Epizootic Hemorrhagic Disease Virus Infection Research Articles

Isolation of Epizootic Hemorrhagic Disease Virus Serotype 10 from Culicoides tainanus and Associated Infections in Livestock in Yunnan, China.

Two strains of viruses, JC13C644 and JC13C673, were isolated from Culicoides tainanus collected in Jiangcheng County, Yunnan Province, situated along the border area shared by China, Laos, and Vietnam. JC13C644 and JC13C673 viruses can cause cytopathic effect (CPE) in mammalian cells BHK21 and Vero cells, and cause morbidity and mortality in suckling mice 48 h after intracerebral inoculation. Whole-genome sequencing was performed, yielding complete sequences for all 10 segments from Seg-1 (3942nt) to Seg-10 (810nt). Phylogenetic analysis of the sub-core-shell (T2) showed that the JC13C644 and JC13C673 viruses clustered with the Epizootic Hemorrhagic Disease Virus (EHDV) isolated from Japan and Australia, with nucleotide and amino acid homology of 93.1% to 98.3% and 99.2% to 99.6%, respectively, suggesting that they were Eastern group EHDV. The phylogenetic analysis of outer capsid protein (OC1) and outer capsid protein (OC2) showed that the JC13C644 and JC13C673 viruses were clustered with the EHDV-10 isolated from Japan in 1998, with the nucleotide homology of 98.3% and 98.5%, and the amino acid homology of 99.6% and 99.6-99.8%, respectively, indicating that they belong to the EHDV-10. Seroepidemiological survey results demonstrated that JC13C644 virus-neutralizing antibodies were present in 29.02% (177/610) of locally collected cattle serum and 11.32% (89/786) of goat serum, implying the virus's presence in Jiangcheng, Yunnan Province. This finding suggests that EHDV-10 circulates not only among blood-sucking insects in nature but also infects local domestic animals in China. Notably, this marks the first-ever isolation of the virus in China and its discovery outside of Japan since its initial isolation from Japanese cattle. In light of these results, it is evident that EHDV Serotype 10 exists beyond Japan, notably in the natural vectors of southern Eurasia, with the capacity to infect local cattle and goats. Therefore, it is imperative to intensify the surveillance of EHDV infection in domestic animals, particularly focusing on the detection and monitoring of new virus serotypes that may emerge in the region and pose risks to animal health.

A serologic investigation of epizootic hemorrhagic disease virus in China between 2014 and 2019.

Epizootic hemorrhagic disease virus (EHDV) is a member of the genus Orbivirus, family Sedoreoviridae. It was firstly recognized in 1955 to cause a highly fatal disease of wild white-tailed deer in America. So far, EHDV was detected and isolated in many wild or domestic ruminants, and widely distributed all over the world. Although the domestic cattle and sheep infected by EHDV were usually asymptomatic or subclinical, several outbreaks of epizootic hemorrhagic disease (EHD) in deer and cattle had been reported. Many EHDV strains were isolated and sequenced in last two decades in China, which promoted a general serologic investigation of EHDV in China. In this study, 18,122 sera were collected from asymptomatic or subclinical domestic ruminants (cattle, cow, yaks, sheep, goats, and deer) in 116 regions belonging to 15 provinces in China. All the sera were tested by EHDV C-ELISA, and the results were obtained by big data analysis. EHDV infections were detected in the 14 of 15 provinces, and only Tibet (average altitude ≥ 4000 ​m) which was the highest province in China was free of EHDV. The numbers of seropositive collections in both bovine and goat/sheep were in an inverse proportion to the latitude. However, the seropositive rates in bovine were ranged from 0% to 100%, while the seropositive rates in goat/sheep were no more than 50%. The results suggested that bovine was obviously more susceptive for EHDV infection than goat and sheep, therefore might be a major reservoir of EHDV in China. The prevalence of EHDV was consistent with the distribution of Culicoides which were known as the sole insect vectors of EHDV. In particular, the seropositive rates of EHDV were very high in the southern provinces, which required the enhanced surveillance in the future.

EVIDENCE OF EPIZOOTIC HEMORRHAGIC DISEASE VIRUS AND BLUETONGUE VIRUS EXPOSURE IN NONNATIVE RUMINANT SPECIES IN NORTHERN FLORIDA.

Epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV) are vector-borne viruses of ruminants nearly worldwide. They can affect white-tailed deer (WTD; Odocoileus virginianus), the ranching industry, and nonindigenous hoof stock species managed for conservation. One potential risk factor for ranched WTD is commingling with nonindigenous species on high-fenced properties. Nonindigenous species provide novel viewing and hunting opportunities; however, their presence may create disease hazards. Furthermore, animals within conservation properties may be at a risk from commingling exotics and adjacent wild WTD. Currently, knowledge about EHDV and BTV seroprevalence and transmission is limited in nonindigenous populations in the southeastern United States. The authors conducted a serological survey of 10 Bovidae and 5 Cervidae species residing within two properties in northern Florida. The first site was a conservation property breeding threatened nonindigenous species for conservation. The second property was a private high-fenced game preserve managing WTD and nonindigenous species for breeding, sale, and harvest. Blood samples were tested for titers to three EHDV serotypes (1, 2, and 6) and active circulating viral EHDV and BTV. The private ranch had evidence of EHDV or BTV in one of three (33.3%) Bovidae species and four of five (80%) Cervidae species sampled. At the conservation property, evidence of EHDV infection was found in four of seven (57.1%) Bovidae and one of one (100%) Cervidae species sampled. The presence of antibodies in many nonindigenous species sampled might indicate these species are potential viral hosts and may be a risk to ranched WTD and other species within the same property. Nonindigenous species within the private ranch and conservation properties are at risk of contracting EHDV and BTV, and herd managers should reduce vector-host interactions and consider increased biosecurity measures when translocating animals.

Evaluation of A Baculovirus-Expressed VP2 Subunit Vaccine for the Protection of White-Tailed Deer (Odocoileus virginianus) from Epizootic Hemorrhagic Disease.

Epizootic hemorrhagic disease virus (EHDV) is an arthropod-transmitted RNA virus and the causative agent of epizootic hemorrhagic disease (EHD) in wild and domestic ruminants. In North America, white-tailed deer (WTD) experience the highest EHD-related morbidity and mortality, although clinical disease is reported in cattle during severe epizootics. No commercially licensed EHDV vaccine is available in North America. The objective of this study was to develop and evaluate a subunit vaccine candidate to control EHD in WTD. Recombinant VP2 (rVP2) outer capsid proteins of EHDV serotypes 2 (EHDV-2) and 6 (EHDV-6) were produced in a baculovirus-expression system. Mice and cattle vaccinated with EHDV-2 or EHDV-6 rVP2 produced homologous virus-neutralizing antibodies. In an immunogenicity/efficacy study, captive-bred WTD received 2 doses of EHDV-2 rVP2 or sham vaccine, then were challenged with wild-type EHDV-2 at 30 d post vaccination. None of the rVP2-vaccinated deer developed clinical disease, no viral RNA was detected in their blood or tissues (liver, lung, spleen, kidney), and no EHDV-induced lesions were observed. Sham-vaccinated deer developed clinical disease with viremia and typical EHD vascular lesions. Here, we demonstrate a rVP2 subunit vaccine that can provide protective immunity from EHDV infection and which may serve as an effective tool in preventing clinical EHD and reducing virus transmission.

POSTMORTEM DETECTION OF BLUETONGUE AND EPIZOOTIC HEMORRHAGIC DISEASE VIRUSES IN THE BONE MARROW OF WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS)

We determined the temporal aspects of detecting bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in postmortem bone marrow samples of white-tailed deer (Odocoileus virginianus) using molecular and in vitro cell culture techniques. Bone marrow samples from carcasses were collected and assayed on the day of death and at intervals up to 16 wk after death. We recovered BTV and EHDV from fresh bone marrow collected at day 0 by isolation in Vero and BHK-21 cell cultures. However, attempts to replicate the viruses from aged bone marrow in Vero and BHK-21 cell cultures failed. The real-time quantitative reverse transcriptase PCR (qRT-PCR) results confirmed that EHDV and BTV can be detected in aged bone marrow for up to 12 and 16 wk, respectively, after death. The RNA of BTV and EHDV could be detected by qRT-PCR in white-tailed deer bone marrow for extended periods of time postmortem. This technique will provide a useful tool for retrospective determination of BTV or EHDV infection of white-tailed deer at the time of death.

Vector Competence of Culicoides sonorensis (Diptera: Ceratopogonidae) for Epizootic Hemorrhagic Disease Virus Serotype 2 Strains from Canada and Florida.

Epizootic hemorrhagic disease virus (EHDV), an Orbivirus transmitted by Culicoides spp. vectors, is represented by seven serotypes and numerous strains worldwide. While studies comparing vector competence between serotypes exist, studies between viral strains are lacking. In this study, we examined the rates of infection, dissemination, and transmission of two strains of EHDV-2 orally fed to the known vector, Culicoides sonorensis Wirth & Jones. Culicoides sonorensis cohorts were fed an infectious blood meal containing EHDV-2 strains from either Alberta, Canada (Can-Alberta) or Florida (5.5 log10 PFUe/mL) and tested for the vector’s susceptibility to infection and dissemination. In addition, transmission rates of the virus were assessed and compared using capillary tube and honey card methods. Our results show that the Florida strain had higher infection and dissemination rates than the Can-Alberta strain in spite of the Florida strain having significantly lower viral titers in C. sonorensis bodies, legs, and saliva than the Can-Alberta strain. Overall transmission rates were not significantly different between the two strains but varied significantly between the methods used. These findings suggest that the consequences of EHDV infection in C. sonorensis vary between virus strains and have huge implications in future vector competence studies involving Culicoides species and Orbiviruses.

Dynamics of epizootic hemorrhagic disease virus infection within the vector, Culicoides sonorensis (Diptera: Ceratopogonidae).

Culicoides sonorensis biting midges are confirmed vectors of epizootic hemorrhagic disease virus (EHDV), which causes mortality in white-tailed deer and ruminant populations. Currently, of the seven EHDV serotypes, only 1, 2, and 6 are detected in the USA, and very few studies have focused on the infection time course of these serotypes within the midge. The objective of this current research was to characterize EHDV-2 infection within the midge by measuring infection prevalence, virus dissemination, and viral load over the course of infection. Midges were fed a blood meal containing 106.9 PFU/ml EHDV-2, collected every 12 h from 0–2 days post feeding (dpf) and daily from 3–10 dpf, and cohorts of 20 C. sonorensis were processed using techniques that assessed EHDV infection and dissemination. Cytopathic effect assays and quantitative (q)PCR were used to determine infection prevalence, revealing a 50% infection rate by 10 dpf using both methods. Using immunohistochemistry, EHDV-2 infection was detectable at 5 dpf, and shown to disseminate from the midgut to other tissues, including fat body, eyes, and salivary glands by 5 dpf. Stain intensity increased from 5–8 dpf, indicating replication of EHDV-2 in secondary infection sites after dissemination. This finding is also supported by trends in viral load over time as determined by plaque assays and qPCR. An increase in titer between 4–5 dpf correlated with viral replication in the midgut as seen with staining at day 5, while the subsequent gradual increase in viral load from 8–10 dpf suggested viral replication in midges with disseminated infection. Overall, the data presented herein suggest that EHDV-2 disseminates via the hemolymph to secondary infection sites throughout the midge and demonstrate a high potential for transmission at five days at 25°C after an infective blood-meal.

Tumor necrosis factor-alpha expression in white-tailed deer (Odocoileus virginianus) infected with Epizootic haemorrhagic disease virus.

Epizootic haemorrhagic disease (EHD) is the most important infectious disease of white‑tailed deer (WTD), however little is known about the role of inflammatory mediators in the pathogenesis. We characterized the expression of tumor necrosis factor‑alpha (TNF-α) ex vivo in tissues of WTD experimentally or naturally infected with EHD virus serotype 2 and in WTD peripheral blood mononuclear cells (PBMC) infected with EHD virus serotype 2 in vitro. Circulating levels of TNF-α were evaluated in serum from experimentally infected deer via cytotoxicity assay. The expression of TNF-α in tissues was evaluated via immunohistochemistry (IHC) in both experimentally and naturally infected deer. Semi‑quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to assess the level of TNF-α mRNA in tissues from experimentally infected deer and WTD's PBMC. Circulating levels of TNF-α were not increased in infected animals and TNF-α was not detected in tissues of infected deer. Increased transcription of TNF-α was detected neither in infected WTD nor in the PBMC. Tumor necrosis factor-alpha may not play a significant role in the pathogenesis of EHD virus infection in WTD.

Epizootic haemorrhagic disease

Summary Epizootic haemorrhagic disease (EHD) is an arthropod-transmitted viral disease of certain wild ungulates, notably North American white-tailed deer and, more rarely, cattle. The disease in white-tailed deer results from vascular injury analogous to that caused by bluetongue virus (BTV), to which EHD virus (EHDV) is closely related. There are seven serotypes of EHDV recognised, and Ibaraki virus, which is the cause of sporadic disease outbreaks in cattle in Asia, is included in EHDV serotype 2. The global distribution and epidemiology of BTV and EHDV infections are also similar, as both viruses occur throughout temperate and tropical regions of the world where they are transmitted by biting Culicoides midges and infect a wide variety of domestic and wild ungulates. However, the global distribution and epidemiology of EHDV infection are less well characterised than they are for BTV. Whereas most natural and experimental EHDV infections (other than Ibaraki virus infection) of livestock are subclinical or asymptomatic, outbreaks of EHD have recently been reported among cattle in the Mediterranean Basin, Reunion Island, South Africa, and the United States. Accurate and convenient laboratory tests are increasingly available for the sensitive and specific serological and virological diagnosis of EHDV infection and confirmation of EHD in animals, but commercial vaccines are available only for prevention of Ibaraki disease and not for protection against other strains and serotypes of EHDV.

Transmission and Epidemiology of Bluetongue and Epizootic Hemorrhagic Disease in North America: Current Perspectives, Research Gaps, and Future Directions.

Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) are arthropod-transmitted viruses in the genus Orbivirus of the family Reoviridae. These viruses infect a variety of domestic and wild ruminant hosts, although the susceptibility to clinical disease associated with BTV or EHDV infection varies greatly among host species, as well as between individuals of the same species. Since their initial detection in North America during the 1950s, these viruses have circulated in endemic and epidemic patterns, with occasional incursions to more northern latitudes. In recent years, changes in the pattern of BTV and EHDV infection and disease have forced the scientific community to revisit some fundamental areas related to the epidemiology of these diseases, specifically in relation to virus-vector-host interactions and environmental factors that have potentially enabled the observed changes. The aim of this review is to identify research and surveillance gaps that obscure our understanding of BT and EHD in North America.

Epizootic Hemorrhagic Disease in Brocket Deer, Brazil

Epizootic Hemorrhagic Disease in Brocket Deer, Brazil

Vector competence of Culicoides sonorensis (Diptera: Ceratopogonidae) to epizootic hemorrhagic disease virus serotype 7

BackgroundCulicoides sonorensis (Diptera: Ceratopogonidae) is a vector of epizootic hemorrhagic disease virus (EHDV) serotypes 1 and 2 in North America, where these viruses are well-known pathogens of white-tailed deer (WTD) and other wild ruminants. Although historically rare, reports of clinical EHDV infection in cattle have increased in some parts of the world over the past decade. In 2006, an EHDV-7 epizootic in cattle resulted in economic loss for the Israeli dairy industry. White-tailed deer are susceptible to EHDV-7 infection and disease; however, this serotype is exotic to the US and the susceptibility of C. sonorensis to this cattle-virulent EHDV is not known. The objective of the study was to determine if C. sonorensis is susceptible to EHDV-7 infection and is a competent vector.MethodsTo evaluate the susceptibility of C. sonorensis, midges were fed on EHDV-7 infected WTD, held at 22 ± 1°C, and processed individually for virus isolation and titration on 4–16 days post feeding (dpf). Midges with a virus titer of ≥102.7 median tissue culture infective doses (TCID50)/midge were considered potentially competent. To determine if infected C. sonorensis were capable of transmitting EHDV-7 to a host, a susceptible WTD was then fed on by a group of 14–16 dpf midges.ResultsFrom 4–16 dpf, 45% (156/350) of midges that fed on WTD with high titer viremia (>107 TCID50/ml) were virus isolation-positive, and starting from 10–16 dpf, 32% (35/109) of these virus isolation-positive midges were potentially competent (≥102.7 TCID50/midge). Midges that fed on infected deer transmitted the virus to a susceptible WTD at 14–16 dpf. The WTD developed viremia and severe clinical disease.ConclusionThis study demonstrates that C. sonorensis is susceptible to EHDV-7 infection and can transmit the virus to susceptible WTD, thus, C. sonorensis should be considered a potential vector of EHDV-7. Together with previous work, this study demonstrates that North America has a susceptible ruminant and vector host for this exotic, cattle-virulent strain of EHDV-7.

Epizootic hemorrhagic disease virus serotype 7 in European cattle and sheep: Diagnostic considerations and effect of previous BTV exposure

Epizootic hemorrhagic disease virus (EHDV), an arthropod-borne orbivirus (family Reoviridae), is an emerging pathogen of wild and domestic ruminants that is closely related to bluetongue virus (BTV). The present study examines the outcome of an experimental EHDV-7 infection of Holstein cattle and East Frisian sheep. Apart from naïve animals that had not been exposed to BTV, it included animals that had been experimentally infected with either BTV-6 or BTV-8 two months earlier. In addition, EHDV-infected cattle were subsequently challenged with BTV-8.Samples were tested with commercially available ELISA and real-time RT-PCR kits and a custom NS3-specific real-time RT-PCR assay. Virus isolation was attempted in Vero, C6/36 and KC cells (from Culicoides variipennis), embryonated chicken eggs and type I interferon receptor-deficient IFNAR−/− mice.EHDV-7 productively infected Holstein cattle, but caused no clinical signs. The inoculation of East Frisian sheep, on the other hand, apparently did not lead to a productive infection. The commercial diagnostic kits performed adequately. KC cells proved to be the most sensitive means of virus isolation, but viremia was shorter than 2 weeks in most animals. No interference between EHDV and BTV infection was observed; therefore the pre-existing immunity to some BTV serotypes in Europe is not expected to protect against a possible introduction of EHDV, in spite of the close relation between the viruses.

Epizootic Hemorrhagic Disease Outbreak in a Captive Facility Housing White-Tailed Deer (Odocoileus virginianus), Bison (Bison bison), Elk (Cervus elaphus), Cattle (Bos taurus), and Goats (Capra hircus) in Colorado, USA

An ungulate research facility in Fort Collins, Colorado, U.S.A., experienced mortality in white-tailed deer (Odocoileus virginianus) because of epizootic hemorrhagic disease virus (EHDV) infection from 20 August 2007 through 26 September 2007. Epizootic hemorrhagic disease virus (EHDV) was detected by reverse transcriptase polymerase chain reaction and virus isolation from the spleen and lung tissues of two white-tailed deer. Virus neutralization tests were performed on pre- and postoutbreak sera from other species maintained in the same facility, including bison (Bison bison), elk (Cervus elaphus), domestic cattle (Bos taurus), and domestic goats (Capra hircus), as well as postoutbreak sera from the surviving white-tailed deer. Serum samples that represented all species in the facility neutralized EHDV-1 and EHDV-2 either before or after the outbreak. The animals that neutralized EHDV-1 did not neutralize EHDV-2. No clinical signs attributable to EHDV infection were noted in any of the species other than the deer during the outbreak. Although experimental EHDV infections have been reported in bison and elk, natural exposures have not been previously documented in these species in North America. The roles that elk, bison, cattle, and goats might play in the epidemiology of EHDV in a close-contact multispecies situation remain unknown.

An improved Real-Time Polymerase Chain Reaction for the Simultaneous Detection of All Serotypes of Epizootic Hemorrhagic Disease Virus

Epizootic hemorrhagic disease virus (EHDV) is a significant pathogen of wild and sometimes domestic ungulates worldwide. Rapid and reliable methods for virus detection and identification play an essential part in the control of epizootic hemorrhagic disease (EHD). In the present study, a 1-step real-time polymerase chain reaction (PCR) group-specific assay was developed. The assay detects genome segment 5 (NS1) from all of the 8 serotypes of EHDV. Assay sensitivity was evaluated relative to a conventional gel-based nested PCR using cell culture-derived virus and diagnostic samples from clinically affected white-tailed deer (Odocoileus virginianus). The assay reliably amplified the NS1 gene from any of the EHDV strains tested, including isolates from each of the 8 EHDV serotypes. No cross-reactions were detected when all 24 serotypes of Bluetongue virus, a closely related member of the genus Orbivirus, were tested. A panel of 76 known EHDV-positive clinical samples was used to compare the performance of the assay relative to a previously reported real-time PCR assay. Results indicated that there was no statistically significant difference between the threshold cycle values obtained with both assays. A collection of 178 diagnostic samples submitted for EHD diagnosis was also used for test evaluation. The assay could be applied for rapid detection of EHDV in clinical samples from susceptible ruminants during an outbreak of the disease. In addition, this PCR assay has the benefits of being reliable and simple and could provide a valuable tool for studying the epidemiology of EHDV infection in susceptible ruminants by facilitating the detection of EHDV, regardless of the serotype.

Evaluation of Epizootic Haemorrhagic Disease Virus Infection in Sentinel Calves from the San Joaquin Valley of California

Evaluation of epizootic haemorrhagic disease virus infection in sentinel calves from the San Joaquin Valley of California.

PCR detection of North American and Central African isolates of epizootic hemorrhagic disease virus (EHDV) based on genome segment 10 of EHDV serotype 1.

PCR amplification technology for the detection of epizootic hemorrhagic disease virus (EHDV) ribonucleic acid in cell culture and clinical specimens was developed. With oligoribonucleotide primers selected from genome segment 10 of EHDV serotype 1 (EHDV-1), which codes for two nonstructural proteins (NS3 and NS3a), the PCR-based assay resulted in a 535-bp PCR product. RNAs from North American EHDV-1 prototype, EHDV-2 prototype, and a number of EHDV field isolates, including the Central African isolates of EHDV-5 and EHDV-318 propagated in cell cultures, were detected by this PCR-based assay. The specific 535-bp PCR products were visualized onto agarose gels, and the identity of the PCR products was confirmed by chemiluminescent hybridization with a 352-bp internal probe. The sensitivity of the EHDV PCR assay was increased by chemiluminescent hybridization; by this EHDV-NS3 PCR, 10 fg of EHDV RNA was detected (equivalent to 600 viral particles). Amplification product was not detected when the PCR-based assay was applied to RNAs from North American bluetongue virus prototype serotypes 2, 10, 11, 13, and 17; total nucleic acid extracts from uninfected BHK-21 cells; or unfractionated blood from calves and deer that were EHDV seronegative and virus isolation negative. The described EHDV PCR-based assay with primers derived from segment 10 of EHDV-1 resulted in detection of EHDV RNA from blood and tissues collected from calves and deer with natural and experimental EHDV infections and provides a valuable tool to study the epidemiology of EHDV infection in susceptible ruminants.

Interaction of epizootic hemorrhagic disease virus with bovine erythrocytes in vitro: Electron microscope study

The interaction of epizootic hemorrhagic disease virus (EHDV) with bovine erythrocytes in t'itro has been studied, and the results are presented herein. EHDV, a member of the Orbit, irus genus in the family Reoviridae, causes fatal hemorrhagic disease in North American white-tailed deer (Odocoileus virginianus). Other captive and free ranging ruminants may also be infected with the disease [1, 4]. At least 10 serotypes of EHDV are recognised worldwide, but serotypes 1 and 2 are enzootic in the United States. In white-tailed deer, viremia is short and the disease usually follows a peracute course leading to death. In cattle, EHDV infection is common but typically asymptomatic, and viremia in EHDV infected cattle is usually short. However, infection of European breed of cattle with laboratory adapted strain of EHDV induced viremia which persisted for up to 50 days [3], suggesting that cattle may play a role in the epidemiology of the disease [2]. During the later course of EHDV-infection, the virus is principally associated with erythrocytes. Erythrocyte-associated viremia has been reported with a number of orbiviruses including bluetongue virus (BTV); however, there is no information available on the interaction of EHDV with bovine erythrocytes. The mechanism involved in prolonged viremia in EHDV-infected cattle, despite the presence of circulating neutralising antibodies, has yet to be explained. A plaque-purified EHDV serotype 1 (New Jersey strain) was used to inoculate suspended bovine erythrocytes, obtained from 4-month-old calves, at a multiplicity of infection (MOI) of 1, and the inoculated erythrocytes were incubated at 37~>C for 1 h. Uninoculated erythrocytes were used as controls. The details for collection of blood, preparation of erythrocytes, preparation of sections for transmission electron microscope were described previously [5]. EHDV particles were commonly observed in close association with erythrocyte cell membrane and were typically present in invaginations (Fig. 1). The viral particles measure about 50 70 nm in diameter and were recognized as EHDV particles by their morphological appearance. The viral particles were not observed in the erythrocytes from controls. The results of this study indicated that EHDV becomes associated with the erythrocytic cell membrane soon after in vitro infection of bovine erythrocytes, with the virus particles becoming sequestered in invaginations of the cell membrane. The infection of erythrocytes did not progress beyond the cell membrane invagination, as free virons

Serogrouping and topotyping of Sudanese and United States strains of epizootic hemorrhagic disease virus using PCR

The potential use of the recently reported polymerase chain reaction (PCR) protocol for detection of United States epizootic hemorrhagic disease virus (EHDV) serotype 1 (EHDV-1) and serotype 2 (EHDV-2) ribonucleic acid in cell culture and clinical specimens was evaluated for detection of Sudanese EHDV strains. EHDV serotype 5 (EHDV-5) and EHDV, isolate 318 (untyped) designated (EHDV-318), recovered from sentinel calves at the Khartoum University farm (Sudan) were studied. RNA from EHDV-5 and EHDV-318 and a number of EHDV field isolates, propagated in cell cultures, were detected by the described PCR-based assay. The specific 387 bp PCR products were visualized on ethidium-bromide stained agarose gel. Specificity of the PCR products was confirmed by chemiluminescent hybridization with non-radiolabeled internal probe. Amplification product was not detected when the PCR-based assay was applied to RNA from bluetongue virus (BTV) prototypes serotypes 2, 10, 11, 13, 16 and 17; total nucleic acid extracts from uninfected BHK-21 cells. The results of this study indicated that the previously described EHDV-PCR assay could be applied for detection of Sudanese as well as United States strains of EHDV serogroup. In addition, the described EHDV-PCR assay could be used as a supportive diagnostic assay to the current conventional virus isolation procedures used for detection of EHDV infection in susceptible ruminants.

Systemic adenovirus infection associated with high mortality in mule deer (Odocoileus hemionus) in California.

Seventeen counties in northern California experienced epizootics of high mortality in the mule deer (Odocoileus hemionus) population during the latter half of 1993. Thirteen deer submitted to the California Veterinary Diagnostic Laboratory System as part of this natural die-off had systemic adenovirus infection. Pulmonary edema was present in all 13 deer. Erosions, ulceration, and abscessation of the upper alimentary tract occurred in 7/13 deer. Four of 13 deer had hemorrhagic enteritis. All 13 deer had widespread systemic vasculitis with endothelial intranuclear inclusions. Fluorescein isothiocyanate-labeled antibody directed against bovine adenovirus type 5 bound to antigen in endothelial cells. Adenovirus was identified by transmission electron microscopy within the nuclei of endothelial cells in 6/6 deer examined. An adenovirus was isolated from lung homogenates of one deer that were cultured on black-tailed deer pulmonary artery endothelial cells. With the exception of the intranuclear inclusions evident on histologic evaluation, gross and histologic changes were similar to those described for bluetongue virus infection and epizootic hemorrhagic disease virus infection in white-tailed deer. Nine additional deer were emaciated and had pharyngeal abscesses with focal vasculitis, which may represent the chronic affects of previous nonfatal adenovirus infection.