected sheep but no such antibodies were detected in sheep injected with inactivated BTV vaccines. Tubules are also produced during the replication of other related orbiviruses, including epizootic haemorrhagic disease of deer virus (EHDV). However, antibodies to the EHDV tubules were not detected in antisera from EHDV infected animals using the BTV NS1 specific monoclonal antibody and this assay system, confirming the serogroup-specific nature of this assay. This assay may prove useful not only for confirming the inactivated nature of experimental vaccines but also for the detection of viral replication and hence measurement of protection in vaccine potency trials following virulent BTV challenge. If inactivated BTV vaccines are ever widely adopted this test could provide a valuable means of discriminating between vaccinated and infected animals.

Molecular comparison of VP3 from bluetongue and epizootic hemorrhagic disease viruses.

The complete nucleic acid sequence of gene 3 from epizootic hemorrhagic disease of deer (EHD) virus serotype 1 was determined. The 2768 bp sequence encodes a single protein that contains 899 amino acids and has a molecular weight of 103 kDa. The predicted protein sequence has 94.7% identity with EHD virus serotype 2 and greater than 77% identity with the related bluetongue viruses serotypes 1, 10 and 17 VP3 proteins. The relevance of these data to studies of recombinant DNA diagnostics and genetic relatedness is discussed.

Antibodies against certain bluetongue and epizootic haemorrhagic disease viral serotypes in Indonesian ruminants

The orbiviruses contain several important viruses of livestock including bluetongue (BT) and epizootic haemorrhagic disease of deer (EHD) which share some group antigens. Preliminary screening of sera for antibodies to orbiviruses by the agar gel immunodiffusion (AGID) test has previously revealed widespread infections with the BT group in Indonesia. However serum neutralization (SN) tests give a more accurate estimate of exposure to each serotype in the BT and EHD groups, and in this study were applied to sera that had reacted previously in the AGID test. Five different serotypes of BT and one serotype of EHD virus were studied. Reactors to BT serotype 20 were the most prevalent, followed by EHD type 5 and BT types 21, 12, 1 and 17. Antibodies against BT serotype 20 were present in cattle, buffaloes, goats and sheep, but were most common in buffaloes. Buffaloes showed the highest exposure to the BT serotypes tested. Antibodies to EHD type 5 occurred most frequently in cattle. Antibodies against all BT and EHD serotypes tested were found in buffaloes and cattle while goats had antibodies against BT types 20, 21 and EHD type 5 and sheep had antibodies only against BT type 20.

Epidemiology of bluetongue and related orbiviruses in the Sultanate of Oman

Sentinel herds at 34 farms were used to study the epidemiology of bluetongue and related orbiviruses in Oman. The results indicate that bluetongue virus (BTV) is widespread and is enzootic in Northern Oman. At least three BTV serotypes (3, 4 and 22) were present at the time of the study. Antibodies to epizootic haemorrhagic disease of deer virus (EHDV) type 2 and EHDV-318 were also detected but were less prevalent. Entomological investigations identified the presence of 16 species of Culicoides. The peak seasonal incidence of the BTV vector C. imicola and the EHDV vectors C. schultzei (group) midges at Rumais in Northern Oman correlated closely with the spring rains in that area. However, both species of midge were also present in lower numbers throughout the year. four species of Oman midge, C. arabiensis, C. ibriensis, C. neoschultzei and C. buettikeri are new to science.

Dot immunoperoxidase assay using monoclonal antibody for detection of bluetongue virus antigens

A rapid, simple dot immunoperoxidase assay (DIPA) is described for visual detection and identification of bluetongue virus (BTV) antigens in samples of infected cell culture fluid. The assay was performed using nitrocellulose (NC) paper and ‘dipsticks’. Dots of samples were adsorbed to the NC surface and the remaining non-specific binding sites were blocked with skim milk solution. BTV was detected with either of two murine monoclonal antibodies (4H4, 5G12) to the major group specific antigens of BTV, and the complex was reacted with a peroxidase conjugated anti-mouse immunoglobulin G (heavy- and light-chain specific). Positive reactions were easily visualized as brown spots after enzyme degradation of substrate containing H 2O 2 and diaminobenzidine (DAB). The DIPA was specific in detecting BTV in samples of cell culture fluid from baby hamster kidney (BHK-21) cells infected with U.S.A. isolates of the five BTV serotypes (2, 10, 11, 13 and 17) known to exist in the U.S.A., and South African isolates of 17 BTV serotypes (1–12, 14–16, 18 and 20), but not with two North American isolates of epizootic hemorrhagic disease of deer virus (EHDV) representing serotypes 1 and 2. Attempts to detect BTV directly in infected sheep blood cells and chick embryo tissue suspensions by DIPA were unsuccessful. Of 55 cell culture fluid samples examined from BHK-21 or Vero cell monolayers inoculated with 55 clinical specimens, propagated initially in embryonating chicken egg (ECE) 11 proved positive and 44 were negative by DIPA. The results were in complete agreement with the conventional ECE and tissue culture isolation systems. The DIPA appears to have potential application, especially as a ‘dipstick’ kit, for rapid and inexpensive laboratory diagnosis of bluetongue virus infection.

The complete nucleotide sequence of bluetongue virus serotype l RNA3 and a comparison with other geographic serotypes from Australia, South Africa and the United States of America, and with other orbivirus isolates

The sequence of the RNA segment 3 of bluetongue virus (BTV) serotype l from Australia is presented along with its deduced amino acid sequence. DNA copies of this genome segment were inserted either into the E. coli plasmid pBR322 by homopolymeric tailing or by direct insertion of double-stranded DNA fragments generated by restriction endonuclease cleavage into the appropriate M13 bacteriophage vectors (Vieira J. and Messing J., 1982, Gene 19, 259–268). Direct comparisons were made to the nucleotide sequence data of Purdy M. et al., 1984 (J. Virol. 51, 754–759) and Ghiasi H. et al., 1985 (Virus Res. 3, 181–190) for the United States of America (US) isolates of BTV, serotypes 10 and 17, respectively. A method for the rapid cloning, sequencing and alignment of orbivirus RNA 3 segments was utilised to compare other geographical isolates of BTV, as well as those of other orbivirus serotypes, in particular, epizootic haemorrhagic disease of deer virus (EHDV) and Warrego. The comparison of this sequence data reveals that BTV isolates can be separated into distinct geographical types which in turn are distinct from the other orbivirus isolates studied. The sequence conservation at the amino acid level for the gene product of RNA3 (VP3) does not enable distinctions to be made amongst the BTV isolates at a geographical level, but does afford easy distinction into the different orbivirus groups. A possible evolutionary schematic is presented for the orbiviruses studied.

Bluetongue and related viruses in Nigeria: Experimental infection of West African dwarf sheep with Nigeria strains of the viruses of epizootic haemorrhagic disease of deer and bluetongue

West African dwarf sheep were inoculated by the subcutaneous route with epizootic haemorrhagic disease of deer (EHD) virus or bluetongue (BT) virus. No clinical disease was observed following primary EHD or BT infection, or subsequent challenge with either homologous or heterologous virus. However, viraemia was detected in non-immune sheep exposed to BT virus, but not in BT- or EHD-immune sheep challenged with either virus, or in non-immune sheep exposed to primary EHD virus infection. Complement fixing antibodies developed against both EHD and BT viruses following the primary infection with either virus, or subsequent challenge with homologous or heterologous virus. Following a primary infection, virus-neutralising (VN) antibodies developed only against the inoculated virus, while the detection of VN antibodied to both viruses followed the challenge of an EHD- or BT-immuned sheep with either the homologous or heterologous virus. These findings further support previous reports of a relationship between EHD and BT viruses. between EHD and BT viruses.

Identification of viruses involved in the 1971 outbreak of hemorrhagic disease in southeastern United States white-tailed deer.

Tissues and serum from white-tailed deer (Odocoileus virginianus) in the southeastern United States were taken during an outbreak of hemorrhagic disease. These were subjected to virus isolation attempts in tissue culture and several serological tests (plaque neutralization, complement fixation, agar gel precipitation). Both epizootic hemorrhagic disease of deer virus (EHDV) and bluetongue virus (BTV) were isolated and evidence of their presence demonstrated serologically. All of the evidence supported previous suggestions that these two agents are antigenically distinguishable.

Multiplication of the virus of epizootic haemorrhagic disease of deer in Culicoides species (Diptera, Ceratopogonidae)

A study of the replication of epizootic haemorrhagic disease of deer (EHD) virus inCulicoides variipennis from North America and six species of Culicoides from Britain was made. The virus multiplied to a high titre inC. varriipennis following inoculation and oral ingestion; the growth cycle was similar to that of blue tongue virus. EHD virus also multiplied to a high titre following inoculation into three British species —C. riethi, C. nubeculosus (both closely related toC. variipennis), andC. impunctatus. In view of these results it is suggested thatC. variipennis may be capable of transmitting EHD virus, and that all members of thenubeculosus group ofCulicoides warrant consideration as possible vectors of viruses belonging to the bluetongue group.