Fig 1.
Creation of random mutation library of ALV-J Env.
(A) Schematic of mutation library construction. (B) High-throughput sequencing identified 2,437 high-quality mutations. (C) DF-1 cells were transfected with infectious clone of ALV-J J1 or ALV-J mutant library. Immunofluorescence were applied to detect viral p27 and Env protein. Supernatants were collected for replication kinetics.
Fig 2.
Continuous passage and replication of mutant ALV-J in DF-1 cells.
(A) DF-1 cells were inoculated with ALV-J J1 or ALV-J mutant library (MOI = 0.1) for 5 days. Supernatants were collected for viral titer and capsid p27 ELISA across passages 1–10. (B) Env protein expression in DF-1 cells infected with mutant or wild-type ALV-J over 10 generations, with GAPDH being the housekeeper gene. (C) Viral shedding in cloacal swabs from SPF White Leghorns inoculated with the library virus during weeks 1–4 post-inoculation. (D) Three 1-day-old SPF White Leghorns were inoculated with 5,000 TCID₅₀ ALV-J library virus. Histopathological examination of chicken liver and spleen 30 days post-inoculation.
Fig 3.
Per-codon mutation frequency of different viruses at all positions.
(A-F) Per-codon mutation frequency analysis showing amino acid positions across viral variants, with nonsense (purple), missense (red), and synonymous (blue) mutations annotated. Key mutational hotspots showing preferential patterns are indicated. (G) Total mutation frequency profiles of wild-type ALV-J J1 (virus), plasmid mutant libraries (DNA), derived mutant viruses across generations 1–10 (G1-G10) and those from SPF chicken.
Table 1.
Key nucleotide substitutions identified from ALV-J mutation library (Generation 10).
Fig 4.
Identification of lethal and preferred mutations using quantitative highthroughput genomics. (A) Log10 RC index of all individual point mutations in mutant library virus after 10 generations and from SPF chicken. Reference lines indicate the logarithmic transformations of RC = 1.5, RC = 0.5, and RC = 0.05 for comparison. (B) The amino acid mutation fitness values (AA mutation fitness values) of all point mutations. Based on the calculated values, different amino acid site mutations are classified into three categories: lethal, attenuating and variable. The variable mutations were also marked with ‘*’ on the top.
Fig 5.
DMS reveals selective preferences for glycosylation sites and insertion/deletion mutation mutations in ALV-J SU.
(A) Positional amino acid composition of ALV-J SU among 572 natural isolates. At each position, the top five most abundant amino acids are colored. Variations in column length reflect the presence of indels mutations. The variable regions vr1, vr2 and vr3, hypervariable regions hr1 and hr2 are annotated. N/O-glycosylation sites and their proportions are marked. (B) Deletion mutation profile in the ALV-J mutant library. Deletion mutation frequencies are displayed. In-frame deletions are specifically labeled, showing their corresponding amino acid deletions. (C) Insertion mutation profile in the ALV-J mutant library. Insertion mutation frequencies are displayed. Positions with in-frame insertions that lead to amino acid insertions are highlighted.
Fig 6.
DMS revealed no significant variation in the ALV-J TM.
(A) Positional amino acid composition of ALV-J TM among 340 natural isolates. At each position, the top five most abundant amino acids are colored. Variations in column length reflect the presence of indels mutations. Heptad repeat 1 (HR1) and heptad repeat 2 (HR2) are annotated. N-linked glycosylation sites and their proportions are marked. (B) Point mutation profile in the ALV-J mutant library (G10). Frequencies of mutations are plotted across all sites.
Fig 7.
DMS uncovered naturally selected amino acid preferences in the ALV-J Env and a shift in the viral phylogenetic clustering.
(A) ALV-J Env trimer surface model (J1 strain) showing conservation levels: ≥ 90% (grey), 60–90% (pink), ≤ 60% (red). Substituted residues are highlighted in blue. (B) Maximum-likelihood phylogenetic tree was generated using MEGA X with 1,000 bootstrap replicates. Two major clades are highlighted in red and blue, respectively. Strains closely related to ALV-J J1 or its evolved mutants were specifically labeled. (C-K) Nine amino acid substitutions were chosen with high RC values. 572 ALV-J strains were categorized into 3 groups according to their isolation time (1988–2003, 2004–2017 and 2018–2025). The proportional changes (top two or three most abundant amino acids) were examined in natural isolate.
Fig 8.
The evolved ALV-J carrying all selected mutations showed enhanced viral replication and shedding in vivo.
(A) Body weight of chickens in three groups during weeks 1–4. (B) Viral shedding in cloacal swabs from three groups of chickens during weeks 1–4. (C) Viremia levels in three groups of chickens during weeks 1–4. (D) Viral loads in heart, liver, kidney, and spleen of chickens from three groups at day 30. (E) Detection of ALV-J protein in tissues from three groups by immunohistochemistry (IHC).
Fig 9.
Key amino acid substitutions promote ALV-J replication.
(A) Viral titers of single-point mutant ALV-J strains at 3 days post-infection (MOI = 0.1) were determined by TCID50 assay. (B) Replication kinetics of wild-type J1 and mutant strains (J1-A64T, J1-H304R) were compared at 1–5 days post-infection (MOI = 0.1) using TCID50 titration. (C) Env protein processing was analyzed by western blot. (D) Viral genomic RNA (RT-qPCR) and Env protein (western blot) levels in cell supernatants were quantified. (E) DF-1 cells were co-transfected with plasmid expression of various Env and NHE1-ECL1-rIgG. Co-immunoprecipitation (Co-IP) was performed using anti-Flag antibodies, followed by Western blot to verify protein interaction. (F) Viral attachment was assessed by measuring cell-associated viral RNA (RT-qPCR) and Env protein (western blot) after 2h incubation at 4 °C with equal viral inputs.