Molecular Epidemiology of Avian Leukosis Virus Subgroup J in Layer Flocks in China
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.
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
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. Ap-
proximately 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 sig-
niﬁcantly 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.
vian leukosis viruses (ALVs), which belong to the genus Al-
pharetrovirus of the Retroviridae family, cause neoplastic dis-
eases and other reproduction problems in the poultry industry
worldwide. The ALVs can be classiﬁed as endogenous (ALV-E) or
exogenous viruses according to their mode of transmission. Exoge-
nous ALVs from chickens have been further divided into different
subgroups (A, B, C, D, and J) on the basis of their host range, viral
envelope interference, and cross-neutralization patterns (22).
Avian leukosis virus subgroup J (ALV-J) was isolated from meat-
type chickens in the late 1980s in the United Kingdom (21). ALV-J
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-
mentally infected with ALV-J to induce tumors (22), ﬁeld cases of
ALV-J infection and tumors in commercial layer chickens were
not observed worldwide until 2004 (37).
In China, the ALV-J infection of broilers was ﬁrst detected and
reported in 1999 (12), followed by scattered reports of infection of
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
parent and commercial layer ﬂocks in recent years, especially from
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
several provinces. Dead or sick birds had hemangiomas of various
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
sequences for ALV-J strains isolated from layer chickens have been
reported (6, 18, 36), little is known about the molecular epidemi-
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
the molecular epidemiology of ALV-J layer strains and the molec-
ular characterization of ALV-J isolates that are circulating in
China. A total of 16 ALV-J strains were isolated from clinical sam-
ples of different layer ﬂocks from 7 provinces between 2008 and
2011. The env gene, 3= untranslated region (UTR), and long ter-
minal repeat (LTR) of ALV-J layer isolates (including 16 isolates in
this study and other ALV-J layer isolates in China deposited in
GenBank between 2007 and 2010) were sequenced and phyloge-
MATERIALS AND METHODS
Clinical samples. Between April 2008 and May 2011, a total of 320 clinical
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-
ferent parents or commercial layer ﬂocks. Most of the ﬁeld cases of tumors
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, firstname.lastname@example.org.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
0095-1137/12/$12.00 Journal of Clinical Microbiology p. 953–960 jcm.asm.org 953
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
exogenous ALVs (19). The procedures for the isolation and identiﬁcation
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
(FBS) for two serial passages (5 days for each passage) at 37°C in a 5% CO
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
were harvested for DNA extraction and PCR ampliﬁcation. The DNA was
directly extracted from the positive cultured DF-1 cells or tumors using an
established method. Brieﬂy, the cultured cells were lysed in tissue lysis
buffer (4 M guanidine hydrochloride, 25 mM sodium citrate, and 1%
Triton X-100) and extracted twice with phenol-chloroform-isoamyl alco-
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
detection of subgroup A to E ALVs, which generate a 295- to 326-bp PCR
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
TACAGCTGCTC-3=) was designed for the ampliﬁcation of the ALV-J env
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
for 30 s at 54°C, and an extension for 2 min at 72°C, with a ﬁnal extension
of 7 min at 72°C. Another primer pair (UF [5=-CCGCGAAAGGTGTTA
AGACG-3=] and UR [5=-TTCCCCCTCCCTATGCAAA-3=]) was de-
signed according to the HPRS-103 sequence to amplify a 700-bp fragment
that encompasses a partial region of gp37, the entire 3= UTR (including a
nonfunctional redundant transmembrane region [rTM], the direct repeat
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
to 2011 in Heilongjiang, Henan, Hubei, Jilin, Liaoning, Shandong,
and Jiangsu Provinces in China. The PCR of DNA extracted from
infected DF-1 cells with 16 ALV-J isolates produced an ALV-J-
speciﬁc 545-bp fragment with primers H5 and H7 (Fig. 1A); how-
ever, no speciﬁc fragments were produced with primers H5 and
AD1 (Fig. 1B). Further evidence of ALV-J in the samples was dem-
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
sequence identities ranging from 92.6 to 100%. The analysis of the
deduced amino acid sequences showed that the maximum diver-
gence in the amino acid sequence was 11.2%, with sequence iden-
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).
Compared with the ALV-J broiler isolates, the amino acid sub-
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.
Molecular characterization of 3=UTR of layer isolates. The 3=
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.
954 jcm.asm.org Journal of Clinical Microbiology
rTM region, and 8 bp of nucleotides were deleted from the 5= end
of the DR-1 region in the HLJ08MDJ01 isolate. A total of 113 bp of
nucleotides were deleted from the 3= end of the rTM region, which
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
degree of identity to the E element of the prototype HPRS-103 and
American strains (ADOL-7501 and UD5) (Fig. 4A). Only a single
TABLE 1 ALV-J strains used in the construction of phylogenetic trees
Isolate Origin Yr Accession no. Host
1 LN08SY10 Liaoning, China 2008 HQ634802 CL ML VI
2 JL08CH3-1 Jilin, China 2008 HQ634809 CL He ⫹ ML VI
3 LN08SY31 Liaoning, China 2009 HQ634803 CL ML VI
4 SD09DP04 Shandong, China 2009 HQ634808 CL He ⫹ ML VI
5 HLJ09SH01 Heilongjiang, China 2009 HQ634806 CL He ⫹ ML VI
6 HLJ09SH02 Heilongjiang, China 2009 HQ634807 CL He VI
7 HuB09JY03 Hubei, China 2009 HQ634811 CL ML VI
8 HuB09WH02 Hubei, China 2009 HQ634804 CL He ⫹ ML VI
9 HuB09WH03 Hubei, China 2009 HQ634805 CL He ⫹ ML VI
10 HLJ10SH03 Heilongjiang, China 2010 HQ634813 CL He ⫹ ML VI
11 HLJ10SH04 Heilongjiang, China 2010 HQ634814 CL He ⫹ ML VI
12 HN10PY01 Henan, China 2010 CL He VI
13 JL10HW01 Jilin, China 2010 HQ634800 CL He ⫹ ML VI
14 JL10HW02 Jilin, China 2010 HQ634801 CL He ⫹ ML VI
15 JS11HA94 Jiangsu, China 2011 CL He VI
16 HLJ08MDJ01 Heilongjiang, China 2008 HQ634807 CL He VI
17 JS09GY2 Jiangsu, China 2009 GU982307 CL He 36
18 JS09GY3 Jiangsu, China 2009 GU982308 CL He 36
19 JS09GY5 Jiangsu, China 2009 GU982309 CL He 36
20 JS09GY6 Jiangsu, China 2009 GU982310 CL He 36
21 NHH China 2009 HM235668 He NF
22 SD07LK1 Shandong, China 2007 FJ216405 PL ML 34
23 SX090912J Shanxi, China 2009 HQ386988 CL — NF
24 SX090915J Shanxi, China 2009 HQ386989 CL — NF
25 HA08 China 2009 HM235664 ML NF
26 HN1001-1 Henan, China 2010 HQ260974 He NF
27 HN1001-2 Henan, China 2010 HQ260975 He NF
28 HPRS-103 UK 1988 Z46390 M ML 21
29 ADOL-Hc1 USA 1993 AF097731 BB ML 13
30 ADOL-7501 USA 1997 AY027920 CB ML 13
31 UD5 USA 2000 AF307952 M ML NF
32 10022-2 USA 2006 GU222396 — 20
33 JS-nt China 2003 HM235667 ML NF
34 NX0101 China 2001 AY897227 BB ML 9
35 YZ9901 China 1999 AY897222 CB — 9
36 SD0101 China 2001 AY897225 BB ML 9
37 NM2002-1 China 2002 HM235669 BB ML NF
38 YZ9902 China 1999 HM235670 CB — 12
39 SD9901 China 1999 AY897220 BB ML 9
40 SD9902 China 1999 AY897221 BB ML 9
41 SD0001 China 2000 AY897223 BB ML 9
42 SD0002 China 2000 AY897224 CB ML 9
43 SD0102 China AY897224 — NF
44 HN0001 China 2000 AY897219 BB ML 9
45 SDC2000 China 2000 AY234052 BB ML 38
46 HB0301 China 2003 AY897229 BB ML 35
47 BJ0303 China 2003 AY897230 BB ML 35
Isolates 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.
The host of the ALV-J isolates. CL, commercial layer chicken; PL, parental layer; M, meat-type chicken; BB, broiler breeder; CB, commercial broiler.
Type of tumor induced by ALV-J. ML, myeloma leukosis; He, hemangioma; —, unknown background of the isolate.
VI, viruses isolated and identiﬁed in this study; NF, no reference.
Avian Leukosis Virus Subgroup J in Layer Flocks
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
isolates were aligned with those of the ALV-J reference strains. The
consensus sequence from layer chickens is derived from 15 isolates
and termed “consensus (layer).” The consensus sequence from
broiler chickens is derived from eight Chinese isolates and termed
“consensus (broiler)” (9), as presented in Fig. 4C. The sequence
alignment revealed that most of the sequence of the E element was
conserved in all of the ALV-J layer isolates, and only minor muta-
tions were observed. There was a single nucleotide deletion (bases
29 and 31) and 3 unique nucleotide substitutions at positions 15
(A to G), 79 (G to A), and 125 (A to G) in the E elements of 15 layer
isolates and 2 unique nucleotide substitutions at positions 32 (A to
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
bp). The R and U5 regions of all layer isolates were well conserved
compared with those of the ALV-J broiler isolates. The phyloge-
netic analysis indicated that the U3 sequences of 84.2% (16/19) of
the layer isolates belonged to a new branch (Fig. 4B) and have 96.5
to 100% DNA sequence identity with each other, which is less than
the 92.5% sequence homology to the ALV-J broiler isolates. The
U3 sequences of NHH, HLJ08MDJ01, and SD07LK1 layer isolates
displayed relatively greater homology to the ALV-J broiler isolates
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.
A TATA box with a consensus sequence TATTTAA was located at
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
ALV-J broiler isolates (Fig. 5), which further indicated the impor-
tance of these regulatory elements during viral replication (24).
As shown in Fig. 5, compared with the ALV-J broiler isolates,
14 unique nucleotide substitutions were detected in the U3 region
of most of the ALV-J layer isolates.
Since ALV-J emerged in the 1980s, it has become highly prevalent
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
since 2008 (6, 14). In the present study, the analysis of sequences of
ALV-J layer isolates showed that (i) the env genes of 77.8% (21/27)
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 proto-
type strain of ALV-J, HPRS-103. Squares represent the American ALV-J strains
from meat-type chickens.
Gao et al.
956 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
149 residues. This is the ﬁrst comprehensive report concerning the
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,
33). To characterize the molecular changes of the env gene of these
layer isolates, the env genes were ampliﬁed and compared with
those of the ALV-J broiler isolates (the prototype strain HPRS-103
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
region of the gp85 subunit contains regions of sequence variability
in ﬁve clusters designated hr1, hr2, vr1, vr2, and vr3 (4). Analyses
of ALV env genes have identiﬁed the hr1 and hr2 domains to be the
principal receptor interaction determinants (11); vr3 also plays a
role in determining the speciﬁcity of receptor recognition (10, 31).
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
of the gp85 subunit. These results suggested that these amino acid
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
rTM, DR-1, and E elements of layer ALV-J isolates were analyzed.
The entire rTM region consists of 210 nucleotides. In the present
study, 15 out of 17 layer isolates displayed a 175-bp deletion at the
3= end of the rTM region. Although the number and region of the
deletion in the rTM region varied between ALV-J layer and broiler
isolates, the deletion mutation in the rTM region was common in
most of these ALV-J strains, suggesting that this region is not
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.
Avian Leukosis Virus Subgroup J in Layer Flocks
March 2012 Volume 50 Number 3 jcm.asm.org 957
necessary for viral replication or spreading (3). A previous study
indicated that DR-1 elements are exclusively found in sarcoma
viruses and ALV-J (2), and they seem to contribute to the ﬁtness of
the viruses because these elements have been associated with the
efﬁcient accumulation of unspliced RNA in the cytoplasm and the
selective increase of spliced src mRNA in avian sarcoma virus
(ASV). However, a 30-bp deletion at the 5= end of the DR-1 region
in most of ALV-J layer isolates suggested that at least the 5= end
might be dispensable for viral ﬁtness.
Recent reports have demonstrated substantial deletions
(⬎50%) in the E element of ALV-J proviruses that were isolated
from breeding stocks and broiler chickens between 1988 and 2007
(39), and the E element had an almost identical deletion of 127 bp
in earlier Chinese strains (8) and the recently isolated strain JS-nt
from meat-type chickens. In contrast, almost all layer isolates in
this study contained an intact E element. An analysis of the
E-element sequence revealed unique nucleotide substitutions
(A15G, G79A, and A125G) and deletions (bases 29 and 31) in this
region in most of the ALV-J layer isolates. Previous studies indi-
cated that the E element might exert a tissue-speciﬁc effect on the
transcription of cellular oncogenes in the vicinity of the integrated
provirus and might also be involved in determining the oncogenic
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.
Gao et al.
958 jcm.asm.org Journal of Clinical Microbiology
spectrum of avian retrovirus-induced disease (15, 31). Although
an intact E element was not a critical requirement for the ALV-J
induction of tumors, the presence of the E element could be asso-
ciated with a higher frequency of tumors in susceptible chickens
(8). The ALV-Js isolated from layer ﬂocks induced a high inci-
dence of hemangioma in China (6, 36). However, previous studies
indicated that the dominant tumor induced by ALV-J infection in
meat-type chickens was a myelocytoma (ML) (21). Whether these
properties of the E element of ALV-J layer isolates are related to
the change in tumor spectrum in the layer ﬂocks in China is un-
known and should be further investigated.
The U3 sequences of the ALV-J broiler strains have been pre-
viously compared (16, 36). In the present study, we analyzed the
U3 sequences of ALV-J layer isolates. The phylogenetic analysis
indicated that the U3 sequences of 16 layer isolates were highly
homologous with each other and showed relatively low homology
(less than 92.5%) with those of the ALV-J broiler isolates. Four-
teen unique nucleotide substitutions in the U3 region were found
in most of the layer isolates. These results indicated that the U3
region of the ALV-J layer isolates has evolved rapidly. The U3
region of the LTR contained a transcriptional promoter and en-
hancer elements and determined both the level of viral transcrip-
tion and the oncogenic potential of ALVs (10, 25). These nucleo-
tide mutations in the U3 region were likely associated with the
tumor disease of layers that can be induced by ALV-J.
ALV-Js have evolved rapidly in pathogenicity, which results in
a change in host range and tumor spectrum. The genetic diversity
of ALV-J might not be restricted to the env gene; genetic rear-
rangements and high mutation rates were also found in noncod-
ing genomic regions (16, 39). The two noncoding regions of the
ALV-J genome that might activate oncogenes and be involved in
oncogenesis are the U3 sequences of the LTR and the E element
located in the 3= UTR (3). Our results indicated signiﬁcant genetic
variation in these genes. The env gene, U3 region, and E element of
most of the ALV-J layer isolates formed a separate branch from the
prototype meat-type strain HPRS-103 and other ALV-J broiler
strains, and several unique nucleotide substitutions were detected
in these regions. Although the present study did not provide in-
formation concerning the functional characteristics of the genes
described, it provided some interesting information on the mo-
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.
Avian Leukosis Virus Subgroup J in Layer Flocks
March 2012 Volume 50 Number 3 jcm.asm.org 959
lecular characterization of ALV-J layer isolates. These ﬁndings will
contribute to a better understanding of the pathogenic mecha-
nism of layer tumor diseases induced by ALV-J.
The study was supported by the National Natural Science Foundation of
China (31072146), the earmarked fund for Modern Agro-Industry Tech-
nology Research System (no. nycytx-42-G3-01), and Harbin Programs for
Science and Technology Development (no. 2010AA6AN034).
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