Deep sequencing and variant analysis of an Italian pathogenic field strain of equine infectious anaemia virus.

The research article is about the use of long-range PCR and next-generation sequencing for the molecular characterization of the equine infectious anaemia virus, a lentivirus that causes persistent infections in equids.

Equine Infectious Anemia Virus

• The Equine infectious anaemia virus (EIAV) is a lentivirus that affects equids and is prevalent almost globally.
• Due to technical limitations, previous genetic analysis of EIAV was restricted mainly to ‘gag’ sequences, resulting in insufficient information about the variation in ‘pol’, ‘env’, and the three ancillary open reading frames (ORFs).

Techniques Used

• The researchers used long-range Polymerase Chain Reaction (PCR) in conjunction with next-generation sequencing (NGS) to carry out molecular characterization of all viral ORFs and known transcription factor binding motifs within the long terminal repeat of two EIAV isolates from an Italian outbreak in 2006.
• The next-generation sequencing technology allows for high-throughput genome sequencing and is advantageous over traditional sequencing methods as it avoids potential artifacts generated by composite sequencing strategies.

Key Findings

• The two isolates analyzed were from foals believed to have been exposed to the same source material but with different clinical histories.
• Nucleotide sequence identity between the isolates was 99.358%, confirming infection with the same EIAV strain, with most differences comprising single nucleotide polymorphisms in ‘env’ and the second exon of ‘rev’.
• The study found variations in the synonymous to non-synonymous nucleotide substitution ratio, suggesting the ‘env’ and ORF3 sequences were subjected to host-mediated selective pressure.
• EIAV proviral quasispecies complexity in vivo wasn’t extensively explored in previous research, but the analysis in this study suggests it was relatively low in one of the isolates (SA) at the time of death.

Conclusion

• This research underlines the benefits of using NGS for the molecular characterization of EIAV, as it not only eliminates potential artifacts that may occur with traditional sequencing strategies, but can also provide information about viral quasispecies complexity.