Perspectives on the Changing Landscape of Epizootic Hemorrhagic Disease Virus Control.

Epizootic hemorrhagic disease (EHD) is an insect-transmitted viral disease of wild and domestic ruminants. It was first described following a 1955 epizootic in North American white-tailed deer (Odocoileus virginianus), a species which is highly susceptible to the causative agent of EHD, epizootic hemorrhagic disease virus (EHDV). EHDV has been detected globally across tropical and temperate regions, largely corresponding to the presence of Culicoides spp. biting midges which transmit the virus between ruminant hosts. It regularly causes high morbidity and mortality in wild and captive deer populations in endemic areas during epizootics. Although cattle historically have been less susceptible to EHDV, reports of clinical disease in cattle have increased in the past two decades. There is a pressing need to identify new methods to prevent and mitigate outbreaks and reduce the considerable impacts of EHDV on livestock and wildlife. This review discusses recent research advancements towards the control of EHDV, including the development of new investigative tools and progress in basic and applied research focused on virus detection, disease mitigation, and vector control. The potential impacts and implications of these advancements on EHD management are also discussed.

Modeling Abundance of Culicoides stellifer, a Candidate Orbivirus Vector, Indicates Nonrandom Hemorrhagic Disease Risk for White-Tailed Deer (Odocoileus virginianus).

(1) Background: Hemorrhagic diseases in white-tailed deer (Odocoileus virginianus) are caused by orbiviruses and have significant economic impact on the deer ranching industry in the United States. Culicoides stellifer is a suspected vector of epizootic hemorrhagic disease virus (EHDV), with recent field evidence from Florida, but its natural history is poorly understood. Studying the distribution and abundance of C. stellifer across the landscape can inform our knowledge of how virus transmission can occur locally. We may then target vector management strategies in areas where viral transmission can occur. (2) Methods: Here, we used an occupancy modeling approach to estimate abundance of adult C. stellifer females at various physiological states to determine habitat preferences. We then mapped midge abundance during the orbiviral disease transmission period (May-October) in Florida. (3) Results: We found that overall, midge abundance was positively associated with sites in closer proximity to large-animal feeders. Additionally, midges generally preferred mixed bottomland hardwood and agricultural/sand/water habitats. Female C. stellifer with different physiological states preferred different habitats. (4) Conclusions: The differences in habitat preferences between midges across states indicate that disease risk for deer is heterogeneous across this landscape. This can inform how effective vector management strategies should be implemented.

A Mortality-Based Description of EHDV and BTV Prevalence in Farmed White-Tailed Deer (Odocoileus virginianus) in Florida, USA.

Hemorrhagic disease (HD) caused by bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) is the most important viral disease of farmed and wild white-tailed deer (WTD; Odocoileus virginianus) and can cause substantial mortality in susceptible hosts. Captive cervid farming is an emerging industry in Florida, an HD-enzootic region. Morbidity and mortality due to HD are major concerns among deer farmers, but the impact of HD on Florida's cervid farming industry is unknown. Our primary objective was to determine the prevalence of epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV) among WTD submitted to the University of Florida Institute of Food and Agricultural Sciences Cervidae Health Research Initiative (CHeRI) for post-mortem diagnostics. Our secondary objectives were to identify the predominant circulating EHDV serotypes during each sampling year and to determine the age class with the greatest proportion of EHDV- and BTV-positive post-mortem specimens. From 2016 to 2020, spleen samples from 539 farmed WTD with unexplained mortality were tested for the presence of EHDV and BTV by RT-qPCR. Overall, the prevalence of EHDV, BTV, or EHDV/BTV coinfection was 26%, 16%, and 10%, respectively, and 44% of deer (237/539) were diagnosed with HD by RT-qPCR. The predominant circulating EHDV serotype varied by year. Overall, EHDV-2 was the most commonly identified serotype (55% of PCR-positive cases), and EHDV-1 was the least frequently identified serotype (16% of PCR-positive cases). The greatest proportion of EHDV/BTV positives among mortality cases was observed in young WTD aged 3-6 months (50%-82% positive). There was a significant difference in the prevalence of EHDV/BTV by age when comparing specimens from WTD over 1 year old (p = 0.029, n = 527). Among these samples, the number of reported mortalities and the prevalence of EHDV/BTV were highest in yearling animals (56%). These data provide the first estimate of EHDV and BTV prevalence and virus serotypes among farmed WTD in Florida, identify the WTD age groups with the greatest proportions of EHDV- and BTV-positive specimens, and suggest that HD caused by these two viruses may be a major source of mortality challenging the captive cervid farming industry in Florida.

Spatial Analysis of the 2017 Outbreak of Hemorrhagic Disease and Physiographic Region in the Eastern United States.

Hemorrhagic disease (HD) is considered one of the most significant infectious diseases of white-tailed deer in North America. Investigations into environmental conditions associated with outbreaks suggest drought conditions are strongly correlated with outbreaks in some regions of the United States. However, during 2017, an HD outbreak occurred in the Eastern United States which appeared to be associated with a specific physiographic region, the Appalachian Plateau, and not drought conditions. The objective of this study was to determine if reported HD in white-tailed deer in 2017 was correlated with physiographic region. There were 456 reports of HD from 1605 counties across 26 states and 12 physiographic regions. Of the 93 HD reports confirmed by virus isolation, 76.3% (71/93) were identified as EHDV-2 and 66.2% (47/71) were from the Appalachian Plateau. A report of HD was 4.4 times more likely to occur in the Appalachian Plateau than not in 2017. Autologistic regression models suggested a statistically significant spatial dependence. The underlying factors explaining this correlation are unknown, but may be related to a variety of host, vector, or environmental factors. This unique outbreak and its implications for HD epidemiology highlight the importance for increased surveillance and reporting efforts in the future.

Bluetongue and epizootic hemorrhagic disease viruses: recent developments with these globally re-emerging arboviral infections of ruminants.

Bluetongue (BT) and epizootic hemorrhagic disease (EHD) are globally re-emerging diseases of domestic and wild ruminants, respectively caused by BT virus (BTV) and EHD virus. Both viruses are transmitted by hematophagous midges; however, newly recognized BTV serotypes may be transmitted horizontally without requirement for any biological vector. The global range of these viruses and/or their associated diseases have changed remarkably in recent years, most notably with the invasion of Europe by multiple serotypes of BTV since 1998. Although not zoonoses, the unanticipated emergence of BT and EHD in several different areas of the world provides a uniquely sobering and unambiguous reminder of the potential consequences of climate change on the distribution and severity of vector-borne diseases. Recent experiences with these viruses have also emphasized the need for effective, DIVA-compatible vaccines to combat anticipated future incursions, as existing vaccines have serious inherent deficiencies.

A Review of Knowledge Gaps and Tools for Orbivirus Research.

Although recognized as causing emerging and re-emerging disease outbreaks worldwide since the late 1800 s, there has been growing interest in the United States and Europe in recent years in orbiviruses, their insect vectors, and the diseases they cause in domestic livestock and wildlife. This is due, in part, to the emergence of bluetongue (BT) in northern Europe in 2006-2007 resulting in a devastating outbreak, as well as severe BT outbreaks in sheep and epizootic hemorrhagic disease (EHD) outbreaks in deer and cattle in the United States. Of notable concern is the isolation of as many as 10 new BT virus (BTV) serotypes in the United States since 1999 and their associated unknowns, such as route of introduction, virulence to mammals, and indigenous competent vectors. This review, based on a gap analysis workshop composed of international experts on orbiviruses conducted in 2013, gives a global perspective of current basic virological understanding of orbiviruses, with particular attention to BTV and the closely related epizootic hemorrhagic disease virus (EHDV), and identifies a multitude of basic virology research gaps, critical for predicting and preventing outbreaks.

The host specific NS3 glycosylation pattern reflects the virulence of Ibaraki virus in different hosts.

The non-structural protein NS3 was investigated in Ibaraki virus (IBAV), an epizootic hemorrhagic disease virus, serotype 2. Degree of NS3 glycosylation, cytopathic effect, and virus release efficiency were compared between mammalian and insect cells. The molecular weight of synthesized NS3 was compared in Western blot analysis following the removal of the glycochain by PNGase F treatment and revealed that glycosylation of NS3 occurred only in mammalian cells. Also, it was revealed that the amount of infectious IBAV in the extracellular fraction continued to increase for insect cells even after 60h post infection without disrupting cells. These results suggested that glycosylation of NS3 controls pathogenicity of IBAV in host cells to protect vector insects by altering the release pathway of assembled progeny viruses.

Experimental infection of Australian freshwater fish with epizootic haematopoietic necrosis virus (EHNV).

The ranavirus, epizootic hematopoietic necrosis virus (EHNV), is endemic to southern Australia with natural outbreaks resulting in mass mortality events in wild Redfin Perch Perca fluviatilis (also known as Eurasian Perch) and less severe disease in farmed Rainbow Trout Oncorhynchus mykiss. To further investigate the host range for EHNV, 12 ecologically or economically important freshwater fish species from southeastern Australia were exposed experimentally to the virus. A bath-challenge model at 18 ± 3°C was employed with limited use of intraperitoneal inoculation to determine if a species was likely to be susceptible to EHNV. Of the species tested, Murray-Darling Rainbowfish Melanotaenia fluviatilis and Dewfish Tandanus tandanus (also known as Freshwater Catfish) were considered to be potentially susceptible species. EHNV was isolated from approximately 7% of surviving Eastern Mosquitofish Gambusia holbrooki, indicating this widespread alien fish species is a potential carrier. The infection of Silver Perch Bidyanus bidyanus and Macquarie Perch Macquaria australasica and the lack of infection in Murray Cod Maccullochella peelii peelii and Golden Perch Macquaria ambigua ambigua after exposure to EHNV via water confirmed earlier data from Langdon (1989). Five other species of native fish were potentially not susceptible to the virus or the fish were able to recover during the standard 35-d postchallenge observation period. Overall, it appeared that EHNV was less virulent in the present experimental model than in previous studies, but the reasons for this were not identified. Received May 21, 2012; accepted November 1, 2012.

Serosurveillance for livestock pathogens in free-ranging mule deer (Odocoileus hemionus).

Routine disease surveillance has been conducted for decades in mule deer (Odocoileus hemionus) in California for pathogens shared between wildlife and domestic ruminants that may have implications for the animal production industry and wildlife health. Deer sampled from 1990 to 2007 (n = 2,619) were tested for exposure to six pathogens: bluetongue virus (BTV), epizootic hemorrhagic disease virus (EHDV), bovine viral diarrhea virus (BVDV), Leptospira spp., Anaplasma spp. and Brucella spp. We evaluated the relationship between exposure to these pathogens and demographic risk factors to identify broad patterns in seroprevalence across a large temporal and spatial scale. The overall seroprevalence for the entire study period was 13.4% for BTV, 16.8% for EHDV, 17.1% for BVDV, 6.5% for Leptospira spp., 0.2% for Brucella spp., and 17% for Anaplasma spp. Antibodies against BTV and EHDV were most prevalent in the deer populations of southern California. Antibodies against Leptospira spp. and Anaplasma spp. were most prevalent in coastal and central northern California whereas antibodies against BVDV were most prevalent in central-eastern and northeastern California. The overall seroprevalence for Anaplasma spp. was slightly lower than detected in previous studies. North and central eastern California contains large tracts of federal land grazed by livestock; therefore, possible contact between deer and livestock could explain the high BVDV seroprevalence found in these areas. Findings from this study will help to establish baseline values for future comparisons of pathogen exposure in deer, inform on long-term trends in deer population health and provide relevant information on the distribution of diseases that are shared between wildlife and livestock.

Susceptibility of white-tailed deer (Odocoileus virginianus) to experimental infection with epizootic hemorrhagic disease virus serotype 7.

During the fall of 2006, in Israel, epizootic hemorrhagic disease virus (EHDV) serotype 7 caused an intense and widespread epizootic in domestic cattle that resulted in significant economic losses for the dairy industry. The susceptibility of potential North American vector and ruminant hosts to infection with EHDV-7 is not known but is essential to understanding the potential for establishment of this exotic orbivirus in North America if it were introduced. Our primary objective was to determine whether white-tailed deer (WTD; Odocoileus virginianus) are susceptible to infection with EHDV-7. Six, 8-mo-old WTD were experimentally infected with EHDV-7, and all became infected and exhibited varying degrees of clinical disease. Clinical signs, clinicopathologic abnormalities, and postmortem findings were consistent with previous reports of orbiviral hemorrhagic disease (HD) in this species. Four of six animals died or were euthanized because of the severity of disease, one on postinoculation day (PID) 5 and the remaining WTD on PID 7. All deer had detectable viremia on PID 3, which peaked on PID 5 or 6 and persisted for as long as PID 46 in one animal. Deer surviving the acute phase of the disease seroconverted by PID 10. Based on the 67% mortality rate we observed, this strain of EHDV-7 is virulent in WTD, reaffirming their role as a sentinel species for the detection of endemic and nonendemic EHDV. Further, the observed disease was indistinguishable from previous reports of disease caused by North American EHDV and bluetongue virus serotypes, highlighting the importance of serotype-specific diagnostics during suspected HD outbreaks.

Infection kinetics of Epizootic Haemorrhagic Disease virus serotype 6 in Holstein-Friesian cattle.

Epizootic Haemorrhagic Disease virus serotype 6 (EHDV-6) has recently caused serious outbreaks of Epizootic Haemorrhagic Disease (EHD) on the edges of Europe, in Turkey, Israel and Morocco. The aim of this study was to assess the pathogenicity and infection kinetics of EHD in Holstein-Friesian cattle infected with the two distinct strains of EHDV-6 isolated from the recent Turkish and Moroccan outbreaks. Samples taken throughout the study were used to validate two recently developed diagnostic assays that detect EHDV antibodies and viral genome. Two groups of five Holstein-Friesian cattle were experimentally infected with either the Moroccan or the Turkish isolate of EHDV-6. Cattle in both groups remained clinically unaffected throughout the study, but displayed high levels of viral RNA and virus in their blood, confirming that sub-clinical infection of cattle is likely to play an important role in EHDV transmission. A recently developed and commercialised real-time RT-PCR assay detected viral RNA as early as 2 days post infection (dpi) in both infection studies and viral RNA persisted for the course of the study. Antibodies against EHDV were first detected by 9dpi using a recently developed EHDV blocking ELISA and antibodies persisted up to the end of the study. All animals developed high levels of neutralising antibodies to EHDV-6, measured by a serum neutralisation test (SNT), with titres (log(10)) ranging from 2.20 to 2.38 at the end of the study. Virus was isolated from the blood of infected animals from as early as 2dpi up to 28dpi.

Epizootiology of an epizootic hemorrhagic disease outbreak in West Virginia.

An outbreak of epizootic hemorrhagic disease virus, serotype 2 (EHDV-2) was responsible for localized white-tailed deer (Odocoileus virginianus) mortality in Hardy and Hampshire counties, West Virginia (USA), in the summer and fall of 1993. Using available historical data on regional herd immunity, data opportunistically collected during the epizootic, and postepizootic sampling of hunter-harvested deer, we grossly estimate certain epidemiologic parameters and compare findings to a hypothesis about hemorrhagic disease outbreaks in the Appalachian Mountains. During the epizootic, 57.9 km(2) were actively searched and 228 dead deer were found. Epizootic hemorrhagic disease virus, serotype 2 was isolated from seven of nine deer sampled in Hardy and Hampshire counties. Preepizootic exposure of deer to EHD viruses was unknown, but available data suggest that it was negligible. The geographic distribution of the outbreak was defined by plotting the locations of dead deer found during the outbreak, as well as the locations of deer harvested by hunters after the outbreak that had antibodies to EHDV-2 on a map sectioned into 16.65 km(2) rectangular sections. Sections that included one or more dead deer or hunter-harvested deer with antibodies to EHDV-2 were included in the defined outbreak area. Postoutbreak sampling revealed monospecific EHDV-2 antibodies in 12% of deer harvested by hunters within the defined outbreak area. Based on the available data and accepting certain assumptions, gross calculations suggest that this outbreak appears to have been isolated and probably killed a high percentage of the deer that were infected. This is consistent with the hypothesis that sporadic hemorrhagic disease outbreaks in the Appalachian Mountains are usually localized and severe.

Innate resistance to epizootic hemorrhagic disease in white-tailed deer.

Differences in innate disease resistance at the sub-species level have major implications for wildlife management. Two subspecies of white-tailed deer, Odocoileus virginianus borealis and O. virginianus texanus were infected with epizootic hemorrhagic disease (EHD) viruses. These viruses are highly virulent pathogens of white-tailed deer and are endemic within the range of O. virginianus texanus but not within the range of O. virginianus borealis. Two experimental infections were performed. Five O. virginianus texanus fawns and five O. virginianus borealis fawns were infected with 10(7.1) median tissue culture infective doses (TCID50) of EHD virus, serotype 1 and five of each subspecies were infected with 10(7.1) TCID50 of EHD virus, serotype 2. Infections with both EHD virus serotypes caused severe clinical disease and mortality in O. virginianus borealis fawns, whereas disease was mild or nondetectable in O. virginianus texanus fawns. Virus titers and humoral immune response were similar in both subspecies suggesting that differences in innate disease resistance explain the differences seen in clinical disease severity. In white-tailed deer, innate disease resistance may vary at the subspecies level. Should this phenomenon occur in other species, these findings have major implications for managing wildlife populations, both endangered and non-endangered, using tools such as translocation and captive propagation.

Bluetongue virus infection in pregnant ewes.

Inoculation of 53 ewes after 35, 45, 60, or 80 days of gestation with bluetongue virus serotypes 10, 11, 13, or 17, or with epizootic hemorrhagic disease virus serotypes 1 or 2, resulted in overt clinical disease in the 47 ewes inoculated with bluetongue virus but not in the 6 ewes inoculated with epizootic hemorrhagic disease virus. None of the lambs produced by these ewes had developmental defects or any evidence of persistence of viremia.

Contamination of genetically engineered CHO-cells by epizootic haemorrhagic disease virus (EHDV).

The characterization of a contaminating virus which was detected in genetically-engineered Chinese hamster ovary (CHO) cells during the production of biologicals is described in the present paper. Under electron microscopy, the contaminating virus had a morphology resembling that of an orbivirus. The relationship was confirmed by nucleic acid analysis which showed a RNA segment pattern characteristic of orbiviruses. With an immunoperoxidase staining of monolayer cells and through sero-neutralization tests the virus was identified as being identical to Epizootic Haemorrhagic Disease Virus (EHDV), isolate 318 (untyped) from Bahrain. Potential sources of the contaminating virus and feasible procedures to avoid adventitous virus infections in cell cultures are discussed.