Molecular Epidemiology of Avian Leukosis Virus Subgroup J in Layer Flocks in China

Article (PDF Available)inJournal of clinical microbiology 50(3):953-60 · December 2011with55 Reads
DOI: 10.1128/JCM.06179-11 · Source: PubMed
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.
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.
A
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-
netically analyzed.
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, xmw@hvri.ac.cn.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JCM.06179-11
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
size.
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
2
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
80°C.
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.
RESULTS
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
No.
a
Isolate Origin Yr Accession no. Host
b
Tumor type
c
Reference or
source
d
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
a
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.
b
The host of the ALV-J isolates. CL, commercial layer chicken; PL, parental layer; M, meat-type chicken; BB, broiler breeder; CB, commercial broiler.
c
Type of tumor induced by ALV-J. ML, myeloma leukosis; He, hemangioma; —, unknown background of the isolate.
d
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.
4C) (9).
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
(Fig. 4B).
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.
DISCUSSION
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
selection pressure.
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.
ACKNOWLEDGMENTS
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).
REFERENCES
1. Bagust TJ, Fenton SP, Reddy MR. 2004. Detection of subgroup J avian
leukosis virus infection in Australian meat-type chickens. Aust. Vet. J.
82:701–706.
2. Bai J, Howes K, Payne LN, Skinner MA. 1998. Sequence analysis of an
infectious proviral clone of HPRS-103 shows that it represents a new avian
retrovirus envelope subgroup (designated J). Avian Pathol. 27(Suppl 1):
S92–S93.
3. Bai J, Payne LN, Skinner MA. 1995. HPRS-103 (exogenous avian leuko-
sis virus, subgroup J) has an env gene related to those of endogenous
elements EAV-0 and E51 and an E element found previously only in sar-
coma viruses. J. Virol. 69:779 –784.
4. Bova CA, Manfredi JP, Swanstrom R. 1986. Env genes of avian retrovi-
ruses: nucleotide sequence and molecular recombinants define host range
determinants. Virology 152:343–354.
5. Cheng ZQ, Zhang L, Liu SD, Zhang LJ, Cui ZZ. 2005. Emerging of avian
leukosis virus subgroup J in a flock of Chinese local breed. Acta Microbiol.
Sinica 45:584 –587. (In Chinese.)
6. Cheng Z, Liu J, Cui Z, Zhang L. 2010. Tumors associated with avian
leukosis virus subgroup J in layer hens during 2007 to 2009 in China. J.
Vet. Med. Sci. 72:1027–1033.
7. Chesters PM, et al. 2002. The viral envelope is a major determinant for
the induction of lymphoid and myeloid tumours by avian leukosis virus
subgroups A and J, respectively. J. Gen. Virol. 83:2553–2561.
8. Chesters PM, Smith LP, Nair V. 2006. The E (XSR) element contributes
to the oncogenicity of avian leukosis virus (subgroup J). J. Gen. Virol.
87:2685–2692.
9. Cui Z, Du Y, Zhang Z, Silva RF. 2003. Comparison of Chinese field
strains of avian leukosis subgroup J viruses with prototype strain HPRS-
103 and United States strains. Avian Dis. 47:1321–1330.
10. Cullen BR, Raymond K, Ju G. 1985. Functional analysis of the transcrip-
tion control region located within the avian retroviral long terminal re-
peat. Mol. Cell. Biol. 5:438 447.
11. Dorner AJ, Coffin JM. 1986. Determinants for receptor interaction and
cell killing on the avian retrovirus glycoprotein gp85. Cell 45:365–374.
12. Du Y, Cui Z, Qin A. 1999. Subgroup J avian leukosis viruses in China.
China Poult. Sci. 3:1– 4. (In Chinese.)
13. Fadly AM, Smith EJ. 1999. Isolation and some characteristics of a sub-
group J-like avian leukosis virus associated with myeloid leukosis in meat-
type chickens in the United States. Avian Dis. 43:391– 400.
14. Gao YL, et al. 2010. Avian leukosis virus subgroup J in layer chickens,
China. Emerg. Infect. Dis. 16:1637–1638.
15. Hayward WS, Neel BG, Astrin SM. 1981. Activation of a cellular onc gene
by promoter insertion in ALV-induced lymphoid leukosis. Nature 290:
475–480.
16. Hue D, et al. 2006. Major rearrangements in the E element and minor
variations in the U3 sequences of the avian leukosis subgroup J provirus
isolated from field myelocytomatosis. Arch. Virol. 151:2431–2446.
17. Kumar S, Tamura K, Nei M. 2004. MEGA3: integrated software for
molecular evolutionary genetics analysis and sequence alignment. Brief.
Bioinform. 5:150 –163.
18. Lai H, et al. 2011. Isolation and characterization of emerging subgroup J
avian leukosis virus associated with hemangioma in egg-type chickens.
Vet. Microbiol. 151:275–283.
19. Maas R, van Zoelen D, Oei H, Claassen I. 2006. Replacement of primary
chicken embryonic fibroblasts (CEF) by the DF-1 cell line for detection of
avian leukosis viruses. Biologicals 34:177–181.
20. Pandiri AR, et al. 2010. Subgroup J avian leukosis virus neutralizing
antibody escape variants contribute to viral persistence in meat-type
chickens. Avian Dis. 54:848 856.
21. Payne LN, et al. 1991. A novel subgroup of exogenous avian leukosis virus
in chickens. J. Gen. Virol. 72:801– 807.
22. Payne LN, Howes K, Gillespie AM, Smith LM. 1992. Host range of Rous
sarcoma virus pseudotype RSV(HPRS-103) in 12 avian species: support
for a new avian retrovirus envelope subgroup, designated J. J. Gen. Virol.
73:2995–2997.
23. Robinson HL, Blais BM, Tsichlis PN, Coffin JM. 1982. At least two
regions of the viral genome determine the oncogenic potential of avian
leukosis viruses. Proc. Natl. Acad. Sci. U. S. A. 79:1225–1229.
24. Ruddell A. 1995. Transcription regulatory elements of the avian retroviral
long terminal repeat. Virology 206:1–7.
25. Ryden TA, Mde Mars Beemon K. 1993. Mutation of the C/EBP binding
sites in the Rous sarcoma virus long terminal repeat and gag enhancers. J.
Virol. 67:2862–2870.
26. Silva RF, Fadly AM, Hunt HD. 2000. Hypervariability in the envelope
genes of subgroup J avian leukosis viruses obtained from different farms in
the U.S. Virology 272:106 –111.
27. Smith LM, et al. 1998. Development and application of polymerase chain
reaction (PCR) tests for the detection of subgroup J avian leukosis virus.
Virus Res. 54:87–98.
28. Sun S, Cui Z. 2007. Epidemiological and pathological studies of subgroup
J avian leukosis virus infections in Chinese local “yellow” chickens. Avian
Pathol. 36:221–226.
29. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG.
1997. The ClustalX Windows interface: flexible strategies for multiple se-
quence alignment aided by quality analysis tools. Nucleic Acids Res. 24:
4876 4882.
30. Thu WL, Wang CH. 2003. Phylogenetic analysis of subgroup J avian
leucosis virus from broiler and native chickens in Taiwan during 2000-
2002. J. Vet. Med. Sci. 65:325–328.
31. Tsichlis PN, Coffin JM. 1980. Recombinants between endogenous and
exogenous avian tumor viruses: role of the C region and other portions of
the genome in the control of replication and transformation. J. Virol.
33:238–249.
32. Venugopal K, Howes K, Barron GS, Payne LN. 1997. Recombinant
env-gp85 of HPRS-103 (subgroup J) avian leukosis virus: antigenic char-
acteristics and usefulness as a diagnostic reagent. Avian Dis. 41:283–288.
33. Venugopal K, Smith LM, Howes K, Payne LN. 1998. Antigenic variants
of J subgroup avian leukosis virus: sequence analysis reveals multiple
changes in the env gene. J. Gen. Virol. 79:757–766.
34. Wang H, Cui Z. 2008. The identification and sequence analysis of ALV-J
isolated from layers. Chin. J. Virol. 24:369 –370. (In Chinese.)
35. Wang Z, Cui Z, Zang Z, Wu Y. 2005. The mutation tendency of the gp85
gene of Chinese field strains of ALV-J from 1999 to 2003. Vitologica Siniga
20:393–398.
36. Wu X, et al. 2010. Recombinant avian leukosis viruses of subgroup J
isolated from field infected commercial layer chickens with hemangioma
and myeloid leukosis possess an insertion in the E element. Vet. Res. Com-
mun. 34:619 632.
37. Xu B, et al. 2004. Occurrence of avian leukosis virus subgroup J in com-
mercial layer flocks in China. Avian Pathol. 33:13–17.
38. Yang Y, Yie J, Zhao Z, Qin A, Gu Y. 2003. Isolation and identification of
inner Mongolia Strain of ALV subgroup J. Vitologica Siniga 18:454 458.
(In Chinese.)
39. Zavala G, Cheng S, Jackwood MW. 2007. Molecular epidemiology of
avian leukosis virus subgroup J and evolutionary history of its 3= untrans-
lated region. Avian Dis. 51:942–953.
Gao et al.
960 jcm.asm.org Journal of Clinical Microbiology
    • "Dead or sick birds generally have hemangiomas of various sizes distributed on the surface of their heads, claws, and wings. Some infected birds have gray–white nodules in the liver, spleen, or kidneys, and the liver and spleen can be enlarged to several times their normal size [32]. Developed countries and China have made efforts to eradicate ALV-J from chicken breeding flocks in the poultry industry [33]. "
    [Show abstract] [Hide abstract] ABSTRACT: Avian leukosis virus subgroup J (ALV-J) is a retroviruses that induces neoplasia, hepatomegaly, immunosuppression and poor performance in chickens. The tumorigenic and pathogenic mechanisms of ALV-J remain a hot topic. To explore anti-tumor genes that promote resistance to ALV-J infection in chickens, we bred ALV-J resistant and susceptible chickens (F3 generation). RNA-sequencing (RNA-Seq) of liver tissue from the ALV-J resistant and susceptible chickens identified 216 differentially expressed genes; 88 of those genes were up-regulated in the ALV-J resistant chickens (compared to the susceptible ones). We screened for significantly up-regulated genes (P < 0.01) of interest in the ALV-J resistant chickens, based on their involvement in biological signaling pathways. Functional analyses showed that overexpression of GADD45? inhibited ALV-J replication. GADD45? could enhance defense against ALV-J infection and may be used as a molecular marker to identify ALV-J infections.
    Article · Sep 2016
    • "1988 [11], many studies have examined its molecular epidemiology in many parts of the 9 world [6, 8, 9, 11, 14, 15, 18, 19, 20, 24] ). that circulate in a flock. Similar gp85 sequence comparisons were also conducted for ALV-J [20]. "
    [Show abstract] [Hide abstract] ABSTRACT: The genomic diversity of avian leukosis virus subgroup J (ALV-J) was studied in an experimentally infected chicken. ALV-J variants in tissue samples from four different organs of the same bird were re-isolated in cultured DF-1 chicken fibroblast cells, and their gp85 gene was polymerase chain reaction-amplified and cloned. Ten clones from each organ were sequenced and compared with the original inoculum strain NX0101. The minimum homology of the 10 clones from each organ ranged from 96.7 to 97.6%, and the lowest homology among the 40 clones from different organs was only 94.9%, much lower than the 99.1% homology of the inoculum NX0101 stock, which indicated the high diversity of ALV-J, even within the same bird. The gp85 mutations in ALV-J clones from the left kidney, which contained tumors, and the right kidney, which was tumor-free, had higher non-synonymous to synonymous mutation ratios than those in the tumor-bearing liver and lungs. Additionally, the sites of the gp85 mutations in the kidney clones were similar, and they differed from those in the liver and lung clones, which implies that organ- or tissue-specific selective pressure had a greater influence on the evolution of ALV-J diversity. The results suggest that more ALV-J clones from different organs and tissues of infected birds should be sequenced and compared to better understand viral evolution and molecular epidemiology in the field.
    Full-text · Article · Jul 2016
    • "In chickens, ALVs can be further classified into six subgroups: A, B, C, D, E, and J. Notably, ALV-J, which was first isolated in chickens in 1988, has been shown to induce myeloid leukosis in broiler chickens (Payne et al., 1992; Payne and Nair, 2012 ). In fact, the high mortality rate associated with tumour formation and progression as well as with decreased fertility in these animals has caused major economic losses in the poultry industry worldwide, including that in China (Gao et al., 2012 ). Although detection of ALV-J infection in chickens was first reported in 1997, ALV-J has become more widespread in both commercially and locally bred chickens in various regions of the country in recent decades (Cui et al., 2006). "
    [Show abstract] [Hide abstract] ABSTRACT: Subgroup J avian leukosis virus (ALV-J) is an oncogenic retrovirus known to induce tumor formation and immunosuppression in infected chickens. One of the organs susceptible to ALV-J is the bone marrow, from which specialized antigen-presenting cells named dendritic cells (BM-DCs) are derived. Notably, these cells possess the unique ability to induce primary immune responses. In the present study, a method of cultivating and purifying DCs from chicken bone marrow in vitro was established to investigate the effects of ALV-J infection on BM-DC differentiation or generation. The results indicated that ALV-J not only infects the chicken bone marrow mononuclear cells but also appears to inhibit the differentiation and maturation of BM-DCs and to trigger apoptosis. Moreover, substantial reductions in the mRNA expression of TLR1, TLR2, TLR3, MHCI, and MHCII and in cytokine production were detected in the surviving BM-DCs following ALV-J infection. These findings indicate that ALV-J infection disrupts the process of bone marrow mononuclear cell differentiation into BM-DCs likely via altered antigen presentation, resulting in a downstream immune response in affected chickens.
    Full-text · Article · Jun 2016
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