Multi-clade co-evolution and differential replication efficiencies of subgroup A avian leukosis virus in Chinese guinea fowl

Avian leukosis virus (ALV) has a wide range of hosts and is Susceptible to commercial chickens, local chickens and rare birds. To assess the presence of ALV-A infection in guinea fowl and characterize its viral genetics, PCR testing of guinea fowl embryosfrom four provinces revealed an ALV-A positive rate of 68.1% (32/47) in China. Furthermore, phylogenetic analysis of the 15 ALV-A gp85 sequences obtained from guinea fowl-fertilized chicken embryos revealed four evolutionary clades (1, 2, 3, 4). Notably, no clade 1 isolates were detected in this study. Comparative sequence analysis demonstrated that amino acid variations among different clades primarily localized to host range determinant regions 1 and 2 (hr1 and hr2) of the gp85 protein. To systematically investigate the replicative potential of ALV-A across distinct evolutionary clades, envelope genes from three representative strains: HN23A16 (clade2), HN24Axx (clade4), and HN23A09 (clade3) were cloned into the RCASBP (replication-competent avian sarcoma-leukosis virus) retroviral vector to generate recombinant ALV-A viruses. Binding affinity between ALV-A gp85 (surface glycoprotein) and the chicken TVA receptor (the receptor of ALV-A, a protein of the low-density lipoprotein receptor family) was subsequently validated through co-immunoprecipitation (co-IP) assays. Comparative analysis of replication kinetics revealed marked differences among clades. Both HN23A16 (clade2) and HN23A09 (clade3) exhibited robust replication efficiency in vitro and in vivo, whereas HN24Axx (clade4) showed significantly reduced replicative capacity. Co-IP quantification demonstrated that the HN24Axx gp85 variant bound to TVA with 3.3-fold lower affinity compared to clade 2 and 3 counterparts. Here, we report for the first time the viral genetic characteristics of 15 ALV-A strains from Chinese guinea fowl, and the proposed classification method will facilitate future studies of ALV-A epidemiology and the comparison of sequences obtained.