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.

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.

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.

The emergence of EHDV in Europe during the autumn of 2022 reinforces the need for molecular tools (RT-PCR) for rapid detection of animals infected with this virus. Viral genome testing can be performed on whole blood under anticoagulant, spleen, and bloody organ homogenates from ruminants. It can also be performed on cell culture following viral isolation tests. Various so-called classical or end-point RT-PCRs will be described, which permit the amplification of a part of the viral genome (targeting segment 7) allowing the detection of EHDV whatever the serotype (pan-RT-PCR) and also to amplify a portion of the gene coding the viral protein (VP) 2 enabling serotyping. The PCR amplification products are visualized by agarose gel electrophoresis. Sequencing of the type-specific RT-PCR amplification products allows for the serotype of the virus to be determined.

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.

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 10 years. 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.

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 4 h.

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.

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.

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.