A premature stop codon within the tvb receptor gene results in decreased susceptibility to infection by avian leukosis virus subgroups B, D, and E

Avian leukosis virus (ALV) is an oncogenic virus causing a variety of neoplasms in chickens. The group of avian leukosis virus in chickens contains six closely related subgroups, A to E and J. The prevalence of ALVs in hosts may have imposed strong selective pressure toward resistance to ALVs infection. The tvb gene encodes Tvb receptor and determines susceptibility or resistance to the subgroups B, D, and E ALV. In this study, we characterized a novel resistant allele of the tvb receptor gene, tvbr3, which carries a single-nucleotide substitution (c.298C>T) that constitutes a premature termination codon within the fourth exon and leads to the production of a truncated TvbR3 receptor protein. As a result, we observed decreased susceptibility to infection by ALV-B, ALV-D and ALV-E both in vitro and in vivo, and decreased the binding affinity of the TvbR3 receptor for the subgroups B, D, and E ALV envelope glycoproteins. Additionally, we found that the tvbr3 allele was prevalent in Chinese broiler lines. This study demonstrated that premature termination codon in the tvb receptor gene can confer genetic resistance to subgroups B, D, and E ALV in the host, and indicates that tvbr3 could potentially serve as a resistant target against ALV-B, ALV-D and ALV-E infection.


INTRODUCTION
Avian leukosis viruses (ALVs) are members of avian retroviruses that induce diverse pathotypes of neoplastic diseases in chickens [1]. The spread of ALVs in poultry flocks worldwide has caused enormous economic losses [2]. Currently, there are no effective vaccines or drugs against ALVs. The eradication managements and biosecurity strategies are carried out to control ALVs infection [3]. However, these conventional methods cannot completely eliminate the spread of ALVs in chickens in China and Southeast Asia [4,5]. A more effective antiviral www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 62), pp: 105942-105956 Research Paper www.impactjournals.com/oncotarget strategy, therefore, may lie in breeding for resistance to infection by ALVs [6,7].
The group of highly related avian leucosis viruses in chickens contains (A-E and J) six subgroups. ALVs infection is mediated by the interactions of viral envelope proteins with specific host receptors [8,9]. The tva, tvb, and tvc loci in chickens encode the Tva, Tvb, and Tvc proteins, which are receptor for ALV-A, ALV-B/ D/E, and ALV-C, respectively [10][11][12][13][14]. Two naturally occurring tvb susceptible alleles in chickens have been identified. The tvb s1 allele encodes the Tvb S1 receptor and confers susceptibility to ALV-B, ALV-D, and ALV-E, while the tvb s3 allele encodes Tvb S3 receptor and confers susceptibility to only ALV-B and ALV-D [15]. The chicken Na+/H+ exhanger type 1 (chNHE1), which is encoded by the tvj locus, was identified as the receptor for ALV-J [16]. In addition, chicken Annexin A2 (chANXA2) was also identified as a receptor for ALV-J [17]. The resistance to infection by a particular ALV subgroup can be caused by genetic alterations of specific receptor gene. Resistant alleles, tva r , tvb r , tvc r , and tvj r , have been identified in all four receptor loci. The genetic defects in the resistant alleles can result in premature stop codons, frame-shift mutation or intronic deletions in the receptor genes [14,[17][18][19][20][21], and substitutions of the critical amino acid residues in the receptor protein sequence [15,22]. Additionally, the deletion of the tryptophan 38 in the first extracellular loop of NHE1 receptor accounts for the resistance to ALV-J in galliform species [23,24]. Consequently, the resistant alleles not only confer host resistance to ALVs infection, but also provide valuable insight into antiviral strategies.
To further identify resistant alleles of ALV receptor genes, we screened the natural polymorphisms in specific receptor genes as a source of the host resistance in Chinese broiler lines [25]. In our previous study, we have identified two tva resistant alleles with decreased susceptibility to infection by ALV-A [21]. Here, we describe the decreased susceptibility to subgroups B, D, and E ALV in Chinese commercial broilers and respective molecular defect in tvb receptor gene.

Identification of the tvb r3 allele that introduces a premature stop codon
In order to identify the natural mutations critical for resistance to subgroups B, D, and E ALVs infection, we amplified and sequenced the whole genomic region of the tvb receptor gene using 4 sets of specific primers (Supplementary Figure 1A), and dissected genetic mutations in the tvb receptor gene of a panel of 15 Chinese commercial broiler lines. During this process, we identified a single-nucleotide substitution within exon 4 of the tvb gene in Chinese chickens surveyed. In detail the sequence revealed the nucleotide change C>T located at 298 position of the tvb cDNA (c.298C>T) ( Figure 1A and 1B). Since this c.298C>T substitution causes an in-frame stop codon in the open reading frame (ORF) of the tvb gene, implying a role in the resistance to subgroups B, D, and E ALVs infection [14,18]. Thus, we designated this novel variant as tvb r3 allele. To confirm the c.298C>T substitution in tvb r3 transcript, we amplified the entire tvb coding sequence from the cDNA of tvb s1/s1 (wild-type) and tvb r3/r3 birds, and sequenced the RT-PCR products of the complete transcript (Supplementary Figure 1B). We then determined the nucleotide and deduced amino acid sequences of the tvb cDNA products and compared the nucleotide sequences of tvb s1 with the nucleotide sequences of tvb r3 allele. Only a single nucleotide difference was found, which changed the cytosine residue located 298 nucleotides downstream of the start methionine codon in tvb s1 to be a thymidine residue in tvb r3 ( Figure 1C). Accordingly, these findings further indicated this single-nucleotide substitution changed codon 100 (CAG, glutamine) to a termination codon (UAG) in the Tvb S1 receptor protein.

Mutant tvb mRNAs are targeted by the nonsense-mediated mRNA decay (NMD)
Since the c.298C>T mutation causes a premature termination codon in the 4 st of the 10-exon tvb receptor gene. Thus, we assume that mutant tvb mRNAs may undergo NMD [26]. In order to verify this assumption, we firstly compared the tvb transcript levels between wildtype and mutant-type in blood of tvb s1/r3 birds by direct sequencing of RT-PCR products that encompassed the c.298C>T substitution. The results shown that mutant tvb mRNA was scarcely detectable (Figure 2A). Then, we compared the mRNA levels of tvb gene in blood of birds of tvb s1/s1 , tvb s1/r3 and tvb r3/r3 genotypes using quantitative RT-PCR (qRT-PCR). In contrast to homozygous wild-type birds, highly significant reductions in tvb mRNA levels in tvb s1/r3 individuals were observed, 68%±5% and 52%±4% of control values for the 5' and 3' systems, respectively ( Figure 2B). As for tvb r3/r3 birds, the tvb mRNA levels were less than 5% that of wild-types ( Figure 2B). Altogether, the allelic imbalance test and qRT-PCR experiments both supported the notion that the mutant tvb transcripts were degraded by NMD.

The tvb r3 allele generates a truncated Tvb receptor protein product
Since the tvb r3 introduces a premature stop codon, it is predicted to generate a truncated Tvb receptor. To confirm this prediction, we estimated the effect of the tvb r3 allele on the expression of Tvb protein. To this aim, Tvb expression vectors with wild-type (pEGFPC1-tvb s1 ) and mutant tvb (pEGFPC1-tvb r3 ) were constructed and transfected into www.impactjournals.com/oncotarget 293FT cells ( Figure 3A). The expression of Tvb protein was analyzed by Western blotting analyses ( Figure 3B). The GFP protein and Tvb/GFP fusion protein migrate as broad bands (27 to 70 KD). The band corresponding to Tvb S1 /GFP fusion protein with a molecular weight of approximately 70 KD was observed (43 KD for Tvb S1 plus 27 KD for GFP). However, the band corresponding to Tvb R3 /GFP fusion protein with a smaller molecular weight of ca.37 KD was detected (10 KD for Tvb R3 plus 27 KD for GFP). The endogenous Tvb expression was hardly visible, as the signal in mock-transfected 293FT cells ( Figure 3B). Consequently, these results formally demonstrated that the tvb r3 allele generates a truncated Tvb R3 protein product at cysteine-rich domains 2 (CRD2) level (NP_989446.2:p.Thr99 * ).

The tvb r3 allele reduces the susceptibility of Chinese chickens to infection by subgroups B, D, and E ALV in vitro
To determine the effects of the tvb r3 allele on ALVs susceptibility, we first evaluated the infection and spread of the subgroups B, D, and E ALV reporter viruses in wildtype tvb s1/s1 , tvb s1/r3 and tvb r3/r3 chicken embryo fibroblasts (CEFs). To this purpose, we constructed the RCASBP(B)-EGFP, RCASBP(D)-EGFP and RCASBP(E)-EGFP reporter vectors, transducing the enhanced green fluorescent protein (EGFP) reporter gene, based on the RCAS retrovirus vectors (Supplementary Figure 2A) [27]. The resulting replication-competent ALV recombinant viruses, RCASBP(B)-EGFP, RCASBP(D)-EGFP, and RCASBP(E)-EGFP were obtained as previously described (Supplementary Figure 2B) [21]. CEFs of different genotypes were infected with RCASBP(B)-EGFP, RCASBP(D)-EGFP, and RCASBP(E)-EGFP reporter viruses, and the time course of infection were followed as the percentage of GFP-positive cells quantified by fluorescence-activated cell sorting (FACS) on seven subsequent days. The tvb s1/s1 CEFs were used as a positive control since they are susceptible to subgroups B, D, and E ALV. As expected, RCASBP(B)-EGFP, RCASBP(D)-EGFP, and RCASBP(E)-EGFP infected the tvb s1/s1 CEFs efficiently, with almost one-half of the cells being infected by the day 1, and spread quickly, reaching virtually complete infection of cells on day 7 ( Figure 4A, 4B, 4C). However, when the tvb r3/r3 CEFs were infected with RCASBP(B)-EGFP, RCASBP(D)-EGFP, or RCASBP(E)-EGFP reporter viruses, very different results were observed. RCASBP(B)-EGFP, RCASBP(D)-EGFP, and RCASBP(E)-EGFP infected the tvb r3/r3 CEFs inefficiently, with only 3.9%, 4.5% and 4.3% of the cells being infected on day 1, and the virus spread slowly, only approaching 21.2%, 23.6%, and 21.3% of infected cells on day 7 ( Figure 4A, 4B, 4C). The GFP-negative and GFP-positive cells are obviously disparate, which were shown by the presence of two independent peaks in the FACS histogram ( Figure 4D). We also determined the susceptibility of tvb s1/ Comparing tvb mRNA levels in the blood of wild-type, tvb s1/r3 and tvb r3/r3 birds. Data are shown for two amplicons at the 5' and 3' ends of the tvb mRNA, respectively. Error bars correspond to standard errors over three replicates per sample. www.impactjournals.com/oncotarget r3 CEFs to subgroups B, D, and E ALV reporter viruses, which exhibits an infection property between the tvb r3/r3 CEFs and its wild-type counterpart ( Figure 4A, 4B, 4C).
In order to further verify these results, we next infected the CEFs with ALV-B wild virus and determined its growth kinetics in CEFs of defined origin by RT-PCR over a period of 6 days. The results showed that ALV-B strain SDAU09C2 has a significant growth advantage in tvb s1/s1 CEFs over in tvb r3/r3 CEFs, and a moderate growth advantage in tvb s1/r3 CEFs over in tvb r3/r3 CEFs ( Figure  5). These findings were consistent with the infection by reporter viruses and suggested that the tvb r3 allele reduces the susceptibility of tvb r3/r3 CEFs to infection by ALV-B. Taken these results together, we clearly demonstrated that the tvb r3 allele resulted in inefficient infection and slow spread of subgroups B, D, and E ALV in tvb r3/r3 chickens in vitro.

The tvb r3 allele reduces the susceptibility of Chinese chickens to infection by ALV-B in vivo
To further evaluate the effects of the tvb r3 allele on the infection and replication of the ALV-B in vivo, 1-day-old chicks were infected with ALV-B strain SDAU09C2 through the abdominal cavity. At one month postinfection, the infection status of ALV-B in each chick was determined. As shown in Table 1, the tvb r3/r3 chicks showed a significantly lower infection proportion than that of tvb s1/s1 chicks, and a lower infection proportion than that of tvb s1/r3 chicks. In contrast to 100% of infected tvb s1/s1 chicks and 50% of infected tvb s1/r3 chicks, only 26.3% of the tvb r3/r3 chicks being infected by ALV-B strain SDAU09C2. These results demonstrated that the in vivo infection and replication coincides with spread of the virus in vitro. The tvb r3 allele decreases the binding affinity for the ALV envelope glycoproteins Mutations in tvb gene may either abrogate or lower virus envelope-receptor binding affinity. To test this notion, chimeric immunoadhesins SU(B)-rIgG, SU(D)-rIgG and SU(E)-rIgG, which composed of the subgroups B, D and E-specific surface (SU) glycoprotein fused to the constant fragment of rabbit IgG, were constructed ( Figure  6A), and transfected into DF-1 cells. The expression of fusion proteins SU(B)-rIgG, SU(D)-rIgG and SU(E)-rIgG was detected by direct immunofluorescence assay using Alex Fluor-conjugated goat anti-rabbit IgG antibody ( Figure 6B). The integrity of the immunoadhesin proteins SU(B)-rIgG, SU(D)-rIgG and SU(E)-rIgG was verified by Western immunoblot analysis ( Figure 6C). The binding of the SU(B)-rIgG, SU(D)-rIgG or SU(E)-rIgG for Tvb receptors expressed on the surfaces of tvb s1/s1 , tvb s1/r3 and tvb r3/r3 CEFs were detected by fluorescein-conjugated goat anti-rabbit antibody and determined by flow cytometry as described previously [14,23]. As excepted, the SU(B)-rIgG, SU(D)-rIgG or SU(E)-rIgG fusion proteins bound the Tvb S1 receptor expressed on the surfaces of tvb s1/s1 CEFs efficiently, with 21.1%, 20% and 19.2% tvb s1/s1 CEFs being bound at 2 h after incubation ( Figure 7A, 7B, 7C). However, binding of all three soluble SU proteins to the Tvb R3 receptor expressed on the surfaces of tvb r3/r3 CEFs with significantly lower affinities than those of Tvb S1 receptor (p<0.01), at 3-to 4-fold lower affinity, with only 7.1%, 6.4% and 5.8% tvb r3/r3 CEFs being bound ( Figure  7A, 7B, 7C). The fluorescein-negative and fluoresceinpositive cells are obviously disparate, which were shown by the presence of two independent peaks in the FACS histogram ( Figure 7D). Collectively, these findings provided key evidence that the reduction in the binding affinity of Tvb R3 is relevant to the decreased infection efficiency of ALV-B, ALV-D, and ALV-E in tvb r3/r3 CEFs.

The tvb r3 allele are widely distributed in Chinese commercial broilers
Finally, we investigated the distribution of tvb r [18], tvb r2 [22] and tvb r3 alleles in Chinese commercial broiler lines. To this end, the sequence of the tvb genomic region, including these 3 resistant alleles, was amplified and sequenced. Sequencing revealed that the genotypes and their frequency among these 3 resistant alleles were obviously different in Chinese broiler lines ( Table 2). Compared to tvb r and tvb r2 alleles, tvb r3 was prominently distributed in Chinese commercial broilers, suggesting that the tvb r3 allele can used to be a biomarker to improve the resistance of Chinese chickens to infection by ALV-B, ALV-D, and ALV-E through genetic selection.

DISCUSSION
We previously identified intronic deletions within the tva receptor gene resulted in reduced susceptibility to subgroup A ALV [21], which implies the presence of similar genetic mutations within other ALV receptor genes. Indeed, we herein identified that the c.298C>T substitution in the tvb gene reduces the susceptibility of Chinese commercial broilers to subgroups B, D, and E ALV infection in vitro and in vivo. This is the first example to report a genetic mutation in the tvb gene that accounts for the quantitative effect on susceptibility to ALV-B, ALV-D, and ALV-E in Chinese chickens.   Premature termination codons (PTCs), which could result from nonsense mutations or frame-shifts, lead to a reduction of mRNA level. This phenomenon is known as nonsense-mediated mRNA decay (NMD), which generally requires that PTCs at least located 55 bases upstream of the last exon-exon junction [28]. Thus, the PTC in the tvb r3 transcript located 73 bases upstream of the exon 4-exon 5 junction may be a proper target. We assume that aberrant tvb mRNAs results from the c.298C>T substitution may be targeted by NMD. The quantification of gene expression results showed a minimal tvb expression compared to the normal expression ( Figure 2). This aspect is consistent with the role of NMD in degrading mRNAs that contain a premature termination codon. Thus, in our case, NMD pathway acts in the control of tvb receptor gene expression by strongly reducing the aberrant mRNAs.
Some specific regions and residues in the Tvb receptor have been identified to be crucial to efficiently mediate ALV-B, ALV-D and ALV-E infection. The Tvb 32-46 domain in the CRD1 was identified as a minimal soluble Tvb receptor, and residues Leu-36, Gln-37, Leu-41, and Tyr-42 in this region have been reported to be the interaction determinants for ALV-B and ALV-D [29]. By contrast, CRD1, CRD2, and CRD3 of Tvb receptor are required for binding and entry of ALV-E [15,22,30]. The structural integrity of the Tvb protein, including residues Tyr-67, Asn-72, and Asp-73 in CRD2, are crucial for proper receptor function [30]. The tvb s3 allele encodes Tvb S3 receptor, which contains the substitution of Cys62Ser, likely alters the folding and final structure of the CRD2, abrogating the binding and entry of ALV-E [15]. Published study has also demonstrated the Tvb R2 protein with the substitution of Cys125Ser in CRD3, presumably alters the structure of at least CRD3, and may further lead to alterations in other CRDs structure of the Tvb R2 protein, such as CRD1 and CRD2, significantly reducing the binding and entry of ALV-B and ALV-D, and virtually eliminating the protein functions as a receptor for ALV-E [22]. Since the c.298C>T substitution in tvb receptor gene, namely tvb r3 allele, generates a truncated Tvb R3 protein, which consists of only the 99 N-terminal amino acid residues ( Figure 1C). As discussed above, it seems obvious to propose that this c.298C>T substitution may exert the same effects and also cause alterations in structure of the Tvb S1 receptor protein.
In this scenario, the c.298C>T substitution likely alters the final structure of CRD2, and may interfere with and/ or alter the structure of CRD1. Alternatively, the c.298C>T substitution may cause an alteration in the structures of both CRD1 and CRD2. Therefore, if the c.298C>T mutation alters both CRD2 and CRD1, thereby changing the structure of the Tvb protein in a way that reduces but not eliminates ALV-B, ALV-D and ALV-E interaction and subsequent virus entry, which could be plausible explanation for the phenotype of Tvb R3 protein. The tvb r allele contains a premature stop codon at codon 58 in Tvb S1 receptor, which explains the resistance to infection by ALV-B, ALV-D and ALV-E [23]. c The tvb r2 allele encodes a mutant Tvb S1 receptor with the C125S substitution in CRD3, reduces the susceptibility to infection by ALV-B and ALV-D and virtually eliminates susceptibility to ALV-E infection [27].
From the point of view of coevolution between ALVs and host, it is attractive to speculate that the tvb r3 may evolve from the tvb s1 allele during the selective breeding of Chinese commercial broiler lines to confer resistance to infection by subgroups B, D, and E ALV [31]. As a consequence, the tvb r3 allele is widespread in the commercial chicken populations. In fact, the tvb r3 allele was prevalent in Chinese broiler lines (Table 1), which is consistent with the speculation. Consequently, the tvb r3 allele can used to be a biomarker for breeding chickens genetically resistance to subgroups B, D, and E ALV.
In summary, the present study identified a resistant allele tvb r3 in Chinese commercial broilers. The tvb r3 allele contains a novel c.298C>T substitution, which introduces a premature stop codon and results in the generation of the truncated Tvb R3 protein, significantly reducing the binding to the ALV-B, ALV-D and ALV-E glycoproteins and virus infection efficiency. These findings provide potential target for the development of host resistance to ALV-B, ALV-D and ALV-E infection.

Experimental animals
The Chinese commercial broiler line 01 to 15 (CB01 to CB15) have been maintained at Guangdong Wen's Food Group Co., Ltd [25]. Fertilized eggs were incubated at 37.8°C and 58% relative humidity in a forced-air incubator with a tilting motion through a 90° angle every 2 h. All animal experiments were performed following protocols approved by the Institutional Animal Care and Use Committee at the South China Agricultural University, China (approval ID: 201004152).

Cell culture and virus preparation
The procedure for the preparation of CEFs from tenday-old embryos from tvb s1/s1 , tvb s1/r3 and tvb r3/r3 fertilized eggs has been described previously [32]. CEFs, DF-1 and 293FT cells were grown in Dulbecco's modified Eagle's medium (DMEM; Invitrogen Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen Gibco) and penicillin/streptomycin (100 mg/ml each) at 37°C under 5% CO2. The ALV-B strain SDAU09C2 (kindly provided by Professor Zhizhong Cui of Shandong Agricultural University) was propagated in DF-1 cells. The value of the ratio of the sample to the positive control (S/P) for ALV p27 antigen of ALV-B strain SDAU09C2 was determined by an avian leukosis virus antigen test kit (IDEXX, Inc., Westbrook, MA).

DNA extraction, RNA isolation and cDNA synthesis
Genomic DNA was extracted from blood samples collected from lines CB01 to CB15 chickens, and chicks infected by ALV-B strain SDAU09C2 using phenol-chloroform protocol. The proviral DNA of strain SDAU09C2 was extracted as described previously [33]. The RCASBP(A)-EGFP, RCASBP(B), RCASBP(D) and RCASBP(E) plasmids DNA were extracted using DNA Extraction Kits for Plasmids (Bio-Rad Laboratories, Inc). Total RNA was extracted from blood samples collected from the rabbit, tvb s1/s1 , tvb s1/r3 , tvb r3/r3 birds, as well as chicks and CEFs infected by ALV-B strain SDAU09C2 using Trizol reagent (Invitrogen). cDNA was obtained by reverse transcription of 1 μg of DNA-free RNA using ReverTra Ace® qPCR RT Kit (Toyobo, Tokyo, Japan). The prepared DNA and synthesized cDNA were stored at −20°C for further processing.

Primer design
PCR primers that were specific for the whole genomic region of tvb gene sequence clone, the full-length tvb coding sequence clone and a portion of tvb cDNA clone encompassing the c.298C>T mutation, as well as RT-qPCR primers that were specific for 5' and 3' end of the tvb gene, and chicken glyceraldehyde-3-phosphate dehydrogenase (chGAPDH) were designed using Premier Primer 5.0 software (Premier Biosoft, Palo Alto, CA, USA) (Supplementary Table 1). PCR primers that were specific for the gp85 (SU) coding region of subgroups B, D and E ALV sequence clone and the IgG heavy chain of rabbit clone were also designed using Premier Primer 5.0 software (Supplementary Table 2). All the above primers were synthesized by Sangon Biotech Co., Ltd (Guangzhou, China).

Amplification and analysis of tvb alleles from commercial broiler lines
The sequence of the whole tvb gene genomic region was amplified from genomic DNA of a panel of 15 commercial broiler lines using four specific primer pairs with KOD FX (Toyobo, Tokyo, Japan). The PCR products were directly sequenced by Sangon Biotech Co., Ltd. Four tvb fragments sequence traces from Chinese broiler lines were aligned and compared using Lasergene version 7.1 (DNAStar, Inc., Madison, WI). The sequence of the tvb gene genomic region that contains tvb r , tvb r2 and tvb r3 alleles was amplified using tvb-3 primer pair, and sequenced directly. In total, tvb r , tvb r2 and tvb r3 alleles of 444 birds from these 15 commercial broiler lines were genotyped.

Transcript analysis of tvb alleles by RT-PCR
The whole tvb coding sequence was amplified from the cDNA of tvb s1/s1 or tvb r3/r3 birds using specific primers with KOD-Plus-Neo (Toyobo, Tokyo, Japan). In order to identify the transcripts of the tvb s1/s1 or tvb r3/r3 genotypes, the RT-PCR products were visualized by electrophoresis on 2% agarose gels, and then ligated into the pMD19-T vector (TaKaRa, Dalian, China) for sequencing.

Allelic imbalance test and qRT-PCR test of NMD
A portion of tvb cDNA encompassing the c.298C>T mutation, was amplified from cDNA of tvb s1/r3 birds. The RT-PCR products were ligated into the pMD19-T vector (TaKaRa) and then sequenced. To detect the mRNA expression level associated with 5' and 3' end of the tvb gene in the tvb s1/s1 , tvb s1/r3 and tvb r3/r3 birds. qRT-PCR were performed using an ABI7500 instrument (Applied Biosystems, Foster City, CA, USA) with FastStart SYBR Green Master (Rox) (Roche, Switzerland). The chicken GAPDH was used as an internal control. All samples were analyzed in triplicate. The analysis was carried out using the 2 −ΔΔCT method as previously described [34].

Construction of the tvb expression vectors and Western blot analysis
The entire tvb coding sequence was amplified from the cDNA of tvb s1/s1 or tvb r3/r3 birds, and cloned into the pEGFPC1 vector (Clontech, USA) with the proper orientation by XhoI and HindШ (New England BioLabs) digestion. The resulting expression constructs with wt (pEGFPC1-tvb s1 ) and mutated tvb (pEGFPC1-tvb r3 ), as well as pEGFPC1 and empty vectors were transfected into 293FT cells by Lipofectamine 3000 reagent (Invitrogen). At 48 h posttransfection, 293FT cells were subjected to Western blot analysis as previously described [35]. Briefly, the rabbit polyclonal antibody anti-GFP (1:1000; Cell Signaling Technology) was used as the primary antibody, and horseradish peroxidase (HRP)-conjugated goat antirabbit IgG (1:8,000; Proteintech Group, Inc., USA) was used as the secondary antibody.

Construction of subgroups B, D, E ALV reporter vectors and virus propagation
The replication-competent ALV recombinant virus RCASBP(A)-EGFP, transducing the enhanced green fluorescent protein (EGFP) reporter gene, has been described previously [21]. The EGFP gene was isolated from RCASBP(A)-EGFP as ClaI (New England BioLabs) fragment and cloned into the ClaI site of the RCASBP(B), RCASBP(D), and RCASBP(E) retroviral vectors [27], which were kindly obtained from Stephen H. Hughes (HIV Drug Resistance Program, National Cancer Institute, USA). The resulting expression constructs RCASBP(B)-EGFP and RCASBP(D)-EGFP were transfected into DF-1 cells using JetPRIME Regent (Invitrogen). The RCASBP(E)-EGFP was transfected into CEFs prepared from 10-day-old embryos of specific-pathogen-free (SPF) chickens using Lipofectamine 3000 reagent (Invitrogen).
Infection and spread of reporter virus were observed as an increasing proportion of GFP-positive cells, and virus stocks were harvested on day 7 posttransfection. Virus stocks were generated from cell supernatants cleared of cellular debris by centrifugation at 2,000×g for 10 min at 4°C and stored in aliquots at -80°C. The titers were quantitated by terminal dilution and infection of fresh DF-1 cells, reached 10 6 infection units (IU) per ml.

Virus spread assayed by flow cytometry
The tvb s1/s1 , tvb s1/r3 and tvb r3/r3 CEFs were seeded in triplicate wells at a density of 5×10 4 per well in a 24-well plate, and infected with 5×10 5 IU of either RCASBP(B)-EGFP, RCASBP(D)-EGFP or RCASBP(E)-EGFP viruses 24 h after seeding. After incubation for 1 h, the cells were overlaid with DMEM supplemented with 1% FBS and incubated at 37°C in a 5% CO2 atmosphere for 7 days. The percentage of GFP-positive cells was quantitated by FACS using a Cytomics FC 500 analyzer (Beckman Coulter, Kurt, U.S.A) on days 1, 2, 3 and 7 postinfection. The cells of three wells were trypsinized and washed in phosphate-buffered saline (PBS) before the analysis.

Virus growth kinetics of subgroup B ALV
The tvb s1/s1 , tvb s1/r3 and tvb r3/r3 CEFs were seeded in triplicate wells at a density of 1×10 6 per well in a 6-well plate. 24 h after seeding, CEFs were infected with ALV-B strain SDAU09C2 (S/P: 2.0) at a multiplicity of infection (MOI) of 0.1. After 1h the medium was replaced by fresh medium, and the infected cell cultures were harvested at days 1, 2, 3, 4, 5, and 6 postinfection, which were subjected to RNA extraction and cDNA synthesis. The titer of infectious progeny was determined as the virus copies per milliliter by real-time PCR using ALV-B specific primers pair as previously described [36]. The GAPDH gene was used as an internal control. The mean values and standard deviations were calculated from three independent experiments. 45 1-day-old chicks randomly collected from commercial broiler lines were randomly divided into three groups, with 15 chicks and 2 SPF chicks each group. Chicks were maintained in three negatively-pressured biosecurity isolators under quarantine conditions and provided with water and feed ad libitum. Chicks were inoculated with 0.3 ml of ALV-B strain SDAU09C2 (2.2 S/P) through the abdominal cavity, and inoculated once again at 5 days old. Genomic DNA from each chick at 7 days old was used to genotype the tvb r3 allele by direct sequencing. At one month postinfection, the status of infection in each chick by ALV-B strain SDAU09C2 was determined using the method as previously described [37].