Characterization of EIAV env Quasispecies during Long-Term Passage In Vitro: Gradual Loss of Pathogenicity

Abstract

As the only widely used live lentiviral vaccine, the equine infectious anima virus (EIAV) attenuated vaccine was developed by in vitro passaging of a virulent strain for 121 generations. In our previous study, we observed that the attenuated vaccine was gradually selected under increased environmental pressure at the population level (termed a quasispecies). To further elucidate the potential correlation between viral quasispecies evolution and pathogenesis, a systematic study was performed by sequencing env using several methods. Some key mutations were identified within Env, and we observed that increased percentages of these mutations were accompanied by an increased passage number and attenuated virulence. Phylogenetic analysis revealed that env mutations related to the loss of virulence might have occurred evolutionarily. Among these mutations, deletion of amino acid 236 in the V4 region of Env resulted in the loss of one N-glycosylation site that was crucial for virulence. Notably, the 236-deleted sequence represented a “vaccine-specific” mutation that was also found in wild EIAVLN40 strains based on single genome amplification (SGA) analysis. Therefore, our results suggest that the EIAV attenuated vaccine may originate from a branch of quasispecies of EIAVLN40. Generally, the presented results may increase our understanding of the attenuation mechanism of the EIAV vaccine and provide more information about the evolution of other lentiviruses.

Env diversity-dependent protection of the attenuated equine infectious anaemia virus vaccine

“…Previous research using sequencing techniques revealed that the diversity of the attenuated EIAV vaccine was four times higher than its parent strain (Figure S1). Furthermore, a clear parallel was found between the increased diversity and the increased passaging in vitro during the developing attenuation of virulent strain [ 10 ]. Here, three EIAV strains supposedly varying in env diversity were constructed, including an attenuated vaccine EIAV_HD, a recombinant strain EIAV_MD, and a monoclonal infectious clone EIAV_LD ( Figure 1 (A)) (details are given in materials and methods) [ 17 ].…”

“…Systematic sequencing analysis showed that the genomic diversity of the attenuated vaccine EIAV DLV121 was 2.5 times higher than that of its parental strain (2.07% vs 0.81%). Our previous studies suggested that the vaccine EIAV DLV121 might be evolved from a natural quasispecies [2,3,10]. Such intriguingly high diversity in the EIAV DLV121 vaccine was perhaps acquired through an evolutionary process during the long-time passaging.…”

“…Such intriguingly high diversity in the EIAV DLV121 vaccine was perhaps acquired through an evolutionary process during the long-time passaging. In the genome of the EIAV DLV121 strain, the highest diversity is seen in the env gene, which displays 4 times higher diversity than that of its parent strain (2.4% vs 0.6%) [10]. It has been well documented that the considerably variable env plays a dominant role in virus-to-host immunity [8,[11][12][13], and it has become a target in the development of efficacious lentivirus vaccines [8,[13][14][15][16].…”

Keap1 recognizes EIAV early accessory protein Rev to promote antiviral defense

“…Unlike hosts with infections caused by other lentiviruses, most horses infected with EIAV become lifelong inapparent EIAV carriers by eliciting immune control over virus replication [3][4][5]. An attenuated EIAV vaccine was developed through long-term passaging in equine macrophages in vitro and has successfully controlled the EIA epidemic in China, indicating that protective immunity can be induced after virus infection [5][6][7]. EIAV has therefore been widely accepted as a system for identifying potential immune control over lentiviruses [1,8,9].…”

Keap1 recognizes EIAV early accessory protein Rev to promote antiviral defense

“…Unlike hosts with infections caused by other lentiviruses, most horses infected with EIAV become lifelong inapparent EIAV carriers by eliciting immune control over virus replication [3][4][5]. Additionally, an attenuated EIAV vaccine was developed through longterm passaging in equine macrophages in vitro and has successfully controlled the EIA epidemic in China, indicating that protective immunity can be induced after virus infection [5][6][7]. EIAV has therefore been widely accepted as a system for identifying potential immune control over lentiviruses [1,8,9] .…”