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

Abstract

Equine infectious anaemia virus (EIAV) is a lentivirus with an almost worldwide distribution that causes persistent infections in equids. Technical limitations have restricted genetic analysis of EIAV field isolates predominantly to gag sequences resulting in very little published information concerning the extent of inter-strain variation in pol, env and the three ancillary open reading frames (ORFs). Here, we describe the use of long-range PCR in conjunction with next-generation sequencing (NGS) for rapid molecular characterization of all viral ORFs and known transcription factor binding motifs within the long terminal repeat of two EIAV isolates from the 2006 Italian outbreak. These isolates were from foals believed to have been exposed to the same source material but with different clinical histories: one died 53 days post-infection (SA) while the other (DE) survived 5 months despite experiencing multiple febrile episodes. 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. Although the synonymous:non-synonymous nucleotide substitution ratio was approximately 2:1 in gag and pol, the situation is reversed in env and ORF3 suggesting these sequences are subjected to host-mediated selective pressure. EIAV proviral quasispecies complexity in vivo has not been extensively investigated; however, analysis suggests it was relatively low in SA at the time of death. These results highlight advantages of NGS for molecular characterization of EIAV namely it avoids potential artefacts generated by traditional composite sequencing strategies and can provide information about viral quasispecies complexity.

Equine infectious anemia virus in naturally infected horses from the Brazilian Pantanal

“…This is seen as a major obstacle to the routine implementation of a PCR based diagnostic method [34,46]. Although molecular methods represent a highly sensitive alternative for EIAV detection, these techniques require extensive nucleotide sequence information, thus there is an urgent need for EIAV genome characterization from isolates in different regions of the world [33,48].…”

“…In 2013, two new sequences, the Miyazaki 2011-A, isolated in Japan [55], and the EIAV IRE strain, isolated in Ireland [56] were published. Recently in 2017 Cappelli et al [48] sequenced approximately 95% of the proviral DNA of an EIAV strain from an outbreak that occurred in Italy in 2006. Until now there have been no complete Brazilian EIAV genome sequences available in the public databases.…”

High Genomic Variability in Equine Infectious Anemia Virus Obtained from Naturally Infected Horses in Pantanal, Brazil: An Endemic Region Case

“…Characterization of viral populations in early HIV infection, analysis of viral evolution patterns, and identification of super-infective strains have been studied by next-generation sequencing (NGS), also known as high-throughput sequencing (HTS) [7]. Complete genomic sequences of field-isolated EIAV from five different countries have been published to date: EIAV WY (United States of America), EIAV LIA (China), EIAV MIY (Japan) [8], EIAV IRE (Ireland) [9], EIAV DEV and EIAV CORN (England) [10], and the incomplete sequence EIAV ITA (Italy) [11]. Only the last and most recently obtained sequences, EIAV ITA, EIAV DEV and EIAV CORN , were obtained by NGS.…”

Molecular characterization of equine infectious anaemia virus strains detected in England in 2010 and 2012

“…EIAV has been poorly studied compared with its fellow lentiviruses, with most research concentrating on viruses derived from a North American strain (EIAV WYO ; Cook, Leroux, & Issel, ) or a Chinese strain (EIAV LIA ; Tu et al, ) and focused on viral evolution in an individual host or vaccine design. EIAV's genetic variability together with typically low viral load in hosts, especially for asymptomatic cases, have resulted in the lack of publically available sequences and consequently of an useful molecular diagnostic method for laboratories worldwide (Capelli et al, ; Dong et al, ).…”

“…When underpinned by sufficiently large datasets, phylogenetics has proved to be a powerful tool for studying the epidemiology of many diseases including lentiviruses such as Human Immunodeficiency Virus. Full genome EIAV sequences are currently available for the American (EIAV WYO ; Cook et al, ), Chinese (EIAV LIA ; Tu et al, ), Irish (EIAV IRE ; Quinlivan et al, ), Japanese (EIAV MIYA ; Dong et al, ) and Italian cases (EIAV ITA ; Cappelli et al, ) only. The majority of publically available EIAV sequences are only partial genomes covering individual genes.…”