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

Cong Liu,Zhaohua Zhong,Xiaojun Wang,Yan Wang,Yuezhi Lin,Xue-Feng Wang,Jie Chen

doi:10.3390/v11040380

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.

Highlights

As the simplest member of the lentivirus family, equine infectious anemia virus (EIAV) shares similar features with other lentiviruses, including its genomic structure, life cycle, cell tropism and antigen evolution [1,2]
The uniqueness of EIAV lies in the fact that most infected horses become asymptomatic carriers after recurrent febrile episodes, which are associated with a high viral load and anemia, and the horses achieve natural immunologic control [3]
These samples included a virulent strain (EIAVLN40 ) that caused an infection in horses with 100% mortality at a dose of 1 × 105 TCID50; this strain was initially isolated from an EIA positive horse and passed for 16 generations in horses, resulting in three representative strains (EIAVDLV34, EIAVDLV62, and EIAVDLV92 ) with 100%, 100%, and 9.1% mortality that stemmed from EIAV passage in vitro for 34, 62, and 92 generations, respectively (Figure S1) [16]

Summary

Introduction

As the simplest member of the lentivirus family, equine infectious anemia virus (EIAV) shares similar features with other lentiviruses, including its genomic structure, life cycle, cell tropism and antigen evolution [1,2]. The vaccine was generated by serial passage of a virulent EIAV strain (EIAVLN40 ) for 16 generations in vivo, followed by passaging for as many as 121 passages in vitro [4,5]. For RNA viruses, mutation swarms are generated rapidly through error-prone viral replication due to inaccuracy of the RNA polymerase [6] This mutant swarm, which is termed a viral quasispecies, appears as a whole population with a mutation-selection balance and is significantly involved in viral pathogenesis [7,8]. The relationship between RNA viral quasispecies evolution and viral pathogenesis is an intriguing problem

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