Emergence of epizootic hemorrhagic disease in red deer (Cervus elaphus), Spain, 2022.

Epizootic hemorrhagic disease (EHD) virus serotype 8 (EHDV-8) emerged in Spain in autumn 2022. In this study, we aimed to (1) characterize the clinical and lesional presentation of EHDV infection in European red deer (Cervus elaphus), and (2) study the spatial spread of the virus in wild ruminants in Spain after its introduction, in 2022/2023. We confirmed EHDV infection in two clinically compatible sick red deer by PCR and detection of anti-EHDV specific antibodies. EHDV infection occurred in red deer with hyperacute to acute clinical signs and lesions associated to vascular changes leading to death of the animals. Partial sequences of variable segment 2 (VP2) and segment 5 (NS1) genes of the detected viruses had >99% nucleotide identity with EHDV-8 sequences from Tunisia and Italy. In a cross-sectional serological study of EHDV in 592 wild ruminants, mainly red deer (n=578), in southwestern Spain, we detected anti-EHDV antibodies in 37 of 592 samples (6.3%; 95% confidence interval: 4.3-8.2), all from red deer and from the localities where clinical cases of EHD were confirmed in red deer. We conclude that EHDV-8 infection causes severe EHD in European red deer. The serosurvey revealed a limited spread of EHDV-8 in Spanish wild ruminant populations in the first year of virus detection in Spain.

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.

Development and validation of an RT-qPCR for detection and quantitation of emerging epizootic hemorrhagic disease virus serotype 8 RNA from field samples.

Epizootic hemorrhagic disease virus (EHDV) is a Culicoides-transmitted virus circulating in multiple serotypes. It has become a concern in the European Union as a novel strain of the serotype 8 (EHDV-8) of clear Northern African origin, has been recently discovered in symptomatic cattle in Italy (islands of Sardinia and Sicily), Spain, and Portugal. Current molecular typing methods targeting the S2 nucleotide sequences -coding for the outermost protein of the virion VP2- are not able to detect the novel emerging EHDV-8 strain as they enrolled the S2 sequence of the unique EHDV-8 reference strain isolated in Australia in 1982. Thus, in this study, we developed and validated a novel typing assay for the detection and quantitation of the novel EHDV-8 RNA from field samples, including blood of ruminants and insects. This molecular tool will certainly support EHDV-8 surveillance and control.

Complete genome sequences of epizootic hemorrhagic disease virus serotypes 5 and 6 isolated in Japan.

Here, we report the complete genome sequences of epizootic hemorrhagic disease (EHD) virus serotypes 5 (EHDV-5) and 6 (EHDV-6) isolated in the Yaeyama Islands of Okinawa Prefecture, Japan. The EHDV-5 strain, ON-11/E/16, which was isolated in 2016, is, to our knowledge, the second EHDV-5 strain to be isolated after the first was isolated in Australia in 1977. In each of the genome segments, ON-11/E/16 was most closely related to EHDV strains of different serotypes isolated in Australia and Japan. Our results support the idea that various serotypes of EHDV have been circulating while causing reassortment in the Asia-Pacific region. In all genome segments, the EHDV-6 strain, ON-3/E/14, which was isolated in 2014, was highly similar to EHDV-6 strain HG-1/E/15, which was detected in affected cattle during the EHD epidemic in Hyogo prefecture in 2015. Therefore, these two EHDV-6 strains, ON-3/E/14 and HG-1/E/15, may have the same origin. However, it is unclear whether EHDV-6 was transmitted directly between the locations where those strains were isolated/detected (approx. 1,500 km apart) or whether EHDV-6 strains of the same origin entered each location at different times. In addition, we cannot rule out the possibility that EHDV-6 infection has spread unnoticed through asymptomatic cattle in other areas of Japan. Therefore, further investigation into EHDV infection in cattle is necessary for a more detailed understanding of the ecology of EHDV in Japan.

Characterization of Epizootic Hemorrhagic Disease Virus Serotype 8 in Naturally Infected Barbary Deer (Cervus elaphus barbarus) and Culicoides (Diptera: Ceratopogonidae) in Tunisia.

Epizootic hemorrhagic disease (EHD) is a Culicoides-borne disease of domestic and wild ruminants caused by EHD virus (EHDV). This virus circulates in multiple serotypes. In late September 2021, a novel strain belonging to EHDV-8 was reported in cattle farms in Central-Western Tunisia, and in the fall of 2022, the same virus was also detected in Italy and Spain. In the present study, we described EHDV-8 occurrence in deer and, a preliminary identification of the potential Culicoides species responsible for virus transmission in selected areas of Tunisia. EHDV-8 was identified in deer carcasses found in 2021 and 2022 in the national reserve of El Feidja, Jendouba, Northwestern Tunisia, and isolated on cell culture. Instead, insect vectors were collected in October 2021 only in the areas surrounding the city of Tozeur (Southern Tunisia) where EHDV-8 cases in cattle were confirmed. Morphological identification showed that 95% of them belonged to the Culicoides kingi and Culicoides oxystoma species and both species tested positive for EHDV-8 RNA. C. imicola was not detected in this collection and EHDV-8 RNA was not evidenced in vector pools collected in 2020, prior to official EHDV-8 emergence. EHDV whole genome sequences were also obtained directly from infected biological samples of deer and positive vectors. EHDV-8 sequences obtained from deer and vectors share a nucleotide identity ranging from 99.42 to 100% and amino acid identity from 99.18 to 100% across all genome segments with the EHDV-8/17 TUN2021 reference sequence.

Characterization of two novel reassortant bluetongue virus serotype 1 strains isolated from farmed white-tailed deer (Odocoileus virginianus) in Florida, USA.

Hemorrhagic diseases caused by epizootic hemorrhagic disease virus or by bluetongue virus (BTV) are the most important orbivirus diseases affecting ruminants, including white-tailed deer (WTD). Bluetongue virus is of particular concern for farmed WTD in Florida, given its lethality and its wide distribution throughout the state. This study reports the clinical findings, ancillary diagnostics, and genomic characterization of two BTV serotype 1 strains isolated from two farmed WTD, from two different farms in Florida in 2019 and 2022. Phylogenetic and genetic analyses indicated that these two novel BTV-1 strains were reassortants. In addition, our analyses reveal that most genome segments of these strains were acquired from BTVs previously detected in ruminants in Florida, substantiating their endemism in the Southeastern U.S. Our findings underscore the need for additional research to determine the genetic diversity of BTV strains in Florida, their prevalence, and the potential risk of new BTV strains to WTD and other ruminants.

Seroprevalence of Epizootic Hemorrhagic Disease Virus in Guangdong Cattle Farms during 2013-2017, China.

Epizootic hemorrhagic disease (EHD) is an infectious viral disease caused by epizootic hemorrhagic disease virus (EHDV) and EHDV frequently circulates in wild and domestic ruminants. Sporadic outbreaks of EHD have caused thousands of deaths and stillbirths on cattle farms. However, not much is known about the circulating status of EHDV in Guangdong, southern China. To estimate the seroprevalence of EHDV in Guangdong province, 2886 cattle serum samples were collected from 2013 to 2017 and tested for antibodies against EHDV using a competitive ELISA. The overall seroprevalence of EHDV reached 57.87% and was highest in autumn (75.34%). A subset of positive samples were serotyped by a serum neutralization test, showing that EHDV serotypes 1 and 5-8 were circulating in Guangdong. In addition, EHDV prevalence always peaked in autumn, while eastern Guangdong had the highest EHDV seropositivity over the five-year period, displaying apparent temporal-spatial distribution of EHDV prevalence. A binary logistic model analysis indicated a significant association between cattle with BTV infections and seroprevalence of EHDV (OR = 1.70, p < 0.001). The co-infection of different serotypes of EHDV and BTV raises a high risk of potential genomic reassortment and is likely to pose a significant threat to cattle, thus urging more surveillance to monitor their circulating dynamics in China.

Epizootic Hemorrhagic Disease Virus Serotype 8, Italy, 2022.

We describe the detection of epizootic hemorrhagic disease virus (EHDV) serotype 8 in cattle farms in Sardinia and Sicily in October-November 2022. The virus has a direct origin in North Africa; its genome is identical (>99.9% nucleotide sequence identity) to EHDV serotype 8 strains detected in Tunisia in 2021.

West Nile Virus and Epizootic Hemorrhagic Disease Virus Co-Infection in a Novel Host at the Nashville Zoo.

On August 30, 2017, one of five bontebok in a mixed-species exhibit at the Nashville Zoo at Grassmere exhibited acute hind-limb ataxia and altered demeanor. Pathological examination demonstrated meningoencephalitis and spinal myelitis. Coinfection of West Nile virus (WNV) and epizootic hemorrhagic disease virus (EHDV) was revealed by quantitative real-time and traditional reverse transcription-polymerase chain reaction assays and virus isolation/whole genome sequencing from brain tissue, respectively. Whole genome sequencing was conducted for EHDV. Mosquito testing from September 19 to October 13, 2017, demonstrated a higher WNV infection rate in mosquitoes at the zoo compared with the rest of Nashville-Davidson County. EHDV is endemic in wild white-tailed deer (family Cervidae) in Tennessee, and the prevalence in wildlife depends on environmental influences. This case illustrates the potential susceptibility of exotic zoo animals to endemic domestic arthropod-borne viruses (arboviruses) and reinforces the importance of cooperative antemortem and postmortem surveillance strategies among human, wildlife, and domestic animal health agencies.

The Impact of Variation in the Toll-like Receptor 3 Gene on Epizootic Hemorrhagic Disease in Illinois Wild White-Tailed Deer (Odocoileus virginianus).

Epizootic hemorrhagic disease (EHD) leads to high mortality in white-tailed deer (Odocoileus virginianus) and is caused by a double-stranded RNA (dsRNA) virus. Toll-like receptor 3 (TLR3) plays a role in host immune detection and response to dsRNA viruses. We, therefore, examined the role of genetic variation within the TLR3 gene in EHD among 84 Illinois wild white-tailed deer (26 EHD-positive deer and 58 EHD-negative controls). The entire coding region of the TLR3 gene was sequenced: 2715 base pairs encoding 904 amino acids. We identified 85 haplotypes with 77 single nucleotide polymorphisms (SNPs), of which 45 were synonymous mutations and 32 were non-synonymous. Two non-synonymous SNPs differed significantly in frequency between EHD-positive and EHD-negative deer. In the EHD-positive deer, phenylalanine was relatively less likely to be encoded at codon positions 59 and 116, whereas leucine and serine (respectively) were detected less frequently in EHD-negative deer. Both amino acid substitutions were predicted to impact protein structure or function. Understanding associations between TLR3 polymorphisms and EHD provides insights into the role of host genetics in outbreaks of EHD in deer, which may allow wildlife agencies to better understand the severity of outbreaks.

Characterization of a Novel Reassortant Epizootic Hemorrhagic Disease Virus Serotype 6 Strain Isolated from Diseased White-Tailed Deer (Odocoileus virginianus) on a Florida Farm.

We report an outbreak of a novel reassortant epizootic hemorrhagic disease virus serotype 6 (EHDV-6) in white-tailed deer (WTD) on a Florida farm in 2019. At necropsy, most animals exhibited hemorrhagic lesions in the lung and heart, and congestion in the lung, liver, and spleen. Histopathology revealed multi-organ hemorrhage and congestion, and renal tubular necrosis. Tissues were screened by RT-qPCR and all animals tested positive for EHDV. Tissues were processed for virus isolation and next-generation sequencing was performed on cDNA libraries generated from the RNA extracts of cultures displaying cytopathic effects. Six isolates yielded nearly identical complete genome sequences of a novel U.S. EHDV-6 strain. Genetic and phylogenetic analyses revealed the novel strain to be most closely related to a reassortant EHDV-6 strain isolated from cattle in Trinidad and both strains received segment 4 from an Australian EHDV-2 strain. The novel U.S. EHDV-6 strain is unique in that it acquired segment 8 from an Australian EHDV-8 strain. An RNAscope® in situ hybridization assay was developed against the novel U.S. EHDV-6 strain and labeling was detected within lesions of the heart, kidney, liver, and lung. These data support the novel U.S. reassortant EHDV-6 strain as the cause of disease in the farmed WTD.

Development and evaluation of recombinase polymerase amplification combined with lateral flow dipstick assays for co-detection of epizootic haemorrhagic disease virus and the Palyam serogroup virus.

BACKGROUND: Epizootic haemorrhagic disease virus (EHDV) and the Palyam serogroup viruses (PALV) have led to significant economic losses associated with livestock production globally. A rapid, sensitive and specific method for the detection of EHDV and PALV is critical for virus detection, monitoring, and successful control and elimination of related diseases. RESULTS: In the present study, a recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) assay for the co-detection of genome segment 1 (Seg-1) of EHDV and PALV was developed and evaluated. The analytical sensitivities of the established RPA-LFD assay in the detection of EHDV and PALV were 7.1 copies/µL and 6.8 copies/µL, respectively. No cross-reaction with other members of the genus Orbivirus, including African horse sickness virus, bluetongue virus, Guangxi orbivirus, Tibet orbivirus and Yunnan orbivirus was observed. The established RPA-LFD assay accurately detected 39 EHDV strains belonging to 5 serotypes and 29 PALV strains belonging to 3 serotypes. The trace back results of quantitative real-time polymerase chain reaction (qRT-PCR) and the established RPA-LFD assay on sentinel cattle were consistent. The coincidence rates of qRT-PCR and the established RPA-LFD assay in 56 blood samples from which EHDV or PALV had been isolated and 96 blood samples collected from cattle farms were more than 94.8 %. The results demonstrated that the established RPR-LFD assay is specific, sensitive and reliable, and could be applied in early clinical diagnosis of EHDV and PALV. CONCLUSIONS: This study highlights the development and application of the RPA-LFD assay in the co-detection of EHDV and PALV for the first time. The assay could be used as a potential optional rapid, reliable, sensitive and low-cost method for field diagnosis of EHDV and PALV.

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.

Isolation of epizootic hemorrhagic disease virus serotype 7 from cattle showing fever in Japan in 2016 and improvement of a reverse transcription-polymerase chain reaction assay to detect epizootic hemorrhagic disease virus.

Epizootic hemorrhagic disease (EHD) is an arthropod-borne disease of wild and domestic ruminants caused by the EHD virus (EHDV). To date, seven EHDV serotypes have been identified. In Japan, strain Ibaraki of EHDV serotype 2 has caused outbreaks of Ibaraki disease in cattle. In addition, EHDV serotype 7 (EHDV-7) has caused large-scale EHD epizootics. In mid-September 2016, eight cattle at a breeding farm in Fukuoka Prefecture, Japan developed fever. Since EHDV-7 was detected in sentinel cattle in western Japan in 2016, we suspected that the cause of this fever might be an EHDV-7 infection. In this study, we tested cattle for EHDV-7 and some other viruses. Consequently, EHDV was isolated from washed blood cells collected from three of the eight cattle, and genetic analysis of genome segment 2 revealed that this isolate was EHDV-7. Moreover, all affected cattle tested positive for anti-EHDV-7 neutralizing antibodies. Our results suggest that the fever was caused by EHDV-7 infection. In addition, we modified a conventional reverse transcription polymerase chain reaction assay for the specific detection of EHDV. This modified assay could detect various strains of EHDV isolated in Japan, Australia, and North America. Furthermore, the assay permitted the detection of EHDV-7 in blood cells collected from seven of the eight cattle. We believe that this modified assay will be a useful tool for the diagnosis of EHD.

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.

Novel Serotype of Epizootic Hemorrhagic Disease Virus, China.

In 2018, a strain of epizootic hemorrhagic disease virus (EHDV), named YNDH/V079/2018, was isolated from a sentinel calf in Mangshi County, Yunnan Province, China. Nucleotide sequencing and neutralization tests indicated that the virus belongs to a novel serotype of EHDV that had not been reported previously.

Development and optimization of a DNA-based reverse genetics systems for epizootic hemorrhagic disease virus.

Epizootic hemorrhagic disease virus (EHDV) is a member of the genus Orbivirus, family Reoviridae, and has a genome consisting of 10 linear double-stranded (ds) RNA segments. The current reverse genetics system (RGS) for engineering the EHDV genome relies on the use of in vitro-synthesized capped viral RNA transcripts. To obtain more-efficient and simpler RGSs for EHDV, we developed an entirely DNA (plasmid or PCR amplicon)-based RGS for viral rescue. This RGS enabled the rescue of infectious EHDV from BSR-T7 cells following co-transfection with seven helper viral protein expression plasmids and 10 cDNA rescue plasmids or PCR amplicons representing the EHDV genome. Furthermore, we optimized the DNA-based systems and confirmed that some of the helper expression plasmids were not essential for the recovery of infectious EHDV. Thus, DNA-based RGSs may offer a more efficient method of recombinant virus recovery and accelerate the study of the biological characteristics of EHDV and the development of novel vaccines.

Identification and complete-genome phylogenetic analysis of an epizootic hemorrhagic disease virus serotype 7 strain isolated in China.

An epizootic hemorrhagic disease virus (EHDV) strain designated YN09-04 was isolated from sentinel cattle in China. The length of its complete genome was 19,344 bp in total, consisting of 10 segments ranging in size from 810 bp (S10) to 3942 bp (S1). Based on phylogenetic analysis of the S2 sequence, YN09-04 clusters with EHDV serotype 7 (EHDV-7) strains form a distinct, well-supported subgroup, indicating that YN09-04 belongs to EHDV-7. However, the origin of the YN09-04 genome is very complex. The S2 and S6 of YN09-04 cluster with those of Japanese EHDV-7 strains, whereas the S1, S3, S4, S5 and S7 of YN09-04 share high nucleotide sequence identity and a close relationship with those of Japanese Ibaraki viruses, and the S8, S9 and S10 nucleotide sequences of YN09-04 are more similar to those of some Australian EHDV strains than to those of other isolates. These results suggest that the genome of YN09-04 likely originated from a reassortment event between EHDV strains that were similar to the current Japanese and Australian strains and that YN09-04 and some EHDVs from Japan and Australia share the same ancestors. This is the first report of the isolation, identification and complete-genome phylogenetic analysis of an EHDV-7 strain from China.

Detection of a novel reassortant epizootic hemorrhagic disease virus serotype 6 in cattle in Trinidad, West Indies, containing nine RNA segments derived from exotic EHDV strains with an Australian origin.

Epizootic hemorrhagic disease virus (EHDV) is a Culicoides-transmitted orbivirus that infects domestic and wild ruminants in many parts of the world. Of the eight proposed serotypes, only EHDV-1, 2 and 6 have been reported to be present in the Americas. Following the identification of a virulent EHD-6 reasssortant virus in the USA in 2007 (EHDV-6 Indiana), with outer coat protein segments derived from an Australian strain of EHDV and all remaining segments derived from a locally circulating EHDV-2 strain, questions have remained about the origin of the Australian parent strain and how it may have arrived in the USA. When EHDV-6 was identified in asymptomatic cattle imported into the Caribbean island of Trinidad in 2013, full genome sequencing was carried out to further characterise the virus. The EHDV-6 Trinidad was a reassortant virus, with 8 of its 10 segments, being derived from the same exotic Australian EHDV-6 strain as the VP2 and VP5 present in the EHDV-6 Indiana strain from the USA. Analyses of the two remaining segments revealed that segment 8 showed the highest nucleotide identity (90.4%) with a USA New Jersey strain of EHDV-1, whereas segment 4 had the highest nucleotide identity (96.5%) with an Australian EHDV-2 strain. This data strongly suggests that the Trinidad EHDV-6 has an Australian origin, receiving its segment 4 from a reassortment event with an EHDV-2 also from Australia. This reassortant virus likely came to the Americas, where it received its segment 8 from a locally-circulating (as yet unknown) EHDV strain. This virus then may have gained entry into the USA, where it further reassorted with a known locally-circulating EHDV-2, the resulting strain being EHDV-6 Indiana. This study therefore identifies, for the first time, the likely minor parent virus of the EHDV-6 currently circulating in the USA.

Epidemiology of Bluetongue Virus and Epizootic Hemorrhagic Disease Virus in Beef Cattle on a Ranch in South-Central Florida.

Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) infect a variety of wild and domestic ruminant hosts in the United States, with outcomes ranging from subclinical infection to clinical disease resulting in mortality. Because cattle have been suggested as a temporary reservoir for both BTV and EHDV, ongoing national surveillance for these viruses may benefit from inclusion of domestic cattle as a supplement to current programs, such as surveillance of wild white-tailed deer. To better understand the prevalence of BTV and EHDV in cattle, we surveyed for viral RNA (vRNA) in the blood of 1,604 beef cattle on a south-central Florida cattle ranch over 3 years. While overall prevalence of vRNA in blood was low (<2% for either virus), the occurrence of vRNA was much higher in young animals: in 2016, 24% of animals 2 years old were positive by PCR for either BTV or EHDV. Our results suggest that cattle are a likely temporary reservoir for these viruses in Florida, and could provide additional information on the spatial distribution, viral diversity, and timing of emergence of these viruses, particularly if surveillance was restricted to cattle ≤2 years of age.