Molecular Epidemiology of Avian Leukosis Virus Subgroup J in Layer
Flocks in China
Yulong Gao, Bingling Yun, Liting Qin, Wei Pan, Yue Qu, Zaisi Liu, Yongqiang Wang, Xiaole Qi, Honglei Gao, and Xiaomei Wang
Division of Avian Infectious Diseases, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural
Sciences, Harbin, China
eases and other reproduction problems in the poultry industry
envelope interference, and cross-neutralization patterns (22).
Avian leukosis virus subgroup J (ALV-J) was isolated from meat-
has primarily been associated with myeloid leukosis (ML) in
broiler breeders and has caused more serious damage than any
other subgroup. Although egg-type chickens have been experi-
ALV-J infection and tumors in commercial layer chickens were
not observed worldwide until 2004 (37).
broiler and local chickens in some areas of China (5, 7, 28). How-
ever, field cases of ALV-J infection and tumors in commercial
layer chickens emerged in 2004 (37). ALV-J has been found to
induce various tumors and cause significant economic losses in
2008 to 2010 (6, 14, 18, 36). Many field cases of ALV-J infection
and tumors occurred in 15- to 29-week-old egg-type chickens in
sizes that were distributed on the surface of the head, claws, and
wings; some birds had gray-white nodules in the liver, spleen, or
kidneys, and the liver and spleen were enlarged to several times
their normal size. Some affected flocks had dramatically reduced
egg production and an increased rate of mortality (14).
Although several field cases and complete proviral genomic
reported (6, 18, 36), little is known about the molecular epidemi-
vian leukosis viruses (ALVs), which belong to the genus Al-
pharetrovirus of the Retroviridae family, cause neoplastic dis-
ology of ALV-J layer isolates in China. The first study conducted
by our laboratory between 2007 and 2009 showed that ALV-J is
predominantly responsible for layer avian leukosis in China (14).
The present study completes the previous survey by focusing on
ular characterization of ALV-J isolates that are circulating in
ples of different layer flocks from 7 provinces between 2008 and
2011. The env gene, 3= untranslated region (UTR), and long ter-
this study and other ALV-J layer isolates in China deposited in
GenBank between 2007 and 2010) were sequenced and phyloge-
MATERIALS AND METHODS
samples (including tumor, whole-blood, spleen, kidney, liver, and other
tissue samples) were collected from 6- to 36-week-old diseased egg-type
chickens from 7 provinces in China (Heilongjiang, Henan, Hubei, Jilin,
Liaoning, Shandong, and Jiangsu). The chickens originated from 48 dif-
occurred in 15- to 29-week-old egg-type chickens. The levels of egg pro-
duction were dramatically reduced in the affected flocks. The clinical
symptoms included hemorrhages in the skin of the phalanges and feather
follicles. Some birds had gray-white nodules in the liver, spleen, or kid-
Received 24 October 2011 Returned for modification 5 December 2011
Accepted 15 December 2011
Published ahead of print 28 December 2011
Address correspondence to Xiaomei Wang, email@example.com.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
0095-1137/12/$12.00Journal of Clinical Microbiologyp. 953–960 jcm.asm.org
neys, and the liver and spleen were enlarged to several times the normal
Virus isolation and proviral DNA extraction. All virus isolations
were performed in DF-1 cells, which are known to be susceptible only to
of ALV in cell cultures were preformed according to previously described
studies (1). Briefly, filtered tumor homogenates were inoculated into
DF-1 cells, which were cultured in Dulbecco’s modified Eagle’s medium
(DMEM; Invitrogen, CA) supplemented with 10% fetal bovine serum
incubator with daily monitoring. After the incubation, the infected DF-1
cells were tested for the ALV group-specific antigen (p27) by an antigen-
capture enzyme-linked immunosorbent assay (AC-ELISA) with anti-p27
antibody-coated plates (IDEXX Inc., MA) and tested for the ALV-J-
specific antigen by immunofluorescence assay (IFA) with ALV-J-specific
monoclonal antibody FE9 (32). The positive samples detected by ELISA
established method. Briefly, the cultured cells were lysed in tissue lysis
buffer (4 M guanidine hydrochloride, 25 mM sodium citrate, and 1%
hol (25:24:1). The DNA was precipitated with absolute isopropanol,
washed with 70% isopropanol, and dried at room temperature. Subse-
quently, the DNA was resuspended in nuclease-free water and stored at
PCR cloning and sequencing. PCR was used to test genomic DNA
from the cultured DF-1 cells or tumors for the presence of envelope se-
quences that are specific for ALV-J as previously described (27). The
primer set H5 (5=-GGATGAGGTGACTAAGA-3=) and H7 (5=-CGAACC
AAAGGTAACACACG-3=) was used for the specific detection of ALV-J
proviral DNA, which generates a 544-bp PCR product (27). The primer
set H5 and AD1 (5=-GGGAGGTGGCTGACTGTGT-3=) was used for the
product (27). According to the sequence of the ALV-J prototype strain,
HPRS-103 (GenBank accession number AF097731), the primer pair EF
(5=-CGACACTGATAAGGTTATTTGGGT-3=) and ER (5=-TCGGAACC
gene. The PCR conditions included an initial denaturation cycle of 4 min
at 94°C, followed by 30 cycles of denaturation for 30 s at 94°C, annealing
of 7 min at 72°C. Another primer pair (UF [5=-CCGCGAAAGGTGTTA
AGACG-3=] and UR [5=-TTCCCCCTCCCTATGCAAA-3=]) was de-
that encompasses a partial region of gp37, the entire 3= UTR (including a
element [DR-1], and an E element) and the entire 3= LTR. The PCR
amplification scheme was the same as that for the env gene, but the an-
nealing temperature was 56°C and the extension time was 1 min at 72°C.
All PCRs were carried out with PrimerSTAR HS DNA high-fidelity poly-
merase (TaKaRa, Dalian, China).
The PCR products were excised from a 1.0% agarose gel, purified
using an AxyPrep DNA gel extraction kit (Axygen Scientific, Inc., CA),
and cloned into the TA vector pMD18-T (TaKaRa). Three independent
clones of each ALV-J isolate were sequenced by the Beijing Genomics
Institute (Beijing, China).
DNA alignments and phylogenetic analysis. The nucleotide se-
quences were aligned using the Clustal W program, version 1.8 (17). A
neighbor-joining tree was drawn using the MEGA program, version 3.1
(29), with confidence levels assessed using 1,000 bootstrap replications.
The GenBank sequences of the ALV-J strains that were isolated from
meat-type and layer chickens were included in the multiple-sequence
alignment, and they are summarized in Table 1.
Nucleotide sequence accession numbers. The sequences obtained in
this study have been submitted to GenBank, and the accession numbers
are provided in Table 1.
Virus isolation and identification of ALV-J. A total of 16 ALV-J
strains (detailed information is summarized in Table 1) were iso-
lated from clinical samples from different layer flocks from 2008
and Jiangsu Provinces in China. The PCR of DNA extracted from
infected DF-1 cells with 16 ALV-J isolates produced an ALV-J-
ever, no specific fragments were produced with primers H5 and
onstrated by the positive result in the IFA with ALV-J-specific
monoclonal antibody FE9 (Fig. 1C) and in the AC-ELISA with
anti-p27 antibody-coated plates (Fig. 1D).
Molecular characterization of env gene of layer isolates. A
total of 27 ALV-J layer isolates (including 16 isolates in this study
and 11 isolates from GenBank) were analyzed (Table 1). The pro-
totype ALV-J strain HPRS-103 (21), American ALV-J isolates
(e.g., ADOL-Hc1 and ADOL-7501), and Chinese strains from
meat-type chickens (designated ALV-J broiler isolates) were used
as references for comparisons in the molecular studies. All refer-
ence viruses are summarized in Table 1.
The env genes of layer isolates were 1,512 to 1,518 nucleotides
long. The nucleotide changes that occurred throughout the env
gene showed a maximum divergence of 7.4%, with nucleotide
deduced amino acid sequences showed that the maximum diver-
tities ranging from 88.8 to 100%.
The phylogenetic analysis indicated that 77.8% (21/27) of the
ALV-J layer isolates showed close homology (91.3 to 99.0%) with
each other and belonged to one branch, which was designated
group I (Fig. 2). These isolates were 88.7 to 94.3% identical to
prototype strain HPRS-103, 88.7 to 92.5% identical to American
strains, and 88.1 to 94.3% identical to Chinese broiler isolates
at the amino acid level. However, 22.2% (6/27, including iso-
lates HA08, NHH, HLJ08MDJ1, HuB09JY03, LN08SY10, and
SD07LK1) of the layer isolates showed relatively close homology
(88.7 to 99.0%) with the ALV-J broiler isolates (excluding the
ADOL-7501 isolate), which belonged to the other branch and
which was designated group II (Fig. 2).
stitutions of all layer isolates were distributed throughout the en-
velope glycoprotein, and there was a clustering of sequence varia-
tions near the hr1, hr2, and 48- to 76-amino-acid variable
domains of the gp85 gene. Thirteen amino acids (48S, 49A, 61Q,
63D, 76T, 117G, 123I, 143T/E, 146G, 150H, 189K, 203N, and
218Q) of gp85 were relatively conserved in 21 layer isolates.
UTR and LTR sequences from a total of 19 ALV-J layer isolates
(including 15 isolates in this study and 4 isolates from GenBank)
were analyzed (Table 1). A comparison of the nucleotide se-
quences of 3= UTRs with the ALV-J broiler isolates revealed that
205 bp were deleted in the rTM and DR-1 regions of 89.5% (17/
19) of the ALV-J layer isolates (excluding HLJ08MDJ01 and
SD07LK1) (Fig. 3). A total of 175 bp were deleted from the 3= end
of the rTM region, and 30 nucleotides were deleted from the 5=
end of the DR-1 region. This deletion was similar to the mutation
in the 3= UTRs of American ALV-J isolates (ADOL-7501 and
UD5). Approximately 210 bp were deleted from the 3= end of the
Gao et al.
jcm.asm.orgJournal of Clinical Microbiology
rTM region, and 8 bp of nucleotides were deleted from the 5= end
contained the entire DR-1 region in the SD07LK1 isolate.
Approximately 94.7% (16/17) of the ALV-J layer isolates con-
tained an intact E element (146 to 149 nucleotides) with a high
American strains (ADOL-7501 and UD5) (Fig. 4A). Only a single
TABLE 1 ALV-J strains used in the construction of phylogenetic trees
IsolateOrigin YrAccession no.Hostb
He ? ML
He ? ML
He ? ML
He ? ML
He ? ML
He ? ML
He ? ML
He ? ML
He ? ML
aIsolates 1 to 16 are layer strains identified in this study. Isolates 17 to 27 are layer strains obtained from GenBank. Isolates 28 to 32 are the prototype and American strains. Isolates
33 to 47 are Chinese broiler isolates.
bThe host of the ALV-J isolates. CL, commercial layer chicken; PL, parental layer; M, meat-type chicken; BB, broiler breeder; CB, commercial broiler.
cType of tumor induced by ALV-J. ML, myeloma leukosis; He, hemangioma; —, unknown background of the isolate.
dVI, viruses isolated and identified in this study; NF, no reference.
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March 2012 Volume 50 Number 3 jcm.asm.org 955
layer isolate (HLJ08MDJ01) contained a 12-bp deletion in the E
element. In contrast, nine Chinese broiler strains displayed E ele-
ments with substantial deletions (127-nucleotide deletions) (Fig.
The nucleotide sequences of the E element of the ALV-J layer
and termed “consensus (layer).” The consensus sequence from
“consensus (broiler)” (9), as presented in Fig. 4C. The sequence
29 and 31) and 3 unique nucleotide substitutions at positions 15
G) and 35 (G to T) in the E elements of all layer isolates (Fig. 4C).
Molecular characterization of 3=LTR of layer isolates. The
sequence analysis of 3= LTRs of all layer isolates revealed the same
325-bp sequence, except for the 3= LTR of the NHH isolate (314
compared with those of the ALV-J broiler isolates. The phyloge-
the 92.5% sequence homology to the ALV-J broiler isolates. The
Transcriptional regulation elements were identified in the 3=
U3 region of the LTR in all of the ALV-J layer isolates, including
two CArG, Y, and PRE boxes and one C/EBP, CAAT, and TATA
box (Fig. 5). Two CArG boxes were located at positions 49 to 58
and 123 to 132, respectively. Similarly, two highly conserved in-
verse Y boxes with the sequence 5=-ATTGG-3= were located at
positions 83 to 87 and 158 to 162, respectively. Two copies of the
PRE motif GGTGG were located at positions 86 to 90 and 98 to
102, flanking the nucleotide sequence AAGTAA. The CCAAT/
enhancer (5=-TTATGCAAT-3=) was located at positions 10 to 18.
positions 197 to 203. A key polyadenylation signal with the se-
quence AATAAA was located at the end of the 3= U3 region. Al-
most all of the putative transcription regulatory elements identi-
fied in the U3 region were conserved and homologous to those of
tance of these regulatory elements during viral replication (24).
As shown in Fig. 5, compared with the ALV-J broiler isolates,
of most of the ALV-J layer isolates.
in meat-type chickens and has caused serious problems in meat-
type birds (13). However, in China, the host range of ALV-J is
changing. ALV-J has become a major problem in layer chickens
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.
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 proto-
from meat-type chickens.
Gao et al.
jcm.asm.org Journal of Clinical Microbiology
of the layer isolates with a high degree of genetic variation were
significantly different from the env genes of the ALV-J broiler
isolates, (ii) a 205-bp deletion in the 3= UTR and unique nucleo-
tide mutations in the 3= UTR and 3= LTR were detected, and (iii)
almost all layer isolates contained a complete E element of 147 to
molecular characterization of ALV-J layer isolates.
Genetic and antigenic variations with sequence changes in the
variable regions of the env gene of ALV-J have been observed (26,
layer isolates, the env genes were amplified and compared with
and American and Chinese strains from meat-type chickens). A
total of 21 layer isolates showed significant differences in the nu-
cleotide sequences compared with the ALV-J broiler isolates,
forming a separate group (Fig. 2). The envelope glycoprotein of
ALV primarily functions as a ligand for receptor binding for viral
entry into the susceptible cell and determines the host range (7).
Studies with other ALV subgroups have shown that the central
in five clusters designated hr1, hr2, vr1, vr2, and vr3 (4). Analyses
principal receptor interaction determinants (11); vr3 also plays a
In the present study, an alignment of the deduced amino acid
sequences of 27 layer isolates and ALV-J broiler isolates revealed
13 amino acid substitutions distributed within the central region
substitutions might be associated with changes in the host range
and pathogenicity of ALV-J.
Previous studies have demonstrated that ALV-J is prone to
significant molecular variation because the envelope gene of this
virus displays multiple mutations, resulting in antigenic variants,
possibly as a result of immune pressure (26, 33). Among the vari-
able domains within gp85, the hr1, hr2, and vr3 regions are the
main targets of selection pressure (30). In this study, 7 of the 13
amino acid substitutions were distributed within the hr1 and hr2
regions. The pattern of amino acid substitutions presented here
suggested that the antigenic variation might have resulted from
Because the 3= UTR of ALV, which contains potent regulatory
sequences that influence chromosomal and viral gene expression,
is important in viral pathogenesis and replication (23, 24), the
The entire rTM region consists of 210 nucleotides. In the present
3= end of the rTM region. Although the number and region of the
isolates, the deletion mutation in the rTM region was common in
most of these ALV-J strains, suggesting that this region is not
and elements in the genomic proviral DNA of HPRS-103. The deletions are indicated by empty spaces between the thick black lines.
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