Epizootic Hemorrhagic Disease Research Articles

Seven-year prospective study on yearly incidence of Orbivirus infection of captive white-tailed deer and potential Culicoides vectors.

Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) are arthropod-borne viruses that are transmitted by biting midges in the genus Culicoides (Diptera: Ceratopogonidae) and can cause hemorrhagic disease in certain ruminants. The objectives of this study were to measure the incidence of BTV and EHDV infections in captive white-tailed deer herd as well as tissues and corresponding presence of Culicoides midges at a location near Clinton, LA. During a 7-yr study with yearly outbreaks of hemorrhagic disease in the deer herd, 15 species of Culicoides were captured using Centers for Disease Control (CDC) black light traps. Reverse transcriptase quantitative polymerase chain reaction (PCR) was performed to screen for BTV and EHDV in pools of midges and tissues of deer. From 2012 to 2018, 1,711 pools of midges representing 24,859 specimens were tested, and specimens from 5 of the 15 collected species (Culicoides debilipalpis, Culicoides stellifer, Culicoides venustus, Culicoides haematopotus, and Culicoides crepuscularis) were found to be PCR positive for BTV and EHDV. Most of the BTV-positive pools of biting midges were from specimens of C. debilipalpis and C. stellifer, and most of the EHDV-positive pools were from specimens of C. venustus and C. stellifer. During the 7-yr period, 112 white-tailed deer that died at the study location were PCR positive for BTV or EHDV: detected BTV serotypes were 10 and 12 and EHDV serotypes were 1, 2, and 6. There was a significant increase in BTV/EHDV antibody prevalence in white-tailed deer during the study; antibody-positive rates increased from 15% to 78% in the deer herd of approximately 100 animals.

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.

Viral diversity and blood-feeding patterns of Afrotropical Culicoides biting midges (Diptera: Ceratopogonidae).

Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of arboviral pathogens that primarily affect livestock represented by Schmallenberg virus (SBV), epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV). In Kenya, studies examining the bionomic features of Culicoides including species diversity, blood-feeding habits, and association with viruses are limited. Adult Culicoides were surveyed using CDC light traps in two semi-arid ecologies, Baringo and Kajiado counties, in Kenya. Blood-fed specimens were analysed through polymerase chain reaction (PCR) and sequencing of cytochrome oxidase subunit 1 (cox1) barcoding region. Culicoides pools were screened for virus infection by generic RT-PCR and next-generation sequencing (NGS). Analysis of blood-fed specimens confirmed that midges had fed on cattle, goats, sheep, zebra, and birds. Cox1 barcoding of the sampled specimens revealed the presence of known vectors of BTV and epizootic hemorrhagic disease virus (EHDV) including species in the Imicola group (Culicoides imicola) and Schultzei group (C. enderleni, C. kingi, and C. chultzei). Culicoides leucostictus and a cryptic species distantly related to the Imicola group were also identified. Screening of generated pools (11,006 individuals assigned to 333 pools) by generic RT-PCR revealed presence of seven phylogenetically distinct viruses grouping in the genera Goukovirus, Pacuvirus and Orthobunyavirus. The viruses showed an overall minimum infection rate (MIR) of 7.0% (66/333, 95% confidence interval (CI) 5.5-8.9). In addition, full coding sequences of two new iflaviruses, tentatively named Oloisinyai_1 and Oloisinyai_2, were generated by next-generation sequencing (NGS) from individual homogenate of Culicoides pool. The results indicate a high genetic diversity of viruses in Kenyan biting midges. Further insights into host-vector-virus interactions as well as investigations on the potential clinical significance of the detected viruses are warranted.

IFNAR(-/-) Mice Constitute a Suitable Animal Model for Epizootic Hemorrhagic Disease Virus Study and Vaccine Evaluation.

Epizootic hemorrhagic disease (EHD), caused by Epizootic hemorrhagic disease virus (EHDV), is an emerging and severe livestock disease. Recent incursion and distribution of EHDV in Europe have outlined the emerging character of EHD. Despite its worldwide impact, numerous knowledge gaps exist. A range of inconveniences restricts utilization of natural hosts of EHDV. Here, we show that adult mice deficient in type I IFN receptor (IFNAR(-/-)) are highly susceptible to EHDV-6 and EHDV-8 infection when the virus is administered subcutaneously. Disease was characterized by ruffled hair, reluctance to move, dehydration and conjunctivitis, with viraemia detected from day 5 post-infection. A deeper characterization of EHDV-8 infection showed viral replication in the lung, liver, spleen, kidney, testis and ovaries. Importantly, increased expression levels of pro-inflammatory cytokines IL-1β, IL-6 and CXCL2 were observed in spleen after EHDV-8 infection. Furthermore, IFNAR(-/-) adult mice immunized with a EHDV-8 inactivated vaccine elicited neutralizing antibodies specific of EHDV-8 and full protection against challenge with a lethal dose of this virus. This study also explores the possibilities of this animal model for study of BTV and EHDV coinfection. In summary, the IFNAR(-/-) mouse model faithfully recapitulates EHD and can be applied for vaccine testing, which can facilitate progress in addressing the animal health challenge posed by this virus.

Real-Time RT-PCR for the Diagnosis of Epizootic Hemorrhagic Disease Virus.

Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) has become an essential tool in rapid and reliable detection of animal diseases such as epizootic hemorrhagic disease (EHD). Here we provide a protocol for the detection of epizootic hemorrhagic disease virus (EHDV) genetic material in blood and tissue samples, using a real-time RT-PCR that targets a conserved region in segment 9 of the EHDV genome. This protocol can be used to detect up to approximately 90 samples in a single run and can be completed in less than 4h.

Managing Risk When Working with Orbiviruses with a Special Emphasis on Epizootic Hemorrhagic Disease Virus.

Risk assessment is the cornerstone of working safely with biological agents. The World Health Organization (WHO) Laboratory Biosafety Manual Fourth Edition Monograph on Risk Assessment provides stepwise guidance for completing a risk assessment, from information gathering and identifying hazards to evaluating the risks, developing, and implementing controls and review.To support the development of a mature safety culture within laboratories, it is important that all staff who handle biological agents understand the fundamentals of risk assessment and receive training in identifying hazards created by their work activities (or tasks) and understand how to mitigate the risks arising from carrying out that work. Any "competent" person may be involved in assessing the risks posed by carrying out an activity. Those closest to the work, who understand the details of the task being undertaken, should be involved in creating the risk assessment. The guidance in this chapter is not just applicable to biosafety professionals, laboratory scientists, or facility managers but can be used by any competent worker familiar with the activity being assessed.This chapter uses the guidance from the WHO to apply the principles of risk assessment to working with Epizootic hemorrhagic disease virus (EHDV), using an example activity-virus isolation from EHDV test samples in cell culture.

Virus Neutralization Test for Detecting and Quantifying Serum-Neutralizing Antibodies to Epizootic Hemorrhagic Disease Virus (EHDV) (Serotypes 1, 2, and 4-8).

The virus neutralization test (VNT) is a functional immunoassay which detects the presence and quantity of neutralizing antibodies. It is a highly sensitive and specific test. As with most neutralization assays, the EHDV VNT does not react with all virus-targeting antibodies, but specifically with those antibodies that bind to VP2, the outermost capsid structural protein of the virus. The interaction between VP2 and neutralizing antibodies can block EHDV cell binding, neutralizing its infectivity. The detection and quantification of neutralizing antibodies are indicative of how protected an animal is against reinfection. The EHD VNT can therefore be a useful tool to monitor the efficacy of a vaccination campaign. VP2 is also the main determinant of EHDV serotype specificity, and so EHDV-neutralizing antibodies which target VP2 are also serotype-specific. Throughdetecting and quantifying neutralizing antibodies, the VNT can discriminate the EHDV serotype responsible for an infection and provides insights into the time of infection. It is considered the gold standard test for identifying and quantifying antibodies against EHDV serotypes present in test serum samples. The assay is performed in vitro and is based on inhibition of virus infectivity in the presence of neutralizing antibodies. A neutralizing antibody titer is determined through the presence or absence of cytopathic effect in a cell monolayer. The VNT is a relatively inexpensive assay using standard laboratory equipment; however, to perform the assay, cell cultures, significant time, intensive labor, and technical skill are required.

Molecular Serotyping of Epizootic Hemorrhagic Disease Virus.

Molecular methods are routinely used for the differential diagnosis and genetic characterization of viral disease of livestock. Real-time, quantitative PCR (qPCR) allows RNA/DNA sequence detection and quantification and is considered the gold standard diagnostic method for most viruses. However, Sanger sequencing offers additional information and opportunity to differentiate closely related virus strains and/or serotypes, by providing the full sequence of a genetic region of interest. Therefore, to determine epizootic hemorrhagic disease virus (EHDV) serotype or identify additional genetic markers, end-point RT-PCR can be performed on EHDV-positive clinical samples, followed by Sanger sequencing and data analysis. Here we describe a detailed method for the molecular characterization of EHDV serotype using Sanger sequencing.

Virus Isolation of Epizootic Hemorrhagic Disease Virus in Cell Culture.

Virus isolation is used to assist in the diagnosis and confirmation of viral infections. Successful isolation of a virus is highly dependent upon the quality of starting material. Here we describe the preparation and isolation of epizootic hemorrhagic disease virus (EHDV) from blood and tissue samples in tissue culture flasks (TCFs) through the inoculation of susceptible cell lines including Vero, BHK, and KC cells.

Real-Time RT-PCR Assays for Typing of Epizootic Hemorrhagic Disease Virus.

Real-time RT-PCR for the detection of epizootic hemorrhagic disease virus (EHDV) in clinical samples is a fast and sensitive tool for the diagnosis and confirmation of disease. Several real-time RT-PCR methods have been reported over the last 10years. In this chapter, we describe seven duplex real-time RT-PCR assays to amplify part of genome segment 2 of EHDV to enable serotype identification. The assay includes the detection of an endogenous control gene-beta-actin.

Detection of EHDV Antibodies Using Enzyme-Linked Immunosorbent Assay (ELISA).

Enzyme-linked immunosorbent assay (ELISA) is a relatively inexpensive, rapid, and high-throughput diagnostic tool to detect antibodies raised against epizootic hemorrhagic disease virus (EHDV) in ruminant serum. While the presence of EHDV antibodies only confirms prior exposure to the virus, it does not conclusively determine infection status. The c-ELISA can be used in conjunction with other diagnostic tests (e.g., real-time PCR) to reinforce diagnosis of infection or as a surveillance tool to support disease control. The EHDV competition ELISA (c-ELISA) described here is a commercial diagnostic assay, recommended by the World Organisation for Animal Health (WOAH), that detects ruminant antibodies against the highly conserved EHDV structural protein, VP7.

Infection of Culicoides Biting Midges with Epizootic Hemorrhagic Disease Virus.

Epizootic hemorrhagic disease virus (EHDV) is transmitted by Culicoides biting midges. Studies aiming to predict the likely spread of EHDV require an understanding of the viral infection and replication kinetics within these insects, including the proportion of the insect population that are able to support virus transmission. Here, we describe methods for the infection of Culicoides with EHDV in the laboratory via oral infection using an artificial membrane system or a cotton pledget and intrathoracic (IT) inoculation. Each method can be used to explore determinants of vector competence of Culicoides species and populations for EHDV.

Recognition of Field Signs, Necropsy Procedures, and Evaluation of Macroscopic Lesions of Cervids Infected with Epizootic Hemorrhagic Disease Virus.

Epizootic hemorrhagic disease virus (EHDV) is an arthropod-borne RNA virus in the genus Orbivirus, family Sedoreoviridae. Globally, seven known EHDV serotypes circulate among ruminant hosts and Culicoides species vectors. A variety of domestic and wild ruminant species are susceptible to EHDV infection, but infection outcome is highly variable between species, as well as between individuals of the same species. Thus, this disease system inherently operates at the wildlife-livestock interface. Domestic cattle are important hosts for EHDV, and while inapparent infection is the most common outcome, reports of clinical disease have increased in some parts of the world. However, fatal infection of cattle is rare. Among wildlife, white-tailed deer (Odocoileus virginianus) are highly susceptible to severe and often fatal disease. Considering the paucity of data and poorly characterized pathology of EHD in cattle, white-tailed deer represent a case study for describing the field signs and necropsy lesions associated with EHD. Here we describe the field signs that commonly define EHD outbreaks in North America, a basic approach to a gross necropsy examination of white-tailed deer, description of the gross lesions that may be present, and diagnostic sample collection. Field investigations of large-scale EHD outbreaks are common in North America. The necropsy examination is an essential tool in the study of disease and when coupled with other disciplines (e.g., virology, immunology, epidemiology) has been fundamentally important to understanding EHD in North America.

Rapid Laboratory Diagnosis of Epizootic Hemorrhagic Disease Virus Using a Loop-Mediated Isothermal Amplification Assay.

Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) is a molecular diagnostic assay that is particularly useful for the detection of viral diseases of livestock. A major advantage of RT-LAMP is that it can be used either as a rapid field test or as a high-throughput screening tool in veterinary laboratories, with sensitivity comparable to the real-time RT-PCR assay. Unlike conventional or qPCR, RT-LAMP uses a strand displacement polymerase and a set of four to six primers that bind to several regions of the target nucleic acid. Amplification occurs without thermal cycling, and coupled with the numerous primers, RT-LAMP offers a rapid and highly specific molecular assay. In this chapter, we describe the RT-LAMP protocol for the detection of epizootic hemorrhagic disease virus (EHDV) as a low-cost, specific, and sensitive screening tool in veterinary diagnostic laboratories. We also provide guidance on how to adapt the RT-LAMP assay for rapid field testing.

Purification of Epizootic Hemorrhagic Disease Virus (and Other Orbiviruses) Particles from Infected Mammalian or Insect Cells.

Epizootic hemorrhagic disease virus (EHDV), like other orbiviruses, infects and replicates in mammalian and insect vector cells. Within its ruminant hosts EHDV, like bluetongue virus (BTV), it has mainly been associated with infection of endothelial cells of capillaries as well as leukocyte subsets. Furthermore, EHDV infects and replicates within its biological vector, Culicoides biting midges and Culicoides-derived cells. A wide range of common laboratory cell lines such as BHK, BSR, and Vero cells are susceptible to infection with certain EHDV strains. Cell culture supernatants of infected cells are commonly used for both in vivo and in vitro infection studies. For specific virological or immunological studies, using highly purified virus particles, however, might be beneficial or even required. Here we describe a purification method for EHDV particles, which had been originally developed for certain strains of BTV.

Epizootic Hemorrhagic Disease Virus Titration.

The titration of viruses onto susceptible cell lines is an important virological technique used to quantify infectious viral titers. It forms an integral component of epizootic hemorrhagic disease virus (EHDV) research, including estimating infectivity, calculating multiplicity of infection, and confirming virus propagation in cell culture. However, the ability to quantify infectious EHDV is also critical for disease control, particularly in the event of an outbreak. Routine EHD diagnostics do not accurately quantify infectious virus, which would allow accurate prediction of the onward transmission risk, but instead are typically more qualitative in nature (e.g., virus isolation) or only quantify viral genome copies (e.g., real-time PCR) which often remain detectable long after infectious virus is cleared from the host.Infectious EHDV titers are typically quantified through the detection of visible cytopathic effect (CPE) in the monolayer of susceptible mammalian cell cultures. However, not all susceptible cell lines demonstrate visible CPE upon EHDV infection, including cell lines such as KC cells, which are derived from the EHDV biological insect vector, Culicoides sonorensis. This chapter presents a comprehensive method for the titration of EHDV-positive samples onto relevant, susceptible mammalian (Vero) and insect (KC) cell lines and describes alternative methods that can be used to visualize EHDV infection, by CPE or immunofluorescent labeling of viral proteins, to enable the calculation of infectious EHDV titers.

Whole Genome Sequencing of Epizootic Hemorrhagic Disease Virus.

Next-generation sequencing (NGS) technologies are continuously being developed and are becoming a more cost-effective tool for the characterization of viral genomes. Whole genome sequencing of segmented viruses, such as epizootic hemorrhagic disease virus (EHDV), provides insights into the molecular epidemiology as well as such viral evolutionary mechanisms as genetic reassortment. Here, we present a detailed method for obtaining full genome sequence data for EHDV using Illumina technology. The protocol includes details from RNA extraction and purification, the synthesis of cDNA, sequencing library preparation, to genome assembly.

Experimental infection of cattle, sheep, and goats with the newly emerged epizootic hemorrhagic disease virus serotype 8.

Epizootic hemorrhagic disease virus serotype 8 (EHDV-8) emerged in Europe for the first time in late 2022. In this study, we investigated the kinetics of EHDV-8 infection in cattle, sheep, and goats. Following experimental infection with EHDV-8, four out of five calves displayed fever, while another calf exhibited ulcerative and crusty lesions of the muzzle. RNAemia peaked at day 7 post infection in all calves and remained relatively stable till the end of the study, at 78 days post infection. Infectious virus was isolated up to 21 days post infection in one calf. As far as small ruminants are concerned, one sheep experienced fever and two out of five had consistent RNAemia that lasted until the end of the study. Remarkably, infectious virus was evidenced at day 7 post infection in one sheep. In goats, no RNA was observed. All infected animals seroconverted, and a neutralizing immune response was observed in all species, with calves exhibiting a more robust response than sheep and goats. Our study provides insights into the kinetics of EHDV-8 infection and the host immune responses. We also highlight that sheep may also play a role in EHDV-8 epidemiology. Altogether, the data gathered in this study could have important implications for disease control and prevention strategies, providing crucial information to policy makers to mitigate the impact of this viral disease on livestock.

From forests to fields: investigating Culicoides (Diptera: Ceratopogonidae) abundance and diversity in cattle pastures and adjacent woodlands.

Culicoides Latreille (Diptera: Ceratopogonidae) biting midges are hematophagous flies that feed on wild and domestic ruminants. They can transmit arboviruses, such as bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV), which circulate in the United States. Larvae occupy a range of aquatic and semiaquatic habitats, and disperse short distances from their development sites. In the southeastern United States, there are limited studies on the abundance and diversity of Culicoides in wooded and adjacent livestock pasture habitats. In this study, we characterized Culicoides diversity and abundance within these distinct habitat types. BG-Sentinel and CDC miniature suction traps baited with CO2 or UV-light were placed in wooded and pasture habitats at 2 locations on a university beef farm in Savoy, Arkansas. Traps were set once per week for 9 wk during August-October of 2021 and 2022. Fifteen species were collected during this study, and the 2 most abundant species were Culicoides haematopotus Malloch and Culicoides stellifer Coquillett. There was a significant effect of site and location on C. haematopotus collections, and a significant effect and interaction of site and trap on C. stellifer collections. In the woods, significantly more C. stellifer were collected from CDC-UV traps, while in the pasture significantly more were collected in CDC-CO2 traps. These data suggest that C. stellifer, a putative vector of BTV and EHDV in the southeast, may be traveling into the pasture to host-seek, while C. haematopotus remains primarily in wooded areas. This study reveals community differences between these habitat types and implications for Culicoides control.

A thorny taxonomic issue of Quaternary deer (Cervidae: Mammalia) from the South American Highlands resolved based on the recognition of a paleopathology

ABSTRACTThe diversity of South American deer genera during the Quaternary is considered one of the greatest in the world. However, this was established during the beginning of the twentieth century, when the recognition of new extinct deer taxa was based only on differences in antler morphology. Thus, South American extinct deer taxa need a detailed taxonomic revision, and this is the case of the Andean deer genera Agalmaceros and Charitoceros. First, both taxa are diagnosed by the presence of thorns in their antlers. Consequently, by taxonomic priority, Agalmaceros is a senior synonym of Charitoceros. Second, we recognize here that the thorns of the antlers of Agalmaceros are the symptom of a pathology that also affects some extant deer. Excluding the thorns of the antlers, the antler of Agalmaceros is identical to that of Odocoileus virginianus, indicating that Agalmaceros is a junior synonym to this extant deer. The recognition of epizootic hemorrhagic disease as the possible cause for the thorny antlers of Andean O. virginianus and the ample record of this affecting antlers in deer from the tropical Andes suggests that an epidemic occurred in this region in the Late Pleistocene. Before describing a new taxon, taxonomists must exhaust all possibilities to explain morphological variations. In this regard, this contribution provides an interesting, important and weakly explored area in paleontology, namely taxonomic revision under a paleopathology context. With the taxonomic invalidation of Agalmaceros and Charitoceros proposed here, the diversity of South American deer genera during the Pleistocene is poorer than previously established and includes 11 taxa – seven extant and four extinct.