Molecular Characterization of the Major Open Reading Frames (ORFs) and Enhancer Elements From Four Geographically...

Although the equine lentivirus (equine infectious anemia virus [EIAV]) poses a major threat to equid populations throughout most regions of the world, detailed knowledge concerning its molecular epidemiology is still in its infancy. Such information is important because the few studies conducted to date suggest there is extensive genetic variation between viral isolates that if confirmed has significant implications for future vaccine design and development of newer diagnostic procedures. Here, we avoid potential assembly artifacts inherent in composite sequencing techniques by using long-range PCR in conjunction with next-generation sequencing for the rapid molecular characterization of all major open reading frames (ORFs) and known transcription factor binding motifs within the long terminal repeats (LTRs) of four North American EIAV isolates from Pennsylvania (EIAV_PA), Tennessee (EIAV_TN), North Carolina (EIAV_NC), and Florida (EIAV_FL). These were compared with complete published EIAV field strain genomic sequences from Asia (EIAV_LIA, EIAV_MIY), Europe (EIAV_IRE), and North America (EIAV_WY) plus EIAV_UK a laboratory variant of EIAV_WY. Phylogenetic analysis using the long-range PCR products suggested all the New World EIAV isolates comprised a single monophyletic group associated with EIAV_IRE. This is distinct from the Asian isolates and so consistent with known historical details concerning the reintroduction of equids into North America by European settlers. Nonetheless nucleotide sequence identity for example between EIAV_PA and EIAV_TN, EIAV_NC, EIAV_FL, EIAV_WY, EIAV_UK plus EIAV_IRE was limited to 84.6%, 81.0%, 82.1%, 80.4%, 80.1%, and 77.6%, respectively, with some of these values being not too dissimilar to those between EIAV_PA and EIAV_LIA or EIAV_MIY at 78.0% and 75.4%, respectively. Overall, these results suggest substantial genetic diversity exists even within North American EIAV isolates. Comparative alignment of predicted amino acid sequences from all strains provides increased understanding concerning the extent of permitted substitutions in each viral ORF and known transcriptional LTR control elements.