Phylogenetic analysis of Russian EIAV isolates using an optimized nested PCR

Equine infectious anemia (EIA) is a viral disease wich affects all members of Equine species and is caused by а virus belonging to the genus Lentivirus in the family Retroviridae. This disease has a worldwide distribution, and its epizooty is registered in all continents. AGID positively reacting animals are revealed at planned serological studies in the Russian Federation from year to year. At the moment, a lot of attention is paid to control of the EIA virus prevalence, therefore, all over the world there are numerous studies to identify the virus and determine its molecular and genetic characteristics and origin. This paper presents data on the development of test systems based on PCR, which allows not only to identify the EIA viral genome in various samples of biological material, but also to carry out further sequencing the genome fragments of different EIA strains and isolates. EIA virus genome consists of three major genes, gag, pol, env (5'→3'). Аnd the most useful marker for studying genetic diversity between different EIAV strains is a gag-gene necessary for intracellular virus assembly and release from cells. For carrying out the molecular genetic studies, we optimized nested PCR without reverse transcription and designed specific oligonucleotide primers for the second round of the reaction which flanking 1018 bp of proviral DNA of gag-gene. In the research we also determined the nucleotide sequence and phylogenetic relationships between previously isolated Russian EIAV strain and two modern isolates revealed in Russia. The findings allowed assigning 3-K-VNIITIBP-VIEV EIAV strain to group of North American origin with homology of 98 %. Isolates Nizhniy Novgorod-2011 and Omsk-2012 were classified as isolates of European origin with 82-83 % homology. Thus, application of PCR and a phylogenetic analysis allows to establish the belonging of new virus isolates to a certain genogroups and to assume their geographical origin. This information is necessary to determine the source and ways of distribution of the pathogen for the purpose to predict future epidemic situation and planning measures against the disease.