Main topic : Surveillance and control of emerging diseases
A new pan RT-PCR for the detection of Epizootic Haemorrhagic Disease Virus in cattle and small ruminants
DESPOIS L. 1, SAVARIT L. 1, BIANCHINI E. 1, LIMOZIN A. 1, COMTET L. 1, POURQUIER P. 1
1 Innovative Diagnostics, Grabels, France
Background and Objectives.
Epizootic hemorrhagic disease (EHD) is a Culicoides-borne viral disease caused by the
EHD virus (EHDV), associated with clinical manifestations in domestic and wild ruminants, primarily white-tailed deer and cattle. Along with BlueTongue Virus (BTV), EHDV is classified within the genus Orbivirus. EHDV has a 10-segmented RNA genome encoding for seven structural proteins (VP1-VP7) and five nonstructural proteins (NS1-NS4, NS3a).
In late September 2021, EHDV was reported in cattle farms in central/western Tunisia. It rapidly spread throughout the country with more than 200 confirmed outbreaks. For the first time in 2022, EHDV was detected in continental Europe.
Innovative Diagnostics has developed a qualitative duplex real-time PCR (RT-PCR) for a
pan EHDV detection. The test can be used on whole blood, spleen or organs from bovine or small ruminants species. It simultaneously amplifies a specific target from the EHDV genome as well as an endogenous non-target positive (ruminant housekeeping gene).
Material and Methods.
After RNA denaturation (heating at 95°C for 3 minutes), 5 μl of denatured RNA was added to the ready-to-use mastermix and the amplifications were carried out using a rapid amplification program (65 min).
Specific synthetic RNA was designed to estimate the limit of detection of PCR (LDPCR) and to determine the RT-PCR efficiency. RNAs panels (provided by ANSES, Maisons-Alfort, France) from the 7 EHDV known serotypes, and 27 BTV serotypes or from other pathogens affecting cattle were tested to assess test inclusivity and exclusivity, respectively. Samples were also tested in parallel on a commercially available PCR kit.
The LDPCR (95%) was estimated around 10 copies/PCR and PCR efficiency was determined above 98% with the synthetic RNA. The analytical specificity of RT-PCR assay was evaluated in silico by aligning the target PCR systems with the databases available on NCBI (National Center for Biotechnology Information). The alignments show 100% in silico specificity for the EHDV targeted region, and no homology with other pathogens.
This was confirmed experimentally, as the panEHDV RT-PCR successfully detected all 7 EHDV serotypes, including Tunisia-2021 strains which emerged in Europe, without showing any cross-reactions with neither BTV nor other pathogens.
Compared to a commercially available ELISA, IDvet’s RT-PCR gave earlier Cq values with a mean difference gain of 2,8 Cq.
Along with the ID Screen® EHDV Competition ELISA, which is available for specific EHDV antibody detection, the new panEHDV RT-PCR allows for rapid and accurate detection of EHDV, regardless of the serotype.