Intronic deletions of tva receptor gene decrease the susceptibility to infection by avian sarcoma and leukosis virus subgroup A

The group of avian sarcoma and leukosis virus (ASLV) in chickens contains six highly related subgroups, A to E and J. Four genetic loci, tva, tvb, tvc and tvj, encode for corresponding receptors that determine the susceptibility to the ASLV subgroups. The prevalence of ASLV in hosts may have imposed strong selection pressure toward resistance to ASLV infection, and the resistant alleles in all four receptor genes have been identified. In this study, two new alleles of the tva receptor gene, tvar5 and tvar6, with similar intronic deletions were identified in Chinese commercial broilers. These natural mutations delete the deduced branch point signal within the first intron, disrupting mRNA splicing of the tva receptor gene and leading to the retention of intron 1 and introduction of premature TGA stop codons in both the longer and shorter tva isoforms. As a result, decreased susceptibility to subgroup A ASLV in vitro and in vivo was observed in the subsequent analysis. In addition, we identified two groups of heterozygous allele pairs which exhibited quantitative differences in host susceptibility to ASLV-A. This study demonstrated that defective splicing of the tva receptor gene can confer genetic resistance to ASLV subgroup A in the host.

abrogates binding of the subgroup J envelope glycoprotein to ASLV-J-resistant cells, which discriminates sensitive from resistant avian species 16 .
Although research on host genetic resistance to specific ASLVs has progressed in recent years, the current status of host resistance to infection by specific ASLVs in Chinese chickens is unknown. In order to identify additional resistance alleles of the tva receptor gene, we screened a panel of Chinese commercial broiler lines, which have undergone strict virus eradication management. Here, we characterized two alleles of the tva receptor gene, named tva r5 and tva r6 , respectively, with similar intronic deletions encompassing the deduced branch point signal within the first intron and leading to defective splicing of tva mRNA. We also identified two groups of heterozygous allele pairs which exhibited discrepant susceptibility to subgroup A ASLV. To our knowledge, this study is the first to report genetic variations within the tva receptor gene that result in a quantitative effect on ASLV-A susceptibility and pathogenesis in Chinese chickens.

Results
Polymorphisms in the first intron of tva receptor gene and genotyping. To dissect genetic variations within the tva receptor gene in a panel of Chinese commercial broiler lines, the genomic region of the tva gene in each bird was amplified and sequenced. Four novel variants in the same region within the first intron of the tva gene were identified in the Chinese chickens surveyed. In addition to the tva r3 and tva r4 alleles 14 , sequencing revealed one new variant with a deletion of the sequence CGCTCACCCC (nucleotides 502 to 511) and another new variant with a deletion of the sequence CGCTCACCCCGCCCC (nucleotides 502 to 516) (Fig. 1). We designated these two novel variants as tva r5 and tva r6 alleles. These four variants in the tva receptor gene (tva r3 , tva r4 , tva r5 and tva r6 ) together may be considered a multiple allele, denoted here as tva mut502-516 . The genotypic frequencies of the tva mut502-516 multiple allele within the tva receptor gene in the Chinese commercial broiler lines surveyed are presented in Table 1. All genotypes of the tva mut502-516 multiple allele were identified, including the tva s/s , tva r3/r3 , tva r4/r4 , tva r5/r5 and tva r6/r6 homozygotes, as well as two groups of heterozygous genotypes. One group of heterozygous genotypes is sensitive to ALSV-A, included tva s/r3 , tva s/r4 , tva s/r5 and tva s/r6 , while the other group is resistant to ALSV-A, comprised of tva r3/r4 , tva r3/r5 , tva r3/r6 , tva r4/r5 , tva r4/r6 and tva r5/r6 . This difference between the two groups of heterozygous genotypes was identified in the subsequent analysis. The genotypes and their frequencies in Chinese commercial broiler lines were obviously different ( Table 1).

Identification of novel tva receptor gene splicing variants.
Sequencing revealed that both the tva r5 and tva r6 alleles delete the CTCAC consensus sequence of the branch point signal 17 (Fig. 1). These deletions include the A nucleotide, which is required for the first cleavage-ligation step of the splicing reaction 18 . Therefore, we hypothesized that the deletion mutations may disrupt splicing of the tva precursor mRNA (pre-mRNA) ( Fig. 2A).
To determine whether these specific mutations would interfere with the process of tva pre-mRNA splicing, the full-length tva coding sequence from lives samples of defined origin and from DF-1 cells as a control were amplified by RT-PCR using primers crossing the entire coding sequence of the tva receptor gene (Fig. 2B). The cDNA products from the DF-1 cells and tva s/s samples from lives were of the expected sizes corresponding to the longer and shorter tva isoforms. 13 However, the cDNA products from the tva r5/r5 and tva r6/r6 homozygous samples from lives, as well as those from the tva r3/r4 , tva r3/r5 , tva r3/r6 , tva r4/r5 , tva r4/r6 and tva r5/r6 heterozygous samples, were longer by approximately 200 bp (Fig. 2C), similar to those of the tva r3/r3 and tva r4/r4 homozygous samples 14 . These longer sequences suggest that the first intron is retained (i.e., nucleotides 337 to 530, based on the published tva s allele genomic sequence, GenBank accession number: AY531262.1).Interestingly, the products from tva s/r3 , tva s/r4 , tva s/r5 and tva s/r6 heterozygous samples from lives by RT-PCR have three transcripts. Except for one transcript was the same with the normal shorter tva isoforms, the another two transcripts were longer by approximately 200 bp compared with those of the longer and shorter tva isoforms, respectively (Fig. 2C).
In order to verify the retention of intron 1 in the transcripts of the tva r5 and tva r6 alleles, as well as to identify the heterozygotes, their RT-PCR products were then cloned into the T-vector for sequencing. Separate sequence analysis of PCR products revealed that the retention of the first intron in the RT-PCR products and a deletion of the corresponding sequence (CGCTCACCCC or CGCTCACCCCGCCCC) within the first intron in both the longer and shorter tva isoforms of tva r5/r5 and tva r6/r6 homozygotes, as well as the group of heterozygotes included tva r3/r4 , tva r3/r5 , tva r3/r6 , tva r4/r5 , tva r4/r6 and tva r5/r6 (Fig. 2D). Although these similar sequencing  results were found in the tva s/r3 , tva s/r4 , tva s/r5 and tva s/r6 heterozygotes, the splicing of the shorter tva isoforms in these heterozygotes were normal (Fig. 2D). Therefore, the retention of intron 1 in both the longer and shorter tva isoforms of the tva r5 and tva r6 alleles were confirmed via alternative splicing, as well as identified in the heterozygotes. The position of the consensus sequence (CTCAC) of the putative branch point signal positioned at 23 nucleotides upstream of the 39 splice signal, which is within the optimal distance from the intron-exon boundary [19][20] , suggesting that it is used in splicing out the first intron from the tva pre-mRNA and its deletion would abrogate the splicing event. Retention of intron 1 in the tva r5 allele changes the translational reading frame of both forms of the tva mRNAs immediately after the end of exon 1 and introduces a premature stop codons TGA in exon 4 or 5 of the longer or shorter mRNA, respectively (Fig. 2D). These two nonspliced mRNAs should be translated into proteins of 196 and 177 amino acids, respectively. However, the retention of the first intron in the tva r6 allele changes the translational reading frame of both forms of the tva mRNAs immediately after the end of exon 1 and introduces premature a stop codon TGA in exon 3 of both the longer and shorter mRNA, causing both nonspliced mRNAs to be translated into proteins of 167 amino acids (Fig. 2D). Therefore, we have demonstrated that intronic deletions comprising the deduced branch point signal within the first intron lead to inefficient splicing of tva mRNA.
Construction of ASLV subgroup A reporter virus. To simplify the procedure and improve the quantitative assessment of ASLV-A infection, we constructed an expression vector RCASBP(A)-EGFP to produce the green fluorescent protein (GFP) reporter virus of subgroup A specificity based on the RCAS retrovirus vector 21 (Fig. 3A). The EGFP gene was successfully cloned into the RCASBP(A) vector (Fig. 3B). The resulting replication-competent virus, RCASBP(A)-EGFP, was propagated in DF-1 cells and reached a titer of 10 6 IU/mL. We then checked the subgroup specificity of the RCASBP(A)-EGFP virus by infection of chicken DF-1 cells and human 293 T cells. The proportion of GFP-positive cells in virusinoculated DF-1 cells gradually increased over time, providing evidence for the replication and spread of virus (Fig. 3C). By comparison, less than 0.1% of inoculated as well as non-inoculated 293 T cells were GFP-positive, which may be attributed to the background auto-fluorescence (data not shown).
Reduced susceptibility of chinese chickens to subgroup A ASLV. Since two independent intronic deletions that resulted in defective splicing of tva mRNA were identified, we hypothesized that these deletion mutations may confer their functional significance. To determine the effects of tva r5 and tva r6 alleles, as well as their heterozygous variants, on subgroup A ASLV susceptibility, CEFs of defined origin were infected with the RCASBP(A)-EGFP reporter virus, a replication-competent ASLV vector encoding EGFP, and the time course of infection was followed by quantitating the percentage of green fluorescent cells by flow cytometry over 7 subsequent days. DF-1 cells (tva s/s ) are susceptible to subgroup A ASLV and thus were used as a positive control. As expected, the DF-1 cells was efficiently infected by RCASBP(A)-EGFP, with more than 60% of the cells being infected on day 1 post-infection, and virtually complete infection of cells was achieved by day 3 (Fig. 4A). However, a very different result was observed when the tva r5/r5 and tva r6/r6 CEFs were infected with RCASBP(A)-EGFP. Both the tva r5/r5 and tva r6/r6 CEFs were much less efficiently infected with RCASBP(A)-EGFP, with only 2.0% and 1.0% of the cells infected on day 1, respectively, and the virus spread very slowly, with only 19.8% and 20.5% of the cells infected by day 7, respectively ( Fig. 4C). We also determined the susceptibility of tva r3/r3 and tva r4/r4 CEFs to subgroup A ASLV, and the results were consistent with those reported by Reinišová et al. 14 (date not shown). In a separate experiment, quantitative differences in host susceptibility to ASLV-A were observed between heterozygotes tva s/s tva s/r3 tva s/r4 tva s/r5 tva s/r6 tva r3/r3 tva r4/r4 tva r5/r5 tva r6/r6 tva r3/r4 tva r3/r5 tva r3/r6 tva r4/r5 tva r4/r6 tva r5/r6 202     which could be classified into two groups. The tva s/r5 and tva s/r6 CEFs were efficiently infected with RCASBP(A)-EGFP at a similar rate to that of DF-1 cells (tva s/s ) (Fig. 4B), with 94.3% and 95.8% of the cells infected by day 7, respectively ( Fig. 4C). However, the tva r3/r4 , tva r3/r5 and tva r5/r6 CEFs were inefficiently infected by RCASBP(A)-EGFP (Fig. 3B), with only 27.0%, 34.6% and 18.4% of the cells being infected at 7 days post-infection, respectively (Fig. 4C). These results clearly indicated the inefficient infection and slow spread of the subgroup A ASLV in the tva r5/r5 and tva r6/r6 homozygous CEFs and the tva r3/r4 , tva r3/r5 and tva r5/r6 heterozygous CEFs.
Taken together, these results clearly demonstrated that the deletion of the branch point signal within the first tva intron disrupt mRNA splicing of the tva receptor gene, which explains the decreased susceptibility of the homozygous tva r5/r5 and tva r6/r6 birds and the heterozygous tva r3/r4 , tva r3/r5 , tva r3/r6 , tva r4/r5 , tva r4/r6 and tva r5/r6 birds to infection by subgroup A ASLV.

Discussion
In the present study, we described the identification of two similar intronic deletions encompassing the deduced branch point signal in the tva receptor gene, which were shown to significantly decrease the susceptibility of chickens to infection by ASLV-A. Furthermore, we identified two groups of heterozygotes based on their quantitative differences in host susceptibility to ASLV-A. This study is the first to report genetic defects in the tva receptor gene that account for a quantitative effect on ASLV-A susceptibility and pathogenesis in Chinese chickens. The altered susceptibility to infection by ASLV-A was observed both in cultured cells and in chicks challenged with subgroup A ALV.
Alternative splicing allows individual genes to produce two or more variant mRNAs, which in many cases encode functionally distinct proteins 19,[22][23] . Indeed, two tva receptor gene splicing variants created by alternative splicing were identified previously 7,24 . Binding of more than one receptor, probably two, is needed for entry of virions via the Tva800 receptor that is encoded by the shorter tva forms, whereas binding of just one Tva950 receptor encoded by the longer tva forms is sufficient for successful entry 25 . Alternative splicing can be modulated by variation both in the cis genomic splicing signals and in the cellular pathways that regulate splicing 22,[26][27] . In this study, we identified the alternative splicing of the tva r5 and tva r6 resistant alleles resulting from intronic deletions encompassing the deduced branch point signal, leading to the retention of the first intron and introduction of premature TGA stop codons in the longer and shorter tva forms (Fig. 2). Alternative splicing events generally create a premature termination codon that would cause the resulting www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 9900 | DOI: 10.1038/srep09900 mRNA to be degraded by nonsense-mediated mRNA decay [28][29] . Therefore, alternative splicing of mRNAs that changes the encoded proteins has profound functional effects 23,[30][31] . Experimental analysis of distinct protein isoforms showed that alternative splicing regulates binding between proteins, between proteins and nucleic acids, as well as between proteins and membranes 23,[31][32][33] . In most cases, the binding  www.nature.com/scientificreports affinity is modulated, but the binding is not abolished completely [34][35][36][37] . The tva r3 and tva r4 alleles harbor an intronic deletion of the branch point and disrupt splicing of the tva receptor gene, leading to decreasing binding affinity between the Tva receptor and envelope glycoproteins of ASLV-A and display of the Tva receptor on the cell surface, and consequently reduced host susceptibility to subgroup A ASLV 14 . Given that the tva r3 , tva r4 , tva r5 and tva r6 alleles all contain a deletion of the same branch point signal, the same effects are also likely exerted by the tva r5 and tva r6 alleles in homozygotes, as well as the tva r3/r4 , tva r3/r5 , tva r3/r6 , tva r4/r5 , tva r4/r6 and tva r5/r6 resistant heterozygotes, as evidenced by their significantly decreased susceptibility to infection by ASLV-A. In contrast to resistant heterozygotes, the tva s/r3 , tva s/r4 , tva s/r5 and tva s/r6 susceptible heterozygotes may retain a receptor conformation that is at least partially suitable for binding of ASLV-A envelope glycoproteins and subsequent viral infection. A previously similar study were reported by Kučerová 16 , the deletion of W38 completely abrogates receptor activity and explains the resistance of chukar to the J subgroup of ALV; however, alleles with W38 replaced by G or E conferred susceptibility to the virus when overexpressed in the virus entry assay. Alternative splicing is not only important for normal cellular functions but also frequently is involved in disease pathogenesis 27,[38][39][40][41] . Interestingly, published studies have identified intronic deletions causing exon skipping or intron retention in the human LDLR gene of patients with familial hypercholesterolemia [42][43][44][45][46] . Because the human LDLR gene is homologous to the tva receptor gene, such deletions as described above may be speculated as a common mechanism of mutagenesis within this gene family.
From the point of view of virus-host coevolution, it is tempting to speculate that the tva r5 and tva r6 alleles with decreased susceptibility to ASLV-A in Chinese chickens have been selected by pressure from subgroup A ASLV. Although eradication management strategies at the breeder level and high biosecurity level of flock production are used to control avian leukosis [47][48] , these conventional methods are not a practical means of completely eliminating the occurrence and spread of infection in developing countries [49][50][51] . The prevalence of ASLV in chicken populations may have imposed a strong selection pressure toward resistance or at least decreased susceptibility to ASLV infection. ASLV infection is known to cause a variety of neoplastic disease conditions and other production problems in affected flocks 52 . Under these conditions, a wide prevalence of resistance alleles in the ASLV receptor genes can be expected. To date, resistance alleles in the tva, tvb and tvc genes have been found in particular inbred lines of White Leghorns [11][12][13][14][15] . The resistance conferred by some alleles are caused by premature termination codons or frame shift mutations, such as tva r2 , tvb r and tvc r11-13 , which do not encode any product that can carry out normal cellular functions, but their potentially detrimental impact on resistant birds is unknown. Given the presence of such counter-selection and rapid evolution of ASLV envelope glycoproteins, complete resistance to ASLV entry can appear but cannot prevail and be fixed in the chicken population. Hence, we suggest that genetic variations with modest effects on both ASLV entry and natural receptor functions provided a positive selective advantage in the chicken population. Selection of mutant Tva proteins that have lost the ability to function as an ASLV-A receptor while possibly retaining the''normal'' Tva function may have provided the greatest selective advantage, resulting in fixing of the tva r5 and tva r6 loci in the germ lines of certain lines of chickens.
The tva r3 , tva r4 , tva r5 and tva r6 resistant alleles are prevalent in Chinese commercial broiler lines (Table 1), indicating that the potential for genetic improvement of resistance to ASLV-A is great and selective breeding for chickens genetically resistant to ASLV-A is feasible. Despite the potential benefits to be derived from breeding for enhanced resistance to ASLV-A, evaluating whether trade-offs between disease resistance and other economically important traits of chickens exist would be prudent. In fact, in a separate study we have evaluated the effects of genetic resistance to subgroup A ASLV on growth, feed efficiency and carcass traits using an F2 resource population by reciprocally crossing Huiyang Bearded chickens and fast-growing Chinese yellow broilers. The results demonstrated that genetic selection for tva r3 , tva r4 , tva r5 and tva r6 resistant loci can improve genetic resistance to subgroup A ASLV, but does not compromise production performance (data not shown). Selection for genetic resistance to subgroup B ASLV has been found to have no negative effect on laying performance in White Leghorn hens 53 . Furthermore, genetic selection for tva r and tvb r alleles can improve laying performance and growth rate, as well as greatly reduce lymphoid leukosis in White Leghorn hens [54][55] .
In conclusion, our study identified two novel genetic resistant alleles, tva r5 and tva r6 , in Chinese commercial broilers. These two resistant alleles both harbor an internal intronic deletions comprising the deduced branch point signal within the first intron and leading to alternative splicing of the tva receptor gene, resulting in significantly decreased the susceptibility to subgroups A ASLV. We also demonstrated a difference in the susceptibility to subgroup A ASLV between tva r3/r4 , tva r3/r5 , tva r3/r6 , tva r4/r5 , tva r4/r6 and tva r5/r6 heterozygotes and tva s/r3 , tva s/r4 , tva s/r5 and tva s/r6 heterozygotes. This study provides valuable insight into mechanisms of genetic resistance to ASLV and retrovirus-host coevolution.

Methods
Ethics statement. The animal experiments were conducted in accordance with the guidelines of the Guangdong province on the review of welfare and ethics of laboratory animals approved by the Guangdong province administration office of laboratory animals (GPAOLA). All the animal procedures were approved by the Animal Care Committee of the College of Animal Science, South China Agricultural University, Guangzhou, China (approval ID: 201004152).
Amplification and analysis of tva alleles from commercial broiler lines. Genomic DNA was prepared from blood samples of 22 commercial broiler lines using phenolchloroform extraction. These commercial broiler lines were maintained at Guangdong Wen's Food Group Co., Ltd. The sequence of the genomic region including exon 1, intron 1 and exon 2 of the tva gene was amplified using forward TVA1 primer 59-GTTCAGCAGATCCTCATCTCCCG-39 and reverse TVA2 primer 59-GGCCATTGTGCGATCTAAGAGGG-39. The PCR procedure was as follows: an initial denaturation at 95uC for 5 min, followed by 35 cycles of 94uC for 30 s, 67uC for 45 s and 72uC for 90 s, and a final extension of 72uC for 10 min with KOD-Plus Neo (Toyobo, Tokyo, Japan). In total, 712 birds from different commercial broiler lines were genotyped. The final PCR product with an expected length of 1308 bp was directly sequenced using an ABI 3730 sequencer (Applied Biosystems, Foster City, CA, USA).
Splicing analysis by RT-PCR. Total RNAs from lives of commercial broiler lines 208, 419, 502 and 603 were isolated using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. cDNA was obtained by reverse transcription of 1 mg of total RNA with ReverTra AceH qPCR RT Master Mix with gDNA Remover (Toyobo, Tokyo, Japan). The whole tva coding sequence was amplified using the forward TVA3 primer 59-CCGGCATGGTGCGGTTGTTG-39 containing the tva initiation codon and the reverse TVA4 primer 59-AGCCAGGTTCCACGGTCAGC-39, which is complementary to the 39 untranslated region. The PCR conditions were as follows: an initial denaturation at 95uC for 5 min, followed by 30 cycles of 94uC for 30 s, 58uC for 30 s and 72uC for 50 s, and a final extension of 72uC for 5 min with KOD FX (Toyobo, Tokyo, Japan). To identify the transcripts of the tva alleles and their heterozygotes, the RT-PCR products were visualized by electrophoresis on 2% agarose gels. The purified RT-PCR products were ligated into the pMD19-T vector (TaKaRa, Dalian, China) and sequenced using an ABI 3730 sequencer (Applied Biosystems).
Construction of ASLV subgroup A reporter vector and virus propagation. The EGFP gene was isolated from the pVAX1-EGFP vector (stored at our laboratory) as an EcoRI-XbaI fragment and cloned into the Cla12Nco adapter plasmid at the same restriction sites 21,56 . The resulting clones with the proper orientation of EGFP were isolated as ClaI fragments and subcloned into the corresponding site of the RCASBP (A) vector 21 , which was kindly obtained from Stephen H. Hughes (HIV Drug Resistance Program, National Cancer Institute, USA). The resulting expression construct was designated RCASBP(A)-EGFP. The RCASBP(A)-EGFP virus, transducing the EGFP reporter gene, was propagated by transfection of plasmid DNA containing the reporter vector into DF-1 cells, which are free of closely related endogenous retrovirus loci. Transfection was performed by using the X-tremeGENE 9 transfection reagent (Roche, Basel, Switzerland) according to the manufacturer's instructions. Infection and virus spread were observed as an increasing proportion of GFP-positive cells, and virus stocks were harvested on day www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 9900 | DOI: 10.1038/srep09900 9 post-transfection. The cell supernatants were cleared of debris by centrifugation at 2,000 3 g for 10 min at 10uC, and aliquoted viral stocks were stored at 280uC. The virus titer was determined by terminal dilution and subsequent infection of DF-1 cells, reached 10 6 IU/mL.
Preparation of CEFs and cell culture. Primary CEFs were prepared from 9 to 11-day-old embryos from commercial broilers lines 208, 419, 502 and 603. The procedure was described previously 57 . The genotypes of CEFs were determined by direct sequencing as described above. All embryo fibroblasts, as well as the chicken permanent cell line DF-1 58 , were propagated in growth medium containing Dulbecco's modified Eagle's medium (DMEM) (Gibco/Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (Gibco/Invitrogen, Carlsbad, CA, USA), and penicillin/streptomycin (100 mg/ml each) at 37uC and 5% CO 2 .
RCASBP(A)-EGFP virus spread assayed by fluorescence-activated cell sorting (FACS). CEFs of defined origin were mock infected or infected by incubation with diluted RCASBP(A)-EGFP virus stock. Briefly, the CEFs were seeded in triplicate wells at a density of 5 3 10 4 per well in a 24-well plate and inoculated with 5 3 10 5 IU of RCASBP(A)-EGFP virus 24 h after seeding. The virus was applied in 0.25 mL medium for 1 h. The percentage of GFP-positive cells was quantitated by FACS using a Cytomics FC 500 analyzer (Beckman Coulter, Brea, CA, USA) on days 1, 2, 3 and 7 post-infection. For FACS analysis, the cells of three wells were trypsinized, washed in phosphate-buffered saline (PBS) and then analyzed.
Animal experiment. The animal experiment design included two independent experiments with 100 birds each. Chicks randomly collected from commercial broilers lines 208, 419, 502 and 603 were randomly divided into four groups with 25 birds each. Chicks were maintained in four negatively-pressured biosecurity isolators under quarantine conditions and provided with water and commercial feed ad libitum. One-day-old chicks were inoculated with 2.4 S/P of avian leukosis virus subgroup A (ALV-A) strain GD08 49 , which was kindly provided by Weisheng Cao at South China Agricultural University, P. R. China, in 0.2 mL into the abdominal cavity. These chicks were inoculated once again at five days of age. A whole blood sample from each one-day-old chick was drawn from the wing vein and used for genomic DNA isolation by the phenol-chloroform method for genotyping as above described. To determine whether blood samples of chicks were positive for ASLV-A at one month post-infection, a whole blood sample from each bird was drawn for preparing total RNA using the TRIZOL reagent (Invitrogen, Carlsbad, CA, USA). The coding sequence of the env gene of ALV-A was amplified using the forward H5 primer 59-GGATGAGGTGACTAAGAAAG-39 and reverse H6 primer 59-AGAGAAAGAGGGGTGTCTAAGGAGA-39 59 . The PCR conditions were as follows: a reverse translation at 50uC for 30 min, then an initial denaturation at 94uC for 3 min, followed by 30 cycles of 94uC for 30 s, 56uC for 30 s and 72uC for 60 s, and a final extension of 72uC for 5 min with PrimeScriptH One Step RT-PCR Kit Ver. 2 (TaKaRa, Dalian, China). To identify the infection status of ALV-A, the PCR products were visualized by electrophoresis on 2% agarose gels.