Materials and Methods
In October of 2019, at the Sector Agua Buena Chica, Bulnes commune,
region of Ñuble, central Chile (Latitude: -36.7333 Longitude: -72.3),
four samples (one from a Belgian Ardennes Breeder and three from Mixed
Shooting) were obtained from three females and one male that presented
clinical signology in the genital area compatible with EHV-3 infection.
The samples were taken with a sterile swab from papular or crustal
lesions of the vulvar or prepucial area (Fig. 1a and 1b), and then
transported to -4ºC and stored in the Virology Laboratory of the Faculty
of Veterinary Sciences of the University de Concepción, Chillán Campus.
DNA extraction was carried out using the Dneasy Blood & Tissue kit
(Quiagen, Cat. 69506) following the manufacturer’s specifications, and
the samples were stored at -20 °C until use. To detect EHV-3 we
implemented a conventional PCR with primers targeting a conserved
fragment (520 bp) of the glycoprotein G (gG ) gene (Dynon et al.,
2001), which is homologous in most alpha-herpesviruses sequenced to date
(Hartley et al., 1999).
Positive samples were sequenced in both directions, and then compared
with the GenBank database using BLASTn
(www.ncbi.nlm.nih.gov/BLAST).
The sequences were edited using BioEdit (Hall, 1999), with manual
edition when needed, and aligned with the Clustal W algorithm (Thompson
et al., 1994). Phylogenetic relationships were evaluated with the
Bayesian method with MrBayes v3.1.2 (Huelsenbeck and Ronquist, 2001)
using the General Time Reversible model, with 1,000,000 generations.
Each tree was sampled every 100 generations, beginning with random seeds
and ran four times. The first 25% of the trees was considered burn-in,
and the remaining trees used to calculate Bayesian posterior
probabilities. A sequence of Feline herpesvirus 1 (KR381787) was
employed as out group (Fig. 2).