Detection and molecular characterization of equine herpesviruses 1, 2, and 5 in horses in the Republic of Serbia

The presence of equine herpesviruses 1, 2 and 5 (EHV-1, EHV-2 and EHV-5) was examined in 66 samples of spinal cord, submandibular lymph nodes and spleen of healthy, non-vaccinated abattoir horses from different locations in the Republic of Serbia. Virus isolation was conducted on RK-13 cell line with the confirmation of isolated viral strains by multiplex nested polymerase chain reaction. The cytopathic effect was observed 48-72 h after the first inoculation in 28 (42.4%) organ samples, and after 5 days in 11 other samples (16.7%) that were all confirmed as EHV-1. Four other samples (6.1%) that showed cytopathic effects on day 5 of the third passage were all positive for EHV-5. Additionally, EHV-1, EHV-2, and EHV-5 were directly detected in all organs by multiplex nested PCR in 46 (69.7%), 3 (4.5%), and 7 (10.6%) samples, respectively. The molecular characterization based on nucleotide sequencing of the part of the gB gene showed that Serbian EHV-1 isolates were 100% homogenous and clustered with EHV-1 strains from Turkey, the United Kingdom, the United States, and Japan. The EHV-2 strain from Serbia branched together with Turkish EHV-2 isolates with homogeneity from 96% to 98%. Serbian EHV-5 strains can be separated in one distinct cluster with isolates from Turkey and the United States with homogeneity from 98 to 99%. These data represent the first report of the molecular characterization of EHV-1, EHV-2, and EHV-5 in the horse population of the Republic of Serbia and document the first successful isolation of Serbian EHV-5 strains.