Molecular epidemiology of avian leukosis virus subgroup J in layer flocks in China

Abstract

Avian leukosis virus subgroup J (ALV-J) was first isolated from meat-type chickens in 1988. No field cases of ALV-J infection or tumors in layer chickens were observed worldwide until 2004. However, layer flocks in China have experienced outbreaks of this virus in recent years. The molecular epidemiology of ALV-J strains isolated from layer flocks was investigated. The env genes of 77.8% (21/27) of the ALV-J layer isolates with a high degree of genetic variation were significantly different from the env genes of the prototype strain of ALV-J (HPRS-103) and American and Chinese strains from meat-type chickens (designated ALV-J broiler isolates). A total of 205 nucleotides were deleted from the 3' untranslated region of 89.5% (17/19) of the ALV-J layer isolates. Approximately 94.7% (16/17) of the layer isolates contained a complete E element of 146 to 149 residues. The U3 sequences of 84.2% (16/19) of the ALV-J layer isolates displayed less than 92.5% sequence homology to those of the ALV-J broiler isolates, although the transcriptional regulatory elements that are typical of avian retroviruses were highly conserved. Several unique nucleotide substitutions in the env gene, the U3 region, and the E element of most of the ALV-J layer isolates were detected. These results suggested that the env gene, E element, and U3 region in the ALV-J layer isolates have evolved rapidly and were significantly different from those of the ALV-J broiler isolates. These findings will contribute to a better understanding of the pathogenic mechanism of layer tumor diseases induced by ALV-J.

Fig 1

Identification of 16 ALV-J isolates with PCR (A, primer pair H5/AD1; B, primer pair H5/H7), IFA (C) and AC-ELISA (D). Uninfected DF-1 cells served as the negative control. DF-1 cells infected with Rous-associated virus type 1 (subgroup A) served as the positive control for PCR with primer pair H5/AD1. DF-1 cells infected with HPRS-103 served as the positive control for PCR with primer pair H5/AD1, IFA, and AC-ELISA.

Fig 2

Phylogenetic analysis of the nucleotide sequences of env genes of ALV-J layer and broiler isolates. The tree was constructed on the basis of the minimum-evolution method using MEGA 4 software. Bootstrap values were calculated with 1,000 replicates of the alignment. The two groups are marked. Triangles represent the ALV-J layer isolates. The circle represents the prototype strain of ALV-J, HPRS-103. Squares represent the American ALV-J strains from meat-type chickens.

Fig 3

Comparison of the deletions at the 3′ UTR in the genomic proviral DNA of ALV-J layer and broiler isolates. The top two lines represent the base numbers and elements in the genomic proviral DNA of HPRS-103. The deletions are indicated by empty spaces between the thick black lines.

Fig 4

Phylogeny and alignment of the E elements and U3 region of ALV-J layer and broiler isolates. (A and B) Phylogenetic analysis of E elements and U3 regions. Triangles represent the ALV-J layer isolates. Squares represent the American ALV-J strains from meat-type chickens. The circle represents the prototype strain of ALV-J, HPRS-103. (C) Comparison of nucleotide sequences of the E elements. Consensus (layer) sequences were derived from 16 layer isolates (JS11HA94, HLJ09SH02, HLJ10SH04, JL08CH3-1, HLJ09SH01, HuB09WH02, JL10HW02, LN09SY31, SD09DP04, LN08SY10, JS09GY06, HN10PY01, HuB09WH03, HLJ10SH03, HuB09JY03, JS09GY03). The consensus (broiler) sequences were derived from the nine Chinese ALV-J strains (YZ9901, SD9901, SD9902, SD0001, SD0002, HN0001, SD0101, NX0101, and JS-nt). The dots indicate identical residues, while the letters indicate base substitutions. The dashes indicate gaps produced in the alignment. The shading indicates unique nucleotide substitutions of the ALV-J layer isolates.

Fig 5

Comparison of the nucleotide sequences in the U3 region of the ALV-J layer isolates, prototype strain HPRS-103, American ALV-J strains, and the consensus (broiler) sequence of the nine Chinese ALV-J strains. The dots indicate identical residues, while the letters indicate base substitutions. The dashes indicate gaps produced in the alignment. The locations of putative transcription regulatory elements are indicated in boxes and marked. The shading indicates unique nucleotide substitutions in the ALV-J layer isolates.