Identification of a Novel α-herpesvirus Associated with Ulcerative Stomatitis in Donkeys

An outbreak of ulcerative stomatitis was observed in a donkey (Equus asinus) dairy herd. Similar lesions were also observed on the dams’ udders and, sporadically, in genital areas. The lesions typically resolved in 1–3 weeks. An α-herpesvirus, Varicellovirus, genetically related to equid herpesvirus type 3, was identified.

areas. The lesions typically resolved in 1-3 weeks. The herd owner reported weight loss in foals and interruption of lactation in dams. Two weeks after onset in the original group, the disease was observed in a separate group of animals, comprising 63 adult or yearling females and 5 yearling males. In this group, however, oral lesions were observed only in 5 yearlings and 1 mare. A third group of 4 adult males was kept apart from the other animals and was not affected by the disease.
Oral swab and serum samples collected from 8 animals with acute disease were sent to the laboratories at the University of Bari (Valenzano, Italy) for virologic investigation. Using an electron microscope, we observed herpesvirus-like particles in the oral swabs and detected herpesvirus DNA using consensus herpesvirus primers for the DNA polymerase and inverse terminase (2,3). We used BLASTn (https:// blast.ncbi.nlm.nih.gov) to search the GenBank genetic sequence database and found the virus to be highly related to equid herpesvirus (EHV) 3 in the DNA polymerase (93.35% nt identity) and terminase (90.71% nt identity) regions.
We isolated the virus onto equine dermal cells from oral swab specimens. The virus was titrated and used for screening serum samples collected from the donkeys in virus neutralization assays. We detected specific neutralizing antibodies in the serum samples collected from approximately three quarters (80/106) of the animals 2 months after the beginning of the outbreak but not in the serum samples of 8 animals with acute infection, suggesting seroconversion.
To sequence the DNA of the isolate, we performed next generation sequencing using the Illumina MiSeq platform (https://www.illumina.com) and used Nextera XT (Illumina) for library preparation. We obtained the full genome sequence (147,607 bp) of asinine herpesvirus (AsHV) strain AsHV/ Bari/2011/740 and annotated it using the software ORF Finder (https://www.bioinformatics.org).  (4). We performed phylogenetic analyses with MEGAX software (https://www.megasoftware.net) (5) using the  maximum-likelihood method with the general timereversible model, a proportion of invariant sites, and a discrete gamma distribution (5 categories) to model evolutionary rate differences among sites, and bootstrap analyses with 1,000 pseudoreplicate datasets. In the consensus phylogenetic trees (Figure 2), strain AsHV/Bari/2011/740 was closely related to EHV-3 sequences and distantly related to other members of the genus Varicellovirus. We deposited the full-genome sequence of strain AsHV/Bari/2011/740 in the GenBank database (accession no. MT012704).
We report the detection and isolation of a novel AsHV from an outbreak of vesicular and ulcerative stomatitis and mammillitis in a donkey dairy herd. By comparing it with other herpesvirus sequences from the databases, we identified 3 targets (glycoproteins B, C, and D) for which the sequences were available across all the varicelloviruses listed in the ICTV database and that were used for phylogenetic analysis. In these analyses, the AsHV strain appeared similar to EHV-3. By reviewing the literature, we found another donkey herpesvirus, AsHV-1, genetically related to EHV-3 on the basis of restriction enzyme and hybridization analyses (6). AsHV-1 was originally isolated from the vesicular and erosive lesions of the muzzle of a foal and the external genitalia and udder of its dam (12). Unfortunately, the prototype AsHV-1 is no longer available and it is not possible to determine its genome sequence for precise comparison (G.F. Browning, pers. comm.). It is possible that AsHV/ Bari/2011 is actually an AsHV-1 strain, although this possibility cannot be confirmed.
Overall, based on the chronology of the health events observed in the herd, the tendency of herpesviruses to reactivate from latent infection under stress conditions, and the seroconversion observed in the monitored animals, we hypothesized that the newly introduced mare was the vehicle for herpesvirus infection in the herd, although this possibility could not be conclusively confirmed. Also, we screened only 8 animals during the acute phase of the disease, and we do not have an exact picture of the immunological status of the animals before the onset of the disease.
In conclusion, we identified a novel AsHV, genetically related to EHV-3, from an outbreak of infectious ulcerative stomatitis in donkey foals. These findings extend the spectrum of pathologies potentially attributable to herpesviruses in donkeys. Considering the nature and shape of the lesions, the virus should be included in the differential diagnosis of vesicular and ulcerative stomatitis among equids. Also, it needs to be determined whether the novel AsHV can be transmitted to horses.
is focused on diagnosis, epidemiology, and characterization of human and animal viruses, with particular interest for zoonotic viruses.