Equine herpesviruses 8 and 9: two emerging respiratory viruses in equids.

International audiencePurpose of the study: In equids, nine herpesviruses have been described. They belong to the subfamilies Alphaherpesvirinae (EHV-1/3/4/6/8 and 9) and Gammaherpesvirinae (EHV-2/5 and 7) and are able to infect equids in the wild or in captivity. Equine herpesvirus 1 (EHV-1) is by far the most dangerous, causing the most serious forms of disease in horses (respiratory infection, abortion and myeloencephalopathy). While most of these herpesviruses have been known for many years, two of them (EHV-8 and EHV-9) have recently been identified as a potential risk to equine health. Equine herpesvirus type 8 (EHV-8), formerly known as Asinine herpesvirus type 3 (AHV-3), is primarily isolated in donkeys but has recently been associated with abortion in horses. The equine herpesvirus type 9 (EHV-9), initially described in a Thomson’s gazelle, is closed to EHV-1 and appears to have crossed the species with several infection cases described in zebras but also in onagers, rhinoceros and other animal species kept in captivity. This report describes the first isolation in France of an EHV-9 strain from a captive Grevy’s zebras and the prevalence of EHV-8 in French horse and donkey population.Methods used: Sampling of zebra lungs were performed post-mortem, with the agreement of the ZooParc de Beauval veterinarians. Biological samples from horses and donkeys (185 samples collected between 2005 and 2019 from EHV-induced abortion cases or respiratory diseases) were provided by LABÉO (samples initially tested for diagnostic purposes with the agreement of the owners and the veterinarian in charge).The DNA was extracted using the DNA Mini Kit (Qiagen) for tissues and Viral RNA Mini Kit (Qiagen) for respiratory fluids. Nucleic acids were amplified as previously described by Diallo et al. (2006) for the detection of EHV-1 glycoprotein B (PCR1) and van Devanter et al. (1996) for the detection of mammalian herpesvirus (PCR2). PCR was also performed on ORF30 for phylogeny (PCR3). The analysis was conducted using the Neighbor-Joining method, Maximum Composite Likelihood model. Virus isolation and culture was performed on rabbit kidney cell line (RK13).Summary of results: A mammalian herpesvirus was detected in the zebra lung biopsy (PCR2). Sequencing revealed a 99% homology with several EHV-9 referenced strains. This result was confirmed by the complete sequencing of ORF30 (PCR3). An EHV-9 strain was isolated from this sample on RK13, with a characteristic cytopathic effect observed in culture. ORF30 sequencing confirmed the isolation and culture of EHV-9. All samples from horses and donkeys were analysed by PCR1. 164 were confirmed to contain EHV-1, and 6 samples were associated with EHV-8 (all from donkeys). Further analysis showed that this sample exhibited 99% ORF 30 sequence homology with the EHV-8 strain MF431611.1 strain recently described by Garvey et al. 2018 and isolated from a fetus after abortion. The phylogenetic study of all these sequences (EHV-1, EHV-8 and EHV-9) showed a strong proximity of EHV-9 and EHV-8 with EHV-1.Conclusions: These results are the first confirmation of EHV-8 and EHV-9 in France. Their strong genetic proximity to EHV-1 and the possibility to cross the species barriers warrant future studies. The isolation of these two viruses will allow studying their mechanisms of pathogenicity and the efficacy of several antiviral molecules, which could be a therapeutic option in the context of safeguarding endangered species in captivity, such as the Grevy’s zebra (endangered species according to the IUCN classification)