Molecular characteristics of avian leukosis viruses isolated from indigenous chicken breeds in China

Molecular characteristics of avian leukosis viruses isolated from indigenous chicken breeds in China Abstract To assess the status of avian leukosis virus (ALV) infection in indigenous chicken breeds in China, 121 plasma samples collected from various indigenous chicken breeds were tested for the presence of ALV from 2015 to 2016. A total of 14 ALV strains were isolated and identified, including two ALV-A strains, one ALV-B strain, eight ALV-J strains, and three ALV-K strains. To study the genome structure, biological characteristics, and the evolutionary relationships of the ALV-K strains with other known subgroup strains from infected chickens, we determined the complete genome sequence of the three ALV-K strains and performed comparative analysis using the whole genome sequence or selected sequence elements. The replication rates of the three ALV-K strains were markedly lower than the rates of other ALVs, and they shared a common mutation in the pol gene, which had not been previously observed. In addition, nine putative recombinant events were detected in the genomes of the three newly isolated ALV-K strains, with high statistical support. This was the first report of an ALV-K reorganization event, which has contributed to its genetic evolution. In summary, we established a robust classification system for ALV, especially for ALV-K, and revealed additional genomic diversity for the ALV strains in indigenous chicken breeds. Therefore additional works are warranted to explore ALV genomics and epidemiology. INTRODUCTION Avian leukosis virus (ALV) is a pathogenic virus, belong to the genus alpharetrovirus of the family retroviridae that causes avian leukemia as the first known virus associated with poultry tumors (Weiss and Vogt, 2011). In addition, ALV has also been shown to cause subclinical infection in chickens, and is involved in economically important conditions such as egg drop syndrome, immunosuppression, and growth retardation (Payne and Nair, 2012). Especially in recent years, ALV infections in layers and indigenous chicken breeds have been frequently reported in China (Dong et al., 2015a,b; Lai et al., 2011). Currently, ALVs have been divided into 11 different viral subgroups (designated A to K) based on differences in the envelope sequences. Viruses of subgroups A, B, and J are the main exogenous ALVs that infect chickens in the field. Subgroups C and D have rarely been reported (Morgan, 1973; Sandelin and Estola, 1974). Only subgroup E is an endogenous virus has no relationship to the pathogenicity of the viruses (Smith, 1987). Recently, a homology analysis of the gp85 gene sequences from >20 ALV strains were isolated from several indigenous chicken breeds in China by our lab, and the results showed at least 90% nt identity to each other but relatively low similarity to ALV-A, ALV-B, ALV-C, ALV-D, ALV-E, and ALV-J isolates (<90% nt identity). In addition, the gp85 gene of those ALVs were closely related to ALVs isolated from Taiwan or Japan (∼95% nt identity; Dong et al., 2015b). Obviously, this new subgroup of ALV, ALV-K, has existed in the indigenous chicken breeds of East Asia for a long time. Strain JS11C1, which was isolated from a Chinese native breed “Luhua” chicken, has been named as the prototype strain of ALV-K (Cui et al., 2014). Subsequent epidemiological investigation revealed that ALV-K was widespread among Chinese native chickens. However, there are few reports on the genetic analysis and biological characteristics of ALV-K (Li et al., 2016; Shao et al., 2017). In addition, recombination can occur between exogenous viruses; exogenous and endogenous viruses; and exogenous viruses and non-homologous genomes. It has been reported that ALV-J is a recombinant product of an exogenous ALV and an endogenous virus (Bai et al., 1995; Benson et al., 1998; Venugopal et al., 1998), and recombination events among ALVs of different subgroups may lead to viral mutations (Liu et al., 2011; Cai et al., 2013). Infection by these recombinant exogenous viruses may not be detected, causing extensive spread of ALV. Reports about the possibility of recombination of the new subgroup (ALV-K) appear to be rare. In the present study, we described the characteristics of 14 novel ALV strains that were isolated from plasma samples of Chinese indigenous chickens using DF-1 cell culture and ALV p27 antigen detection. In addition, we analyzed three full-length ALV genome sequences using various phylogenetic methods and explored the putative recombination events within each genome. This comprehensive phylogenetic analysis of ALV-K should pave the way for further research into the prevention of ALV-K infection. MATERIALS AND METHODS Virus Isolation and Identification To estimate the ALV infection status of Chinese indigenous chicken breeds, a total of 121 plasma samples were collected from several different indigenous Chinese chicken breeds from 2015 to 2016. Samples of whole blood were collected in sterile 1.5-mL tubes containing 1% sodium heparin (Transgen Biotech, China), and the tubes were inverted several times to avoid clotting. The tubes containing 1.5 mL whole blood were centrifuged for 2 min at 2,000 rpm and the plasma was transferred to fresh plastic tubes and were stored at −20°C. All virus isolations were performed in DF-1 chicken fibroblast cell line maintained in our laboratory (American Type Culture Collection, Manassas, VA). The DF-1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Shanghai, China) supplemented with 12% fetal bovine serum (FBS; Invitrogen) at 37°C in a 5% CO2 incubator. For virus isolation, lymphocytes from the plasma samples were incubated on DF-1 cell monolayers in 24-well culture plates after centrifugation at 1,500 × g for 2 min. The uninfected DF-1 cells were used as a negative control (NC). The culture supernatant containing the virus was harvested 7 days later. After three blind passages of infected cells, the cell supernatants and cell samples were stored at −80°C until analysis. After three freeze-thaw cycles, the supernatant samples from each well (described previously) were examined for the presence of ALV group-specific P27 antigen using the Avian Leukosis Virus Antigen Test Kit (IDEXX; Yuanheng Laboratories, Beijing, China) as described previously (Qin et al., 2001). For subgroup verification by phylogenetic analysis (MEGA version 5.0), positively infected DF-1 cells were selected as a template for gp85 amplification (using the primer pair shown in Table 1), specifically of a highly conserved region common to all ALV subgroups. Non-infected DF-1cells were used as a negative control. Table 1. Primers used for PCR amplification of gp85 and the whole genome sequence of ALV-K isolates. Primera Sequence Fragment size (bp) gp85-F 5΄-GATGAGGCGAGCCCTCTCTTTG-3΄ 1,127 gp85-R 5΄-TGTTGGGAGGTAAAATGGCGT-3΄ A-F 5΄-GAGATTGTCTGCAGGGCCTAGGGCT-3΄ 2,715 A-R 5΄-TGGCAGCAAGGGTGTCTTCTCCG-3΄ B-F 5΄-CACCACATTGGTGTGCACCTGGGT-3΄ 2,778 B-R 5΄-GAAGGGGCCACTGGTCAATCCACA-3΄ C-F 5΄-GAGGTGACTAAGAAAGATGAGGCGA-3΄ 2,124 C-R 5΄-CATCTCCCCCTCCCTATGCGAAAGC-3΄ D-F 5΄-ATTGGAGCAGTGTAAGCAGTACG-3΄ 1,148 D-R 5΄-CGTTTATGACGCTTCCATGCTTG-3΄ Primera Sequence Fragment size (bp) gp85-F 5΄-GATGAGGCGAGCCCTCTCTTTG-3΄ 1,127 gp85-R 5΄-TGTTGGGAGGTAAAATGGCGT-3΄ A-F 5΄-GAGATTGTCTGCAGGGCCTAGGGCT-3΄ 2,715 A-R 5΄-TGGCAGCAAGGGTGTCTTCTCCG-3΄ B-F 5΄-CACCACATTGGTGTGCACCTGGGT-3΄ 2,778 B-R 5΄-GAAGGGGCCACTGGTCAATCCACA-3΄ C-F 5΄-GAGGTGACTAAGAAAGATGAGGCGA-3΄ 2,124 C-R 5΄-CATCTCCCCCTCCCTATGCGAAAGC-3΄ D-F 5΄-ATTGGAGCAGTGTAAGCAGTACG-3΄ 1,148 D-R 5΄-CGTTTATGACGCTTCCATGCTTG-3΄ aF and R represent upstream and downstream primers, respectively. View Large Table 1. Primers used for PCR amplification of gp85 and the whole genome sequence of ALV-K isolates. Primera Sequence Fragment size (bp) gp85-F 5΄-GATGAGGCGAGCCCTCTCTTTG-3΄ 1,127 gp85-R 5΄-TGTTGGGAGGTAAAATGGCGT-3΄ A-F 5΄-GAGATTGTCTGCAGGGCCTAGGGCT-3΄ 2,715 A-R 5΄-TGGCAGCAAGGGTGTCTTCTCCG-3΄ B-F 5΄-CACCACATTGGTGTGCACCTGGGT-3΄ 2,778 B-R 5΄-GAAGGGGCCACTGGTCAATCCACA-3΄ C-F 5΄-GAGGTGACTAAGAAAGATGAGGCGA-3΄ 2,124 C-R 5΄-CATCTCCCCCTCCCTATGCGAAAGC-3΄ D-F 5΄-ATTGGAGCAGTGTAAGCAGTACG-3΄ 1,148 D-R 5΄-CGTTTATGACGCTTCCATGCTTG-3΄ Primera Sequence Fragment size (bp) gp85-F 5΄-GATGAGGCGAGCCCTCTCTTTG-3΄ 1,127 gp85-R 5΄-TGTTGGGAGGTAAAATGGCGT-3΄ A-F 5΄-GAGATTGTCTGCAGGGCCTAGGGCT-3΄ 2,715 A-R 5΄-TGGCAGCAAGGGTGTCTTCTCCG-3΄ B-F 5΄-CACCACATTGGTGTGCACCTGGGT-3΄ 2,778 B-R 5΄-GAAGGGGCCACTGGTCAATCCACA-3΄ C-F 5΄-GAGGTGACTAAGAAAGATGAGGCGA-3΄ 2,124 C-R 5΄-CATCTCCCCCTCCCTATGCGAAAGC-3΄ D-F 5΄-ATTGGAGCAGTGTAAGCAGTACG-3΄ 1,148 D-R 5΄-CGTTTATGACGCTTCCATGCTTG-3΄ aF and R represent upstream and downstream primers, respectively. View Large Genomic DNA Amplification and Sequencing To obtain the complete proviral genome of the three ALV-K strains, four pairs of overlapping primers were designed based on the sequences of JS11C1 (GenBank accession no. KF746200). The primers used are shown in Table 1. The complete sequence of strain was amplified by polymerase chain reaction (PCR) using genomic DNA extracted from infected DF-1 cells as a template with Premix LA Taq polymerase (TaKaRa, Dalian, China) in a 50-μL reaction containing 4 μL of dNTP mixture (TaKaRa), 5 μL of 10 × PCR buffer (TaKaRa), 1 μL of Taq polymerase (TaKaRa), 2 μL of DNA solution, 1 μL of forward and reverse primers, and 36 μL of ddH2O. The thermo cycling profiles for the PCR amplification included an initial denaturation step at 95°C for 5 min, followed by 30 cycles of 95°C for 30 s, annealing at the corresponding Tm (Annealing Temperature) for 30 s, and elongation at 72°C for 3 min, with a final extension at 72°C for 10 min. The PCR products were separated by 1% agarose gel electrophoresis and purified using the Omega Gel Extraction Kit (Omega Bio-tek, USA). The purified PCR products were cloned into the pMD-18T vector (Transgen Biotech, China) and sequenced. The resulting construct was then used to transform Escherichia coli DH5α cells (TaRaKa). DNA from positive clones was sequenced directly (Shenggong, Shanghai, China), and each fragment was sequenced three times independently. Sequence Alignment and Analysis The full-length proviral genome sequences of the three ALV-K isolates were assembled using DNAStar (version 7.0), and a multiple sequence alignment was obtained using Clustal X (BioEdit version 7.0). Nucleotide and deduced amino acid sequence similarity searches were performed using MEGA (version 5.0). The sequences obtained in this study have been deposited in GenBank, and the ALV reference strains (with origin and accession numbers) that were used in this study are shown in Table 2. Table 2. Avian leukosis virus strains used in this study. No. Sub group Isolate Origin Accession no. No. Sub group Isolate Origin Accession no. 1 A B53 USA DQ412727 16 E Ev-1 USA AY013303 2 A RAV-A France M37980 17 E Ev-3 USA AY013304 3 A SDAU09E1 China HM452341 18 E ALVE-B11 Canada KC610517 4 A RAV-1 USA M19113 19 E SD0501 China EF467236 5 A MQNCSU USA DQ365814 20 J ADOL7501 USA AY027920 6 A MAV-1 USA L10922 21 J GD1109 China JX254901 7 A SDAU09C3 China HM452340 22 J HPRS-103 UK Z46390 8 A SDAU09E2 China HM452342 23 J NX0101 China AY897227 9 A SDAU09C1 China HM452339 24 J 0661 USA AF247566 10 B RSV-2 USA M14902 25 J SD07LK1 China FJ201640 11 B RSV-SR-B USA AF052428 26 J HN0001 China AY897219 12 B SDAU09E3 China JF826241 27 K JS11C1 China KF746200 13 B SDAU09C2 China HM446005 28 K GD14LZ China KU605754 14 C RSV-Prague C USA J02342 29 K GDFX0601 China KP686142 15 D RSV-SR-D USA D10652 30 K GDFX0602 China KP686143 No. Sub group Isolate Origin Accession no. No. Sub group Isolate Origin Accession no. 1 A B53 USA DQ412727 16 E Ev-1 USA AY013303 2 A RAV-A France M37980 17 E Ev-3 USA AY013304 3 A SDAU09E1 China HM452341 18 E ALVE-B11 Canada KC610517 4 A RAV-1 USA M19113 19 E SD0501 China EF467236 5 A MQNCSU USA DQ365814 20 J ADOL7501 USA AY027920 6 A MAV-1 USA L10922 21 J GD1109 China JX254901 7 A SDAU09C3 China HM452340 22 J HPRS-103 UK Z46390 8 A SDAU09E2 China HM452342 23 J NX0101 China AY897227 9 A SDAU09C1 China HM452339 24 J 0661 USA AF247566 10 B RSV-2 USA M14902 25 J SD07LK1 China FJ201640 11 B RSV-SR-B USA AF052428 26 J HN0001 China AY897219 12 B SDAU09E3 China JF826241 27 K JS11C1 China KF746200 13 B SDAU09C2 China HM446005 28 K GD14LZ China KU605754 14 C RSV-Prague C USA J02342 29 K GDFX0601 China KP686142 15 D RSV-SR-D USA D10652 30 K GDFX0602 China KP686143 View Large Table 2. Avian leukosis virus strains used in this study. No. Sub group Isolate Origin Accession no. No. Sub group Isolate Origin Accession no. 1 A B53 USA DQ412727 16 E Ev-1 USA AY013303 2 A RAV-A France M37980 17 E Ev-3 USA AY013304 3 A SDAU09E1 China HM452341 18 E ALVE-B11 Canada KC610517 4 A RAV-1 USA M19113 19 E SD0501 China EF467236 5 A MQNCSU USA DQ365814 20 J ADOL7501 USA AY027920 6 A MAV-1 USA L10922 21 J GD1109 China JX254901 7 A SDAU09C3 China HM452340 22 J HPRS-103 UK Z46390 8 A SDAU09E2 China HM452342 23 J NX0101 China AY897227 9 A SDAU09C1 China HM452339 24 J 0661 USA AF247566 10 B RSV-2 USA M14902 25 J SD07LK1 China FJ201640 11 B RSV-SR-B USA AF052428 26 J HN0001 China AY897219 12 B SDAU09E3 China JF826241 27 K JS11C1 China KF746200 13 B SDAU09C2 China HM446005 28 K GD14LZ China KU605754 14 C RSV-Prague C USA J02342 29 K GDFX0601 China KP686142 15 D RSV-SR-D USA D10652 30 K GDFX0602 China KP686143 No. Sub group Isolate Origin Accession no. No. Sub group Isolate Origin Accession no. 1 A B53 USA DQ412727 16 E Ev-1 USA AY013303 2 A RAV-A France M37980 17 E Ev-3 USA AY013304 3 A SDAU09E1 China HM452341 18 E ALVE-B11 Canada KC610517 4 A RAV-1 USA M19113 19 E SD0501 China EF467236 5 A MQNCSU USA DQ365814 20 J ADOL7501 USA AY027920 6 A MAV-1 USA L10922 21 J GD1109 China JX254901 7 A SDAU09C3 China HM452340 22 J HPRS-103 UK Z46390 8 A SDAU09E2 China HM452342 23 J NX0101 China AY897227 9 A SDAU09C1 China HM452339 24 J 0661 USA AF247566 10 B RSV-2 USA M14902 25 J SD07LK1 China FJ201640 11 B RSV-SR-B USA AF052428 26 J HN0001 China AY897219 12 B SDAU09E3 China JF826241 27 K JS11C1 China KF746200 13 B SDAU09C2 China HM446005 28 K GD14LZ China KU605754 14 C RSV-Prague C USA J02342 29 K GDFX0601 China KP686142 15 D RSV-SR-D USA D10652 30 K GDFX0602 China KP686143 View Large Replication of the ALV Isolates in DF-1 Cells The titers of the three ALV-K strains are presented as TCID50 mL−1 and were measured by ELISA using the Reed-Muench method. Briefly, DF-1 cells were plated (at approximately 106 cells per dish) in 60-mm dishes 1 day before infection with 1,000 TCID50 of virus. Three exogenous ALVs are maintained in our laboratory, including a subgroup A strain (strain SDAU09C1), a subgroup B strain (strain SDAU09C2), and a subgroup J strain (strain NX0101), were used as controls. The infections were carried out in the presence of 1% FBS at 37°C under 5% CO2, and aliquots (approximately 400 μL) were harvested on days 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 post-infection. At various times points, the harvested supernatant was replaced with an equal volume of fresh DMEM. After three freeze-thaw cycles, the harvested samples were examined for ALV group-specific p27 antigen by ELISA to determine the replication kinetics. Each sample was tested independently three times. Sequencing to Test for Recombination Events Putative recombination events within each ALV genome were detected using the Recombination Detection Program 4(RDP4; Martin, 2009). Other detection methods, including RDP, GENECONV, BootScan, MaxChi, Chimaera, SiScan, Phyl-Pro, LARD, and 3Seq, were also employed for comparison (Martin et al., 2005a,b). Only recombination events supported by no fewer than six independent methods were regarded as positive. These putative recombination events were further confirmed and visualized using SimPlot (Lole et al., 1999). Ethics Statement The animal care and use protocol was approved by the Shandong Agricultural University Animal care and use Committee (SDAUA-2016–002). All of the experimental animals of this study were cared for and maintained throughout of the experiments strictly following the ethics and biosecurity guidelines approved by the Institutional Animal Care and Use Committee of Shandong Agricultural University. RESULTS Isolation and Identification of ALVs Fourteen ALV strains were isolated from different indigenous chicken breeds in Shandong, Zhejiang, and Jiangsu province from 2015 to 2016 (Table 3). DF-1 cells infected with these strains showed a positive result (the S/P (Samples OD value-negative control value)/(positive control value-negative control value) values of the infected group were between 0.65 and 1.23, positive critical value was 0.2) by measuring the ALV-27 antigen level with an ELISA, whereas mock-infected DF-1 cells showed a negative result (the S/P values of the control group were between 0.00 and 0.08), these results indicated the presence of exogenous ALV in the samples. The gp85 genes of the ALV isolates were amplified by PCR and sequencing. Phylogenetic analysis was carried out on 30 other ALV strains of known subgroups (Figure 1). The results showed that among the new isolates were two ALV-A strains, one ALV-B strain, eight ALV-J strains, and three ALV-K strains. The accession numbers are listed in Table 3. Figure 1. View largeDownload slide Phylogenetic relationships among the gp85 sequences of 14 ALV strains and other ALVs of different subgroups. ALVs isolated in this survey are marked with a star symbol. Figure 1. View largeDownload slide Phylogenetic relationships among the gp85 sequences of 14 ALV strains and other ALVs of different subgroups. ALVs isolated in this survey are marked with a star symbol. Table 3. Avian leukosis virus strains isolated in this study. Accession no. Strain name Year Province Host Length (bp) KY773915 SDAUAB-1 2014 Shandong HY-LINE VARIETY BROWN 1,824 KY773913 SDAUAA-2 2014 Hebei HY-LINE VARIETY BROWN 1,798 KY767730 SDAUAJ-3 2014 Hebei HY-LINE VARIETY BROWN 1,791 KY773914 SDAUAA-4 2014 Hebei HY-LINE VARIETY BROWN 1,788 KY773916 SDAUAJ-5 2015 Jiangsu LANGYA 1,686 KY773917 SDAUAJ-6 2015 Jiangsu LANGYA 1,671 KY773918 SDAUAJ-7 2015 Jiangsu BEIJING YOU 1,689 KY773919 SDAUAJ-8 2015 Jiangsu BEIJING YOU 1,686 KY773920 SDAUAJ-9 2015 Jiangsu LUHUA 1,686 KY773921 SDAUAJ-11 2015 Jiangsu LANGYA 1,662 KY773922 SDAUAJ-12 2015 Jiangsu LANGYA 1,692 KY773911 SDAUAK-11 2015 Jiangsu LANGYA 7,814 KY773912 SDAUAK-12 2015 Jiangsu LANGYA 7,811 KY767731 SDAUAK-13 2015 Jiangsu LANGYA 7,813 Accession no. Strain name Year Province Host Length (bp) KY773915 SDAUAB-1 2014 Shandong HY-LINE VARIETY BROWN 1,824 KY773913 SDAUAA-2 2014 Hebei HY-LINE VARIETY BROWN 1,798 KY767730 SDAUAJ-3 2014 Hebei HY-LINE VARIETY BROWN 1,791 KY773914 SDAUAA-4 2014 Hebei HY-LINE VARIETY BROWN 1,788 KY773916 SDAUAJ-5 2015 Jiangsu LANGYA 1,686 KY773917 SDAUAJ-6 2015 Jiangsu LANGYA 1,671 KY773918 SDAUAJ-7 2015 Jiangsu BEIJING YOU 1,689 KY773919 SDAUAJ-8 2015 Jiangsu BEIJING YOU 1,686 KY773920 SDAUAJ-9 2015 Jiangsu LUHUA 1,686 KY773921 SDAUAJ-11 2015 Jiangsu LANGYA 1,662 KY773922 SDAUAJ-12 2015 Jiangsu LANGYA 1,692 KY773911 SDAUAK-11 2015 Jiangsu LANGYA 7,814 KY773912 SDAUAK-12 2015 Jiangsu LANGYA 7,811 KY767731 SDAUAK-13 2015 Jiangsu LANGYA 7,813 View Large Table 3. Avian leukosis virus strains isolated in this study. Accession no. Strain name Year Province Host Length (bp) KY773915 SDAUAB-1 2014 Shandong HY-LINE VARIETY BROWN 1,824 KY773913 SDAUAA-2 2014 Hebei HY-LINE VARIETY BROWN 1,798 KY767730 SDAUAJ-3 2014 Hebei HY-LINE VARIETY BROWN 1,791 KY773914 SDAUAA-4 2014 Hebei HY-LINE VARIETY BROWN 1,788 KY773916 SDAUAJ-5 2015 Jiangsu LANGYA 1,686 KY773917 SDAUAJ-6 2015 Jiangsu LANGYA 1,671 KY773918 SDAUAJ-7 2015 Jiangsu BEIJING YOU 1,689 KY773919 SDAUAJ-8 2015 Jiangsu BEIJING YOU 1,686 KY773920 SDAUAJ-9 2015 Jiangsu LUHUA 1,686 KY773921 SDAUAJ-11 2015 Jiangsu LANGYA 1,662 KY773922 SDAUAJ-12 2015 Jiangsu LANGYA 1,692 KY773911 SDAUAK-11 2015 Jiangsu LANGYA 7,814 KY773912 SDAUAK-12 2015 Jiangsu LANGYA 7,811 KY767731 SDAUAK-13 2015 Jiangsu LANGYA 7,813 Accession no. Strain name Year Province Host Length (bp) KY773915 SDAUAB-1 2014 Shandong HY-LINE VARIETY BROWN 1,824 KY773913 SDAUAA-2 2014 Hebei HY-LINE VARIETY BROWN 1,798 KY767730 SDAUAJ-3 2014 Hebei HY-LINE VARIETY BROWN 1,791 KY773914 SDAUAA-4 2014 Hebei HY-LINE VARIETY BROWN 1,788 KY773916 SDAUAJ-5 2015 Jiangsu LANGYA 1,686 KY773917 SDAUAJ-6 2015 Jiangsu LANGYA 1,671 KY773918 SDAUAJ-7 2015 Jiangsu BEIJING YOU 1,689 KY773919 SDAUAJ-8 2015 Jiangsu BEIJING YOU 1,686 KY773920 SDAUAJ-9 2015 Jiangsu LUHUA 1,686 KY773921 SDAUAJ-11 2015 Jiangsu LANGYA 1,662 KY773922 SDAUAJ-12 2015 Jiangsu LANGYA 1,692 KY773911 SDAUAK-11 2015 Jiangsu LANGYA 7,814 KY773912 SDAUAK-12 2015 Jiangsu LANGYA 7,811 KY767731 SDAUAK-13 2015 Jiangsu LANGYA 7,813 View Large Sequencing and Analysis of the Complete Proviral Genome of the Three ALV-K Strains Sequence analysis for the complete proviral genomes of the three ALV-K strains were determined by PCR and sequencing, and were named SDAUAK-11, SDAUAK-12, and SDAUAK-13. The lengths of the genomes were 7,814, 7,813, and 7,811 bp, respectively, with a genetic organization typical of replication-competent type C retroviruses lacking viral oncogenes (5΄-LTR-leader-gag-pol-env-3΄-LTR). The nucleotide and amino acid sequences of different regions were analyzed in comparison to the published reference ALVs from GenBank (Tables 4 to 6). The results showed that the gag and pol genes of the three ALV-K strains were highly conserved, and shared at least 97.0% and 91% nt identity, respectively. Analysis of the env gene and LTR showed that the strains were divided into two different situations, respectively. The env genes of the three ALV-K strains were closely related to those of the ALV-A, ALV-B, ALV-C, ALV-D, and ALV-E subgroups (with ∼90% nt identity), but showed only 50% nt identity to env from ALV-J. The LTR of the three ALV-K strains exhibited high sequence similarity to ALV-A and ALV-D strains, with 75.5 to 91.8% nt identity, but showed relatively low similarity to ALV-B, ALV-C, ALV-E, and ALV-J strains, with 32.5 to 42.1% nt identity. In a nutshell, the strains that were most similar to SDAUAK-11, SDAKAK-12, and SDAUAK-13 are GD14LZ, GDFX0601, and JS11C1, respectively, and the three ALV-K strains showed 90.8 to 97.7%, 90.8 to 97.7%, and 91.2 to 97.3% nt identity, respectively, to other ALV-K reference strains. Sequence of three newly identified ALV-K strains, the homology of their full-length genome, LTR, pol, gag, and env gene were 96.5 to 98.3%, 84.8 to 99.4%, 99.4 to 99.7%, 99.1 to 99.8%, 96.6 to 97.2%, respectively. Table 4. Segmental sequence comparison between isolate SDAUAK-11 and other ALV strains from different subgroups. UTR POL GAG ENV GENOME SDAUAK-11 Na N AAb N AA N AA N A 75.5% to 84.9% 97.2% to 99.3% 98.0% to 99.2% 95.4% to 97.3% 96.7% to 97.4% 87.1% to 90.9% 86.3% to 89.2% 87.4% to 94.4% B 32.5% to 38% 97.1% to 98.2% 98.0% to 98.5% 94.7% to 96.4% 96.6% to 97.9% 88.2% to 88.6% 85.1% to 86.6% 82.2% to 90.8% C 36.8% 98.5% 99.0% 96.3% 96.4% 91.3% 89.3% 88.4% D 76.1% 98.0% 98.7% 95.2% 96.4% 89.2% 87.3% 88.9% E 40.3% to 41.4% 99.2% to 99.5% 98.3% to 99.7% 98.7% to 99.1% 98.2% to 98.7% 93.1% to 93.2% 90.1% to 90.6% 94.9% to 95.8% J 36.0% to 37.3% 97.2% to 98.3% 97.4% to 98.9% 95.4% to 98.5% 96.2% to 97.7% 52.3% to 54.8% 48.5% to 50.3% 78.6% to 82.1% K 74.7% to 85.4% 98.8% to 99.6% 99.0% to 99.7% 96.7% to 99.1% 97.3% to 99.1% 95.9% to 99.3% 95.6% to 98.8% 90.8% to 97.7% UTR POL GAG ENV GENOME SDAUAK-11 Na N AAb N AA N AA N A 75.5% to 84.9% 97.2% to 99.3% 98.0% to 99.2% 95.4% to 97.3% 96.7% to 97.4% 87.1% to 90.9% 86.3% to 89.2% 87.4% to 94.4% B 32.5% to 38% 97.1% to 98.2% 98.0% to 98.5% 94.7% to 96.4% 96.6% to 97.9% 88.2% to 88.6% 85.1% to 86.6% 82.2% to 90.8% C 36.8% 98.5% 99.0% 96.3% 96.4% 91.3% 89.3% 88.4% D 76.1% 98.0% 98.7% 95.2% 96.4% 89.2% 87.3% 88.9% E 40.3% to 41.4% 99.2% to 99.5% 98.3% to 99.7% 98.7% to 99.1% 98.2% to 98.7% 93.1% to 93.2% 90.1% to 90.6% 94.9% to 95.8% J 36.0% to 37.3% 97.2% to 98.3% 97.4% to 98.9% 95.4% to 98.5% 96.2% to 97.7% 52.3% to 54.8% 48.5% to 50.3% 78.6% to 82.1% K 74.7% to 85.4% 98.8% to 99.6% 99.0% to 99.7% 96.7% to 99.1% 97.3% to 99.1% 95.9% to 99.3% 95.6% to 98.8% 90.8% to 97.7% aN represents the nucleotide sequence homology, bAA represent the amino acid sequence homology. View Large Table 4. Segmental sequence comparison between isolate SDAUAK-11 and other ALV strains from different subgroups. UTR POL GAG ENV GENOME SDAUAK-11 Na N AAb N AA N AA N A 75.5% to 84.9% 97.2% to 99.3% 98.0% to 99.2% 95.4% to 97.3% 96.7% to 97.4% 87.1% to 90.9% 86.3% to 89.2% 87.4% to 94.4% B 32.5% to 38% 97.1% to 98.2% 98.0% to 98.5% 94.7% to 96.4% 96.6% to 97.9% 88.2% to 88.6% 85.1% to 86.6% 82.2% to 90.8% C 36.8% 98.5% 99.0% 96.3% 96.4% 91.3% 89.3% 88.4% D 76.1% 98.0% 98.7% 95.2% 96.4% 89.2% 87.3% 88.9% E 40.3% to 41.4% 99.2% to 99.5% 98.3% to 99.7% 98.7% to 99.1% 98.2% to 98.7% 93.1% to 93.2% 90.1% to 90.6% 94.9% to 95.8% J 36.0% to 37.3% 97.2% to 98.3% 97.4% to 98.9% 95.4% to 98.5% 96.2% to 97.7% 52.3% to 54.8% 48.5% to 50.3% 78.6% to 82.1% K 74.7% to 85.4% 98.8% to 99.6% 99.0% to 99.7% 96.7% to 99.1% 97.3% to 99.1% 95.9% to 99.3% 95.6% to 98.8% 90.8% to 97.7% UTR POL GAG ENV GENOME SDAUAK-11 Na N AAb N AA N AA N A 75.5% to 84.9% 97.2% to 99.3% 98.0% to 99.2% 95.4% to 97.3% 96.7% to 97.4% 87.1% to 90.9% 86.3% to 89.2% 87.4% to 94.4% B 32.5% to 38% 97.1% to 98.2% 98.0% to 98.5% 94.7% to 96.4% 96.6% to 97.9% 88.2% to 88.6% 85.1% to 86.6% 82.2% to 90.8% C 36.8% 98.5% 99.0% 96.3% 96.4% 91.3% 89.3% 88.4% D 76.1% 98.0% 98.7% 95.2% 96.4% 89.2% 87.3% 88.9% E 40.3% to 41.4% 99.2% to 99.5% 98.3% to 99.7% 98.7% to 99.1% 98.2% to 98.7% 93.1% to 93.2% 90.1% to 90.6% 94.9% to 95.8% J 36.0% to 37.3% 97.2% to 98.3% 97.4% to 98.9% 95.4% to 98.5% 96.2% to 97.7% 52.3% to 54.8% 48.5% to 50.3% 78.6% to 82.1% K 74.7% to 85.4% 98.8% to 99.6% 99.0% to 99.7% 96.7% to 99.1% 97.3% to 99.1% 95.9% to 99.3% 95.6% to 98.8% 90.8% to 97.7% aN represents the nucleotide sequence homology, bAA represent the amino acid sequence homology. View Large Table 5. Segmental sequence comparison between isolate SDAUAK-12 and other ALV strains from different subgroups. UTR POL GAG ENV GENOME SDAUAK-12 Na N AAb N AA N AA N A 85.0% to 91.3% 97.2% to 98.8% 97.8% to 99.0% 95.5% to 97.3% 96.7% to 97.7% 87.4% to 90.5% 85.2% to 87.5% 87.1% to 95.8% B 33.3% to 36.4% 97.3% to 98.3% 97.9% to 98.3% 94.8% to 96.4% 96.7% to 97.9% 88.0% to 88.8% 84.9% to 86.6% 85.9% to 90.5% C 35.0% 98.6% 98.8% 96.5% 96.6% 91.1% 88.6% 89.3% D 86.1% 98.0% 98.2% 95.4% 96.6% 88.8% 86.6% 88.9% E 40.2% to 41.4% 99.4% to 99.6% 98.1% to 99.6% 98.7% to 99.1% 97.9% to 98.6% 92.6% to 93.0% 89.4% to 90.3% 94.9% to 95.8% J 35.4% to 36.7% 97.5% to 98.5% 97.2% to 98.7% 95.6% to 98.0% 96.3% to 98.0% 51.7% to 54.4% 48.0% to 49.9% 78.6% to 82.1% K 85.8% to 99.7% 98.9% to 99.7% 98.5% to 99.4% 97.0% to 99.8% 97.4% to 99.9% 96.0% to 99.2% 94.5% to 99.0% 90.8% to 97.7% UTR POL GAG ENV GENOME SDAUAK-12 Na N AAb N AA N AA N A 85.0% to 91.3% 97.2% to 98.8% 97.8% to 99.0% 95.5% to 97.3% 96.7% to 97.7% 87.4% to 90.5% 85.2% to 87.5% 87.1% to 95.8% B 33.3% to 36.4% 97.3% to 98.3% 97.9% to 98.3% 94.8% to 96.4% 96.7% to 97.9% 88.0% to 88.8% 84.9% to 86.6% 85.9% to 90.5% C 35.0% 98.6% 98.8% 96.5% 96.6% 91.1% 88.6% 89.3% D 86.1% 98.0% 98.2% 95.4% 96.6% 88.8% 86.6% 88.9% E 40.2% to 41.4% 99.4% to 99.6% 98.1% to 99.6% 98.7% to 99.1% 97.9% to 98.6% 92.6% to 93.0% 89.4% to 90.3% 94.9% to 95.8% J 35.4% to 36.7% 97.5% to 98.5% 97.2% to 98.7% 95.6% to 98.0% 96.3% to 98.0% 51.7% to 54.4% 48.0% to 49.9% 78.6% to 82.1% K 85.8% to 99.7% 98.9% to 99.7% 98.5% to 99.4% 97.0% to 99.8% 97.4% to 99.9% 96.0% to 99.2% 94.5% to 99.0% 90.8% to 97.7% aN represents the nucleotide sequence homology, bAA represent the amino acid sequence homology. View Large Table 5. Segmental sequence comparison between isolate SDAUAK-12 and other ALV strains from different subgroups. UTR POL GAG ENV GENOME SDAUAK-12 Na N AAb N AA N AA N A 85.0% to 91.3% 97.2% to 98.8% 97.8% to 99.0% 95.5% to 97.3% 96.7% to 97.7% 87.4% to 90.5% 85.2% to 87.5% 87.1% to 95.8% B 33.3% to 36.4% 97.3% to 98.3% 97.9% to 98.3% 94.8% to 96.4% 96.7% to 97.9% 88.0% to 88.8% 84.9% to 86.6% 85.9% to 90.5% C 35.0% 98.6% 98.8% 96.5% 96.6% 91.1% 88.6% 89.3% D 86.1% 98.0% 98.2% 95.4% 96.6% 88.8% 86.6% 88.9% E 40.2% to 41.4% 99.4% to 99.6% 98.1% to 99.6% 98.7% to 99.1% 97.9% to 98.6% 92.6% to 93.0% 89.4% to 90.3% 94.9% to 95.8% J 35.4% to 36.7% 97.5% to 98.5% 97.2% to 98.7% 95.6% to 98.0% 96.3% to 98.0% 51.7% to 54.4% 48.0% to 49.9% 78.6% to 82.1% K 85.8% to 99.7% 98.9% to 99.7% 98.5% to 99.4% 97.0% to 99.8% 97.4% to 99.9% 96.0% to 99.2% 94.5% to 99.0% 90.8% to 97.7% UTR POL GAG ENV GENOME SDAUAK-12 Na N AAb N AA N AA N A 85.0% to 91.3% 97.2% to 98.8% 97.8% to 99.0% 95.5% to 97.3% 96.7% to 97.7% 87.4% to 90.5% 85.2% to 87.5% 87.1% to 95.8% B 33.3% to 36.4% 97.3% to 98.3% 97.9% to 98.3% 94.8% to 96.4% 96.7% to 97.9% 88.0% to 88.8% 84.9% to 86.6% 85.9% to 90.5% C 35.0% 98.6% 98.8% 96.5% 96.6% 91.1% 88.6% 89.3% D 86.1% 98.0% 98.2% 95.4% 96.6% 88.8% 86.6% 88.9% E 40.2% to 41.4% 99.4% to 99.6% 98.1% to 99.6% 98.7% to 99.1% 97.9% to 98.6% 92.6% to 93.0% 89.4% to 90.3% 94.9% to 95.8% J 35.4% to 36.7% 97.5% to 98.5% 97.2% to 98.7% 95.6% to 98.0% 96.3% to 98.0% 51.7% to 54.4% 48.0% to 49.9% 78.6% to 82.1% K 85.8% to 99.7% 98.9% to 99.7% 98.5% to 99.4% 97.0% to 99.8% 97.4% to 99.9% 96.0% to 99.2% 94.5% to 99.0% 90.8% to 97.7% aN represents the nucleotide sequence homology, bAA represent the amino acid sequence homology. View Large Table 6. Segmental sequence comparison between isolate SDAUAK-13 and other ALV strains from different subgroups. UTR POL GAG ENV GENOME SDAUAK-13 Na N AAb N AA N AA N A 86.2% to 91.8% 97.4% to 99.7% 97.9% to 99.3% 91.3% to 96.6% 96.9% to 97.9% 88.0% to 90.8% 86.1% to 88.9% 87.2% to 95.8% B 34.4% to 36.4% 97.5% to 98.5% 97.9% to 98.4% 95.0% to 96.4% 96.9% to 98.0% 88.1% to 89.0% 85.6% to 87.4% 85.9% to 90.6% C 35.5% 98.9% 99.1% 96.5% 96.7% 91.1% 89.1% 89.9% D 87.4% 98.2% 98.5% 95.4% 96.7% 88.9% 87.6% 89.3% E 40.6% to 42.1% 99.5% to 99.8% 98.1% to 99.6% 98.7% to 99.1% 98.1% to 98.7% 92.9% to 93.2 90.5% to 91.0% 94.8% to 95.8% J 35.4% to 36.7% 97.6% to 98.6% 97.2% to 98.7% 95.6% to 98.9% 96.4% to 98.1% 52.3% to 54.9% 48.0% to 49.7% 78.7% to 82.0% K 87.7% to 99.4% 99.1% to 99.9% 98.9% to 99.6% 97.0% to 99.9% 97.6% to 99.9% 96.2% to 97.5% 95.8% to 96.8% 91.2% to 97.3% UTR POL GAG ENV GENOME SDAUAK-13 Na N AAb N AA N AA N A 86.2% to 91.8% 97.4% to 99.7% 97.9% to 99.3% 91.3% to 96.6% 96.9% to 97.9% 88.0% to 90.8% 86.1% to 88.9% 87.2% to 95.8% B 34.4% to 36.4% 97.5% to 98.5% 97.9% to 98.4% 95.0% to 96.4% 96.9% to 98.0% 88.1% to 89.0% 85.6% to 87.4% 85.9% to 90.6% C 35.5% 98.9% 99.1% 96.5% 96.7% 91.1% 89.1% 89.9% D 87.4% 98.2% 98.5% 95.4% 96.7% 88.9% 87.6% 89.3% E 40.6% to 42.1% 99.5% to 99.8% 98.1% to 99.6% 98.7% to 99.1% 98.1% to 98.7% 92.9% to 93.2 90.5% to 91.0% 94.8% to 95.8% J 35.4% to 36.7% 97.6% to 98.6% 97.2% to 98.7% 95.6% to 98.9% 96.4% to 98.1% 52.3% to 54.9% 48.0% to 49.7% 78.7% to 82.0% K 87.7% to 99.4% 99.1% to 99.9% 98.9% to 99.6% 97.0% to 99.9% 97.6% to 99.9% 96.2% to 97.5% 95.8% to 96.8% 91.2% to 97.3% aN represents the nucleotide sequence homology, bAA represent the amino acid sequence homology. View Large Table 6. Segmental sequence comparison between isolate SDAUAK-13 and other ALV strains from different subgroups. UTR POL GAG ENV GENOME SDAUAK-13 Na N AAb N AA N AA N A 86.2% to 91.8% 97.4% to 99.7% 97.9% to 99.3% 91.3% to 96.6% 96.9% to 97.9% 88.0% to 90.8% 86.1% to 88.9% 87.2% to 95.8% B 34.4% to 36.4% 97.5% to 98.5% 97.9% to 98.4% 95.0% to 96.4% 96.9% to 98.0% 88.1% to 89.0% 85.6% to 87.4% 85.9% to 90.6% C 35.5% 98.9% 99.1% 96.5% 96.7% 91.1% 89.1% 89.9% D 87.4% 98.2% 98.5% 95.4% 96.7% 88.9% 87.6% 89.3% E 40.6% to 42.1% 99.5% to 99.8% 98.1% to 99.6% 98.7% to 99.1% 98.1% to 98.7% 92.9% to 93.2 90.5% to 91.0% 94.8% to 95.8% J 35.4% to 36.7% 97.6% to 98.6% 97.2% to 98.7% 95.6% to 98.9% 96.4% to 98.1% 52.3% to 54.9% 48.0% to 49.7% 78.7% to 82.0% K 87.7% to 99.4% 99.1% to 99.9% 98.9% to 99.6% 97.0% to 99.9% 97.6% to 99.9% 96.2% to 97.5% 95.8% to 96.8% 91.2% to 97.3% UTR POL GAG ENV GENOME SDAUAK-13 Na N AAb N AA N AA N A 86.2% to 91.8% 97.4% to 99.7% 97.9% to 99.3% 91.3% to 96.6% 96.9% to 97.9% 88.0% to 90.8% 86.1% to 88.9% 87.2% to 95.8% B 34.4% to 36.4% 97.5% to 98.5% 97.9% to 98.4% 95.0% to 96.4% 96.9% to 98.0% 88.1% to 89.0% 85.6% to 87.4% 85.9% to 90.6% C 35.5% 98.9% 99.1% 96.5% 96.7% 91.1% 89.1% 89.9% D 87.4% 98.2% 98.5% 95.4% 96.7% 88.9% 87.6% 89.3% E 40.6% to 42.1% 99.5% to 99.8% 98.1% to 99.6% 98.7% to 99.1% 98.1% to 98.7% 92.9% to 93.2 90.5% to 91.0% 94.8% to 95.8% J 35.4% to 36.7% 97.6% to 98.6% 97.2% to 98.7% 95.6% to 98.9% 96.4% to 98.1% 52.3% to 54.9% 48.0% to 49.7% 78.7% to 82.0% K 87.7% to 99.4% 99.1% to 99.9% 98.9% to 99.6% 97.0% to 99.9% 97.6% to 99.9% 96.2% to 97.5% 95.8% to 96.8% 91.2% to 97.3% aN represents the nucleotide sequence homology, bAA represent the amino acid sequence homology. View Large Molecular Characteristics of the Pol Gene The three ALV-K strains share a common mutation, consisting a single nucleotide deletion at position 24 and an 8-nucleotide deletion at positions 32 to 39 (Figure 2), which caused the replacement of the corresponding amino acid PLKWK with RS, and has not been previously reported. This mutation did not prevent the expression and translation of the pol gene. However, its influence on ALV requires further study. Figure 2. View largeDownload slide Comparison of the amino acid sequences of the pol gene of SDAUAK-11, SDAUAK-12, and SDAUAK-13 to those of other ALVs from different subgroups. Figure 2. View largeDownload slide Comparison of the amino acid sequences of the pol gene of SDAUAK-11, SDAUAK-12, and SDAUAK-13 to those of other ALVs from different subgroups. Recombination Analysis The recombination events in the complete proviral genomes were analyzed by RDP4, and several potential recombination events were detected (Table 7), including four events in SDAUAK-11, two in SDAUAK-12, and three in SDAUAK-13, which were supported by Bootscan (Figure 3) and five other methods. The above results suggest that the three ALV-K strains are recombinant isolates. All these events can be divided into four groups; events a, b, and c occurred in the 5΄ LTR gene of the three ALV-K strains, with GDFX0602 (ALV-K) as the major parent and HPRS-103 (ALV-J) as the minor parent; events d and e mapped to the env gene of SDAUAK-11 and SDAUAK-13, with EV-3 (ALV-E) as the major parent and GDFX0602(ALV-K) as the minor parent; events f, g, and h were located in the 3΄ LTR of the three ALV-K strains, with SD07LK1 (ALV-J) as the major parent and JS11C1 (ALV-K) as the minor parent, and event i occurred in the 3΄ LTR of SDAUAK-11, with GD14LZ (ALV-K) as major parent. Figure 3. View largeDownload slide Bootscan analysis of the potential recombinant and major and minor parent sequences (a, b, c, d, e, f, g, h, and i). The Bootscan was based on the pairwise distance model with a window size of 200, step size of 50, and 1,000 bootstrap replicates generated by the RDP4 program. (j) The positon of the recombination events in the three ALV-K isolates. Figure 3. View largeDownload slide Bootscan analysis of the potential recombinant and major and minor parent sequences (a, b, c, d, e, f, g, h, and i). The Bootscan was based on the pairwise distance model with a window size of 200, step size of 50, and 1,000 bootstrap replicates generated by the RDP4 program. (j) The positon of the recombination events in the three ALV-K isolates. Table 7. Detected potential recombination events. Events Beginning breakpoint—ending breakpoint Recombination position Recombinants Major parent Minor parent a 142–239 5΄UTR SDAUAK-11 GDFX0602 HPRS-103 b 161–279 5΄UTR SDAUAK-12 GDFX0602 HPRS-103 c 142–239 5΄UTR SDAUAK-13 GDFX0602 HPRS-103 d 5,597–6,153 ENV SDAUAK-11 EV-3 GDFX0602 e 5,597–5,920 ENV SDAUAK-13 EV-3 GDFX0602 f 6,119–7,321 ENV, 3΄UTR SDAUAK-11 SD07LK1 JS11C1 g 5,199–7,642 POL, ENV, 3΄UTR SDAUAK-12 SD07LK1 JS11C1 h 6,119–7,163 ENV, 3΄UTR SDAUAK-13 SD07LK1 JS11C1 i 7,118–7,814 3΄UTR SDAUAK-11 GD14LZ SDAUAK-13 Events Beginning breakpoint—ending breakpoint Recombination position Recombinants Major parent Minor parent a 142–239 5΄UTR SDAUAK-11 GDFX0602 HPRS-103 b 161–279 5΄UTR SDAUAK-12 GDFX0602 HPRS-103 c 142–239 5΄UTR SDAUAK-13 GDFX0602 HPRS-103 d 5,597–6,153 ENV SDAUAK-11 EV-3 GDFX0602 e 5,597–5,920 ENV SDAUAK-13 EV-3 GDFX0602 f 6,119–7,321 ENV, 3΄UTR SDAUAK-11 SD07LK1 JS11C1 g 5,199–7,642 POL, ENV, 3΄UTR SDAUAK-12 SD07LK1 JS11C1 h 6,119–7,163 ENV, 3΄UTR SDAUAK-13 SD07LK1 JS11C1 i 7,118–7,814 3΄UTR SDAUAK-11 GD14LZ SDAUAK-13 View Large Table 7. Detected potential recombination events. Events Beginning breakpoint—ending breakpoint Recombination position Recombinants Major parent Minor parent a 142–239 5΄UTR SDAUAK-11 GDFX0602 HPRS-103 b 161–279 5΄UTR SDAUAK-12 GDFX0602 HPRS-103 c 142–239 5΄UTR SDAUAK-13 GDFX0602 HPRS-103 d 5,597–6,153 ENV SDAUAK-11 EV-3 GDFX0602 e 5,597–5,920 ENV SDAUAK-13 EV-3 GDFX0602 f 6,119–7,321 ENV, 3΄UTR SDAUAK-11 SD07LK1 JS11C1 g 5,199–7,642 POL, ENV, 3΄UTR SDAUAK-12 SD07LK1 JS11C1 h 6,119–7,163 ENV, 3΄UTR SDAUAK-13 SD07LK1 JS11C1 i 7,118–7,814 3΄UTR SDAUAK-11 GD14LZ SDAUAK-13 Events Beginning breakpoint—ending breakpoint Recombination position Recombinants Major parent Minor parent a 142–239 5΄UTR SDAUAK-11 GDFX0602 HPRS-103 b 161–279 5΄UTR SDAUAK-12 GDFX0602 HPRS-103 c 142–239 5΄UTR SDAUAK-13 GDFX0602 HPRS-103 d 5,597–6,153 ENV SDAUAK-11 EV-3 GDFX0602 e 5,597–5,920 ENV SDAUAK-13 EV-3 GDFX0602 f 6,119–7,321 ENV, 3΄UTR SDAUAK-11 SD07LK1 JS11C1 g 5,199–7,642 POL, ENV, 3΄UTR SDAUAK-12 SD07LK1 JS11C1 h 6,119–7,163 ENV, 3΄UTR SDAUAK-13 SD07LK1 JS11C1 i 7,118–7,814 3΄UTR SDAUAK-11 GD14LZ SDAUAK-13 View Large ALV-K Growth Kinetics in DF-1 Cells The replication rates of the three ALV-K strains were evaluated in vitro using DF-1 cells, which are commonly used as host cells for ALV. DF-1 cells were infected with the three ALV-K strains along with SDAU09C1 (ALV-A), SDAU09C2 (ALV-B), and NX0101 (ALV-J) as controls. As shown in Figure 4, the replication rates of the three ALV-K strains were markedly lower than those of other ALVs, and NX0101(ALV-J) showed the highest replication rate. Figure 4. View largeDownload slide Comparison of the viral replication dynamics of ALV-K to ALV-A, ALV-B, and ALV-J. Growth curves were generated by measuring the viral titers at various time points. The viral titers were determined using samples harvested every day and were expressed as TCID50 mL–1. The mean ± SD values from three independent experiments are shown. Figure 4. View largeDownload slide Comparison of the viral replication dynamics of ALV-K to ALV-A, ALV-B, and ALV-J. Growth curves were generated by measuring the viral titers at various time points. The viral titers were determined using samples harvested every day and were expressed as TCID50 mL–1. The mean ± SD values from three independent experiments are shown. DISCUSSION ALVs can be classified as endogenous (ALV-E) or exogenous according to their mode of transmission. Exogenous ALVs from chickens are classified into five different subgroups (A, B, C, D, and J) based on their host range and viral interference and cross-neutralization activities (Payne et al., 1991, 1992). Recently, a new subgroup, ALV-K, was identified in the in the indigenous chicken breeds of East Asia (Cui et al., 2014). To assess the status of avian leukosis virus infection in indigenous chicken breeds in China, a total of 121 plasma samples collected from various indigenous chicken breeds from 2015 to 2016 were tested for the presence of ALV. In this study, 14 ALVs were isolated and identified from several Chinese indigenous breeds. The phylogenetic analysis showed that the isolates consisted of two ALV-A strains, one ALV-B strain, eight ALV-J strains, and three ALV-K strains, which demonstrated the exist of different subgroups of ALVs in the indigenous chicken breeds of China. A genome analysis of those three ALV-K was performed. The results, in keeping with previous reports (Cui et al., 2014; Dong et al., 2015b; Li et al., 2016; Shao et al., 2017), revealed that SDAUAK-11, SDAUAK-12, and SDAUAK-13 belong a single clade along with other ALV-K and are distantly phylogenetically related to reference strains of other existing ALV subgroups. The nucleotide and amino acid sequences of different regions of SDAUAK-11, SDAUAK-12, and SDAUAK-13 were analyzed, and the results showed that the pol gene was highly conserved (>97% nt identity) among them; however, compared to other ALVs, the genes contained a single nucleotide deletion at position 24 and an 8-nucleotide deletion at positions 32 to 39, which led to the corresponding amino acid sequence PLKWK being replaced by RS, which has not been previously reported. Pol encodes several enzymatic activities. For example, the reverse transcriptase encoded by the pol gene catalyzes DNA synthesis using DNA or RNA as a template. The pol protein also has nuclease H activity, which degrades the RNA in a DNA: RNA heterodimer. Subunit b of pol has an IN domain, specifically an integrase (p32) domain, which could catalyze integration of the provirus DNA into the genome of host cells. This mutation, and the resulting amino acid sequence changes, did not prevent the transcription and translation of the pol gene; however, its effects on the biology of ALV require further study. As a retrovirus, the replication of ALV requires RNA as intermediate in reverse transcription, which lacks a proofreading function and tends to allow recombination. In recent years, recombination events have been frequently detected among the different subgroups of ALV. An exogenous ALV-A strain was isolated from Malik vaccine by Silva et al. (2007), which was a recombinant virus containing the gp85 gene of ALV-A and a partial gene from an endogenous virus. An ALV strain was isolated by Cai et al. (2013) that was identified as a recombinant virus composed of a gp85 gene from ALV-C, a gp37 gene from ALV-E, and a LTR from ALV-J. SDAU09E1 (ALV-A), which was isolated from a Chinese indigenous breed (Zhang, 2010), also has a partial sequence from ALV-E in the LTR region. Those recombination events lead to variation among ALVs. In particular, ALV isolates carrying the endogenous LTR induce lower level p27 antigen expression, thus reducing the relevance ratio, and easily triggering the wide spread of ALV. In this study, potential recombination events were detected in the three new ALV-K isolates, which showed the complex evolutionary processes among ALVs. More importantly, potential recombination events were detected in the env gene (a major determinant of subgroup and pathogenicity) of all three ALV-K strains, which suggested that recombination might be the primary cause of ALV evolution. On the other hand, the replication rates of the three ALV-K strains were markedly lower than those of the other ALVs in DF-1 cells, which may be the reason for the difficulty in their isolation and identification. Future studies should investigate the large geographic distribution of these novel ALVs, and monitor their variation, rearrangements, host range, and associated diseases. Acknowledgements This work was supported by the National Key Research and Development Program of China (2016YFD0501606) and the Shandong “Double Tops” Program (SYL2017YSTD11). AUTHOR CONTRIBUTIONS QS and YL conceived and performed the experiments, analyzed the data, and drafted the manuscript. PZ and SCu supervised the project and edited the manuscript. ST and SCu conducted part of the experiments. SCh and ZC analyzed part of the data. PZ and ZC provided important suggestions. REFERENCES Bai J. , Howes K. , Payne L. N. , Skinner M. A. . 1995 . Sequence of host-range determinants in the env gene of a full-length, infectious proviral clone of exogenous avian leukosis virus HPRS-103 confirms that it represents a new subgroup (designated J) . J. Gen. Virol. 76 : 181 – 187 . 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Comparison of Genome and Biological Characteristic of Different Subgroup A Avian Leukosis Virus Strains . [Dissertation]. [Tai’an (SD)] . University of ShanDong Agriculture . © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Molecular characteristics of avian leukosis viruses isolated from indigenous chicken breeds in China

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Oxford University Press
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© 2018 Poultry Science Association Inc.
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0032-5791
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1525-3171
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10.3382/ps/pex367
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Abstract

Abstract To assess the status of avian leukosis virus (ALV) infection in indigenous chicken breeds in China, 121 plasma samples collected from various indigenous chicken breeds were tested for the presence of ALV from 2015 to 2016. A total of 14 ALV strains were isolated and identified, including two ALV-A strains, one ALV-B strain, eight ALV-J strains, and three ALV-K strains. To study the genome structure, biological characteristics, and the evolutionary relationships of the ALV-K strains with other known subgroup strains from infected chickens, we determined the complete genome sequence of the three ALV-K strains and performed comparative analysis using the whole genome sequence or selected sequence elements. The replication rates of the three ALV-K strains were markedly lower than the rates of other ALVs, and they shared a common mutation in the pol gene, which had not been previously observed. In addition, nine putative recombinant events were detected in the genomes of the three newly isolated ALV-K strains, with high statistical support. This was the first report of an ALV-K reorganization event, which has contributed to its genetic evolution. In summary, we established a robust classification system for ALV, especially for ALV-K, and revealed additional genomic diversity for the ALV strains in indigenous chicken breeds. Therefore additional works are warranted to explore ALV genomics and epidemiology. INTRODUCTION Avian leukosis virus (ALV) is a pathogenic virus, belong to the genus alpharetrovirus of the family retroviridae that causes avian leukemia as the first known virus associated with poultry tumors (Weiss and Vogt, 2011). In addition, ALV has also been shown to cause subclinical infection in chickens, and is involved in economically important conditions such as egg drop syndrome, immunosuppression, and growth retardation (Payne and Nair, 2012). Especially in recent years, ALV infections in layers and indigenous chicken breeds have been frequently reported in China (Dong et al., 2015a,b; Lai et al., 2011). Currently, ALVs have been divided into 11 different viral subgroups (designated A to K) based on differences in the envelope sequences. Viruses of subgroups A, B, and J are the main exogenous ALVs that infect chickens in the field. Subgroups C and D have rarely been reported (Morgan, 1973; Sandelin and Estola, 1974). Only subgroup E is an endogenous virus has no relationship to the pathogenicity of the viruses (Smith, 1987). Recently, a homology analysis of the gp85 gene sequences from >20 ALV strains were isolated from several indigenous chicken breeds in China by our lab, and the results showed at least 90% nt identity to each other but relatively low similarity to ALV-A, ALV-B, ALV-C, ALV-D, ALV-E, and ALV-J isolates (<90% nt identity). In addition, the gp85 gene of those ALVs were closely related to ALVs isolated from Taiwan or Japan (∼95% nt identity; Dong et al., 2015b). Obviously, this new subgroup of ALV, ALV-K, has existed in the indigenous chicken breeds of East Asia for a long time. Strain JS11C1, which was isolated from a Chinese native breed “Luhua” chicken, has been named as the prototype strain of ALV-K (Cui et al., 2014). Subsequent epidemiological investigation revealed that ALV-K was widespread among Chinese native chickens. However, there are few reports on the genetic analysis and biological characteristics of ALV-K (Li et al., 2016; Shao et al., 2017). In addition, recombination can occur between exogenous viruses; exogenous and endogenous viruses; and exogenous viruses and non-homologous genomes. It has been reported that ALV-J is a recombinant product of an exogenous ALV and an endogenous virus (Bai et al., 1995; Benson et al., 1998; Venugopal et al., 1998), and recombination events among ALVs of different subgroups may lead to viral mutations (Liu et al., 2011; Cai et al., 2013). Infection by these recombinant exogenous viruses may not be detected, causing extensive spread of ALV. Reports about the possibility of recombination of the new subgroup (ALV-K) appear to be rare. In the present study, we described the characteristics of 14 novel ALV strains that were isolated from plasma samples of Chinese indigenous chickens using DF-1 cell culture and ALV p27 antigen detection. In addition, we analyzed three full-length ALV genome sequences using various phylogenetic methods and explored the putative recombination events within each genome. This comprehensive phylogenetic analysis of ALV-K should pave the way for further research into the prevention of ALV-K infection. MATERIALS AND METHODS Virus Isolation and Identification To estimate the ALV infection status of Chinese indigenous chicken breeds, a total of 121 plasma samples were collected from several different indigenous Chinese chicken breeds from 2015 to 2016. Samples of whole blood were collected in sterile 1.5-mL tubes containing 1% sodium heparin (Transgen Biotech, China), and the tubes were inverted several times to avoid clotting. The tubes containing 1.5 mL whole blood were centrifuged for 2 min at 2,000 rpm and the plasma was transferred to fresh plastic tubes and were stored at −20°C. All virus isolations were performed in DF-1 chicken fibroblast cell line maintained in our laboratory (American Type Culture Collection, Manassas, VA). The DF-1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Shanghai, China) supplemented with 12% fetal bovine serum (FBS; Invitrogen) at 37°C in a 5% CO2 incubator. For virus isolation, lymphocytes from the plasma samples were incubated on DF-1 cell monolayers in 24-well culture plates after centrifugation at 1,500 × g for 2 min. The uninfected DF-1 cells were used as a negative control (NC). The culture supernatant containing the virus was harvested 7 days later. After three blind passages of infected cells, the cell supernatants and cell samples were stored at −80°C until analysis. After three freeze-thaw cycles, the supernatant samples from each well (described previously) were examined for the presence of ALV group-specific P27 antigen using the Avian Leukosis Virus Antigen Test Kit (IDEXX; Yuanheng Laboratories, Beijing, China) as described previously (Qin et al., 2001). For subgroup verification by phylogenetic analysis (MEGA version 5.0), positively infected DF-1 cells were selected as a template for gp85 amplification (using the primer pair shown in Table 1), specifically of a highly conserved region common to all ALV subgroups. Non-infected DF-1cells were used as a negative control. Table 1. Primers used for PCR amplification of gp85 and the whole genome sequence of ALV-K isolates. Primera Sequence Fragment size (bp) gp85-F 5΄-GATGAGGCGAGCCCTCTCTTTG-3΄ 1,127 gp85-R 5΄-TGTTGGGAGGTAAAATGGCGT-3΄ A-F 5΄-GAGATTGTCTGCAGGGCCTAGGGCT-3΄ 2,715 A-R 5΄-TGGCAGCAAGGGTGTCTTCTCCG-3΄ B-F 5΄-CACCACATTGGTGTGCACCTGGGT-3΄ 2,778 B-R 5΄-GAAGGGGCCACTGGTCAATCCACA-3΄ C-F 5΄-GAGGTGACTAAGAAAGATGAGGCGA-3΄ 2,124 C-R 5΄-CATCTCCCCCTCCCTATGCGAAAGC-3΄ D-F 5΄-ATTGGAGCAGTGTAAGCAGTACG-3΄ 1,148 D-R 5΄-CGTTTATGACGCTTCCATGCTTG-3΄ Primera Sequence Fragment size (bp) gp85-F 5΄-GATGAGGCGAGCCCTCTCTTTG-3΄ 1,127 gp85-R 5΄-TGTTGGGAGGTAAAATGGCGT-3΄ A-F 5΄-GAGATTGTCTGCAGGGCCTAGGGCT-3΄ 2,715 A-R 5΄-TGGCAGCAAGGGTGTCTTCTCCG-3΄ B-F 5΄-CACCACATTGGTGTGCACCTGGGT-3΄ 2,778 B-R 5΄-GAAGGGGCCACTGGTCAATCCACA-3΄ C-F 5΄-GAGGTGACTAAGAAAGATGAGGCGA-3΄ 2,124 C-R 5΄-CATCTCCCCCTCCCTATGCGAAAGC-3΄ D-F 5΄-ATTGGAGCAGTGTAAGCAGTACG-3΄ 1,148 D-R 5΄-CGTTTATGACGCTTCCATGCTTG-3΄ aF and R represent upstream and downstream primers, respectively. View Large Table 1. Primers used for PCR amplification of gp85 and the whole genome sequence of ALV-K isolates. Primera Sequence Fragment size (bp) gp85-F 5΄-GATGAGGCGAGCCCTCTCTTTG-3΄ 1,127 gp85-R 5΄-TGTTGGGAGGTAAAATGGCGT-3΄ A-F 5΄-GAGATTGTCTGCAGGGCCTAGGGCT-3΄ 2,715 A-R 5΄-TGGCAGCAAGGGTGTCTTCTCCG-3΄ B-F 5΄-CACCACATTGGTGTGCACCTGGGT-3΄ 2,778 B-R 5΄-GAAGGGGCCACTGGTCAATCCACA-3΄ C-F 5΄-GAGGTGACTAAGAAAGATGAGGCGA-3΄ 2,124 C-R 5΄-CATCTCCCCCTCCCTATGCGAAAGC-3΄ D-F 5΄-ATTGGAGCAGTGTAAGCAGTACG-3΄ 1,148 D-R 5΄-CGTTTATGACGCTTCCATGCTTG-3΄ Primera Sequence Fragment size (bp) gp85-F 5΄-GATGAGGCGAGCCCTCTCTTTG-3΄ 1,127 gp85-R 5΄-TGTTGGGAGGTAAAATGGCGT-3΄ A-F 5΄-GAGATTGTCTGCAGGGCCTAGGGCT-3΄ 2,715 A-R 5΄-TGGCAGCAAGGGTGTCTTCTCCG-3΄ B-F 5΄-CACCACATTGGTGTGCACCTGGGT-3΄ 2,778 B-R 5΄-GAAGGGGCCACTGGTCAATCCACA-3΄ C-F 5΄-GAGGTGACTAAGAAAGATGAGGCGA-3΄ 2,124 C-R 5΄-CATCTCCCCCTCCCTATGCGAAAGC-3΄ D-F 5΄-ATTGGAGCAGTGTAAGCAGTACG-3΄ 1,148 D-R 5΄-CGTTTATGACGCTTCCATGCTTG-3΄ aF and R represent upstream and downstream primers, respectively. View Large Genomic DNA Amplification and Sequencing To obtain the complete proviral genome of the three ALV-K strains, four pairs of overlapping primers were designed based on the sequences of JS11C1 (GenBank accession no. KF746200). The primers used are shown in Table 1. The complete sequence of strain was amplified by polymerase chain reaction (PCR) using genomic DNA extracted from infected DF-1 cells as a template with Premix LA Taq polymerase (TaKaRa, Dalian, China) in a 50-μL reaction containing 4 μL of dNTP mixture (TaKaRa), 5 μL of 10 × PCR buffer (TaKaRa), 1 μL of Taq polymerase (TaKaRa), 2 μL of DNA solution, 1 μL of forward and reverse primers, and 36 μL of ddH2O. The thermo cycling profiles for the PCR amplification included an initial denaturation step at 95°C for 5 min, followed by 30 cycles of 95°C for 30 s, annealing at the corresponding Tm (Annealing Temperature) for 30 s, and elongation at 72°C for 3 min, with a final extension at 72°C for 10 min. The PCR products were separated by 1% agarose gel electrophoresis and purified using the Omega Gel Extraction Kit (Omega Bio-tek, USA). The purified PCR products were cloned into the pMD-18T vector (Transgen Biotech, China) and sequenced. The resulting construct was then used to transform Escherichia coli DH5α cells (TaRaKa). DNA from positive clones was sequenced directly (Shenggong, Shanghai, China), and each fragment was sequenced three times independently. Sequence Alignment and Analysis The full-length proviral genome sequences of the three ALV-K isolates were assembled using DNAStar (version 7.0), and a multiple sequence alignment was obtained using Clustal X (BioEdit version 7.0). Nucleotide and deduced amino acid sequence similarity searches were performed using MEGA (version 5.0). The sequences obtained in this study have been deposited in GenBank, and the ALV reference strains (with origin and accession numbers) that were used in this study are shown in Table 2. Table 2. Avian leukosis virus strains used in this study. No. Sub group Isolate Origin Accession no. No. Sub group Isolate Origin Accession no. 1 A B53 USA DQ412727 16 E Ev-1 USA AY013303 2 A RAV-A France M37980 17 E Ev-3 USA AY013304 3 A SDAU09E1 China HM452341 18 E ALVE-B11 Canada KC610517 4 A RAV-1 USA M19113 19 E SD0501 China EF467236 5 A MQNCSU USA DQ365814 20 J ADOL7501 USA AY027920 6 A MAV-1 USA L10922 21 J GD1109 China JX254901 7 A SDAU09C3 China HM452340 22 J HPRS-103 UK Z46390 8 A SDAU09E2 China HM452342 23 J NX0101 China AY897227 9 A SDAU09C1 China HM452339 24 J 0661 USA AF247566 10 B RSV-2 USA M14902 25 J SD07LK1 China FJ201640 11 B RSV-SR-B USA AF052428 26 J HN0001 China AY897219 12 B SDAU09E3 China JF826241 27 K JS11C1 China KF746200 13 B SDAU09C2 China HM446005 28 K GD14LZ China KU605754 14 C RSV-Prague C USA J02342 29 K GDFX0601 China KP686142 15 D RSV-SR-D USA D10652 30 K GDFX0602 China KP686143 No. Sub group Isolate Origin Accession no. No. Sub group Isolate Origin Accession no. 1 A B53 USA DQ412727 16 E Ev-1 USA AY013303 2 A RAV-A France M37980 17 E Ev-3 USA AY013304 3 A SDAU09E1 China HM452341 18 E ALVE-B11 Canada KC610517 4 A RAV-1 USA M19113 19 E SD0501 China EF467236 5 A MQNCSU USA DQ365814 20 J ADOL7501 USA AY027920 6 A MAV-1 USA L10922 21 J GD1109 China JX254901 7 A SDAU09C3 China HM452340 22 J HPRS-103 UK Z46390 8 A SDAU09E2 China HM452342 23 J NX0101 China AY897227 9 A SDAU09C1 China HM452339 24 J 0661 USA AF247566 10 B RSV-2 USA M14902 25 J SD07LK1 China FJ201640 11 B RSV-SR-B USA AF052428 26 J HN0001 China AY897219 12 B SDAU09E3 China JF826241 27 K JS11C1 China KF746200 13 B SDAU09C2 China HM446005 28 K GD14LZ China KU605754 14 C RSV-Prague C USA J02342 29 K GDFX0601 China KP686142 15 D RSV-SR-D USA D10652 30 K GDFX0602 China KP686143 View Large Table 2. Avian leukosis virus strains used in this study. No. Sub group Isolate Origin Accession no. No. Sub group Isolate Origin Accession no. 1 A B53 USA DQ412727 16 E Ev-1 USA AY013303 2 A RAV-A France M37980 17 E Ev-3 USA AY013304 3 A SDAU09E1 China HM452341 18 E ALVE-B11 Canada KC610517 4 A RAV-1 USA M19113 19 E SD0501 China EF467236 5 A MQNCSU USA DQ365814 20 J ADOL7501 USA AY027920 6 A MAV-1 USA L10922 21 J GD1109 China JX254901 7 A SDAU09C3 China HM452340 22 J HPRS-103 UK Z46390 8 A SDAU09E2 China HM452342 23 J NX0101 China AY897227 9 A SDAU09C1 China HM452339 24 J 0661 USA AF247566 10 B RSV-2 USA M14902 25 J SD07LK1 China FJ201640 11 B RSV-SR-B USA AF052428 26 J HN0001 China AY897219 12 B SDAU09E3 China JF826241 27 K JS11C1 China KF746200 13 B SDAU09C2 China HM446005 28 K GD14LZ China KU605754 14 C RSV-Prague C USA J02342 29 K GDFX0601 China KP686142 15 D RSV-SR-D USA D10652 30 K GDFX0602 China KP686143 No. Sub group Isolate Origin Accession no. No. Sub group Isolate Origin Accession no. 1 A B53 USA DQ412727 16 E Ev-1 USA AY013303 2 A RAV-A France M37980 17 E Ev-3 USA AY013304 3 A SDAU09E1 China HM452341 18 E ALVE-B11 Canada KC610517 4 A RAV-1 USA M19113 19 E SD0501 China EF467236 5 A MQNCSU USA DQ365814 20 J ADOL7501 USA AY027920 6 A MAV-1 USA L10922 21 J GD1109 China JX254901 7 A SDAU09C3 China HM452340 22 J HPRS-103 UK Z46390 8 A SDAU09E2 China HM452342 23 J NX0101 China AY897227 9 A SDAU09C1 China HM452339 24 J 0661 USA AF247566 10 B RSV-2 USA M14902 25 J SD07LK1 China FJ201640 11 B RSV-SR-B USA AF052428 26 J HN0001 China AY897219 12 B SDAU09E3 China JF826241 27 K JS11C1 China KF746200 13 B SDAU09C2 China HM446005 28 K GD14LZ China KU605754 14 C RSV-Prague C USA J02342 29 K GDFX0601 China KP686142 15 D RSV-SR-D USA D10652 30 K GDFX0602 China KP686143 View Large Replication of the ALV Isolates in DF-1 Cells The titers of the three ALV-K strains are presented as TCID50 mL−1 and were measured by ELISA using the Reed-Muench method. Briefly, DF-1 cells were plated (at approximately 106 cells per dish) in 60-mm dishes 1 day before infection with 1,000 TCID50 of virus. Three exogenous ALVs are maintained in our laboratory, including a subgroup A strain (strain SDAU09C1), a subgroup B strain (strain SDAU09C2), and a subgroup J strain (strain NX0101), were used as controls. The infections were carried out in the presence of 1% FBS at 37°C under 5% CO2, and aliquots (approximately 400 μL) were harvested on days 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 post-infection. At various times points, the harvested supernatant was replaced with an equal volume of fresh DMEM. After three freeze-thaw cycles, the harvested samples were examined for ALV group-specific p27 antigen by ELISA to determine the replication kinetics. Each sample was tested independently three times. Sequencing to Test for Recombination Events Putative recombination events within each ALV genome were detected using the Recombination Detection Program 4(RDP4; Martin, 2009). Other detection methods, including RDP, GENECONV, BootScan, MaxChi, Chimaera, SiScan, Phyl-Pro, LARD, and 3Seq, were also employed for comparison (Martin et al., 2005a,b). Only recombination events supported by no fewer than six independent methods were regarded as positive. These putative recombination events were further confirmed and visualized using SimPlot (Lole et al., 1999). Ethics Statement The animal care and use protocol was approved by the Shandong Agricultural University Animal care and use Committee (SDAUA-2016–002). All of the experimental animals of this study were cared for and maintained throughout of the experiments strictly following the ethics and biosecurity guidelines approved by the Institutional Animal Care and Use Committee of Shandong Agricultural University. RESULTS Isolation and Identification of ALVs Fourteen ALV strains were isolated from different indigenous chicken breeds in Shandong, Zhejiang, and Jiangsu province from 2015 to 2016 (Table 3). DF-1 cells infected with these strains showed a positive result (the S/P (Samples OD value-negative control value)/(positive control value-negative control value) values of the infected group were between 0.65 and 1.23, positive critical value was 0.2) by measuring the ALV-27 antigen level with an ELISA, whereas mock-infected DF-1 cells showed a negative result (the S/P values of the control group were between 0.00 and 0.08), these results indicated the presence of exogenous ALV in the samples. The gp85 genes of the ALV isolates were amplified by PCR and sequencing. Phylogenetic analysis was carried out on 30 other ALV strains of known subgroups (Figure 1). The results showed that among the new isolates were two ALV-A strains, one ALV-B strain, eight ALV-J strains, and three ALV-K strains. The accession numbers are listed in Table 3. Figure 1. View largeDownload slide Phylogenetic relationships among the gp85 sequences of 14 ALV strains and other ALVs of different subgroups. ALVs isolated in this survey are marked with a star symbol. Figure 1. View largeDownload slide Phylogenetic relationships among the gp85 sequences of 14 ALV strains and other ALVs of different subgroups. ALVs isolated in this survey are marked with a star symbol. Table 3. Avian leukosis virus strains isolated in this study. Accession no. Strain name Year Province Host Length (bp) KY773915 SDAUAB-1 2014 Shandong HY-LINE VARIETY BROWN 1,824 KY773913 SDAUAA-2 2014 Hebei HY-LINE VARIETY BROWN 1,798 KY767730 SDAUAJ-3 2014 Hebei HY-LINE VARIETY BROWN 1,791 KY773914 SDAUAA-4 2014 Hebei HY-LINE VARIETY BROWN 1,788 KY773916 SDAUAJ-5 2015 Jiangsu LANGYA 1,686 KY773917 SDAUAJ-6 2015 Jiangsu LANGYA 1,671 KY773918 SDAUAJ-7 2015 Jiangsu BEIJING YOU 1,689 KY773919 SDAUAJ-8 2015 Jiangsu BEIJING YOU 1,686 KY773920 SDAUAJ-9 2015 Jiangsu LUHUA 1,686 KY773921 SDAUAJ-11 2015 Jiangsu LANGYA 1,662 KY773922 SDAUAJ-12 2015 Jiangsu LANGYA 1,692 KY773911 SDAUAK-11 2015 Jiangsu LANGYA 7,814 KY773912 SDAUAK-12 2015 Jiangsu LANGYA 7,811 KY767731 SDAUAK-13 2015 Jiangsu LANGYA 7,813 Accession no. Strain name Year Province Host Length (bp) KY773915 SDAUAB-1 2014 Shandong HY-LINE VARIETY BROWN 1,824 KY773913 SDAUAA-2 2014 Hebei HY-LINE VARIETY BROWN 1,798 KY767730 SDAUAJ-3 2014 Hebei HY-LINE VARIETY BROWN 1,791 KY773914 SDAUAA-4 2014 Hebei HY-LINE VARIETY BROWN 1,788 KY773916 SDAUAJ-5 2015 Jiangsu LANGYA 1,686 KY773917 SDAUAJ-6 2015 Jiangsu LANGYA 1,671 KY773918 SDAUAJ-7 2015 Jiangsu BEIJING YOU 1,689 KY773919 SDAUAJ-8 2015 Jiangsu BEIJING YOU 1,686 KY773920 SDAUAJ-9 2015 Jiangsu LUHUA 1,686 KY773921 SDAUAJ-11 2015 Jiangsu LANGYA 1,662 KY773922 SDAUAJ-12 2015 Jiangsu LANGYA 1,692 KY773911 SDAUAK-11 2015 Jiangsu LANGYA 7,814 KY773912 SDAUAK-12 2015 Jiangsu LANGYA 7,811 KY767731 SDAUAK-13 2015 Jiangsu LANGYA 7,813 View Large Table 3. Avian leukosis virus strains isolated in this study. Accession no. Strain name Year Province Host Length (bp) KY773915 SDAUAB-1 2014 Shandong HY-LINE VARIETY BROWN 1,824 KY773913 SDAUAA-2 2014 Hebei HY-LINE VARIETY BROWN 1,798 KY767730 SDAUAJ-3 2014 Hebei HY-LINE VARIETY BROWN 1,791 KY773914 SDAUAA-4 2014 Hebei HY-LINE VARIETY BROWN 1,788 KY773916 SDAUAJ-5 2015 Jiangsu LANGYA 1,686 KY773917 SDAUAJ-6 2015 Jiangsu LANGYA 1,671 KY773918 SDAUAJ-7 2015 Jiangsu BEIJING YOU 1,689 KY773919 SDAUAJ-8 2015 Jiangsu BEIJING YOU 1,686 KY773920 SDAUAJ-9 2015 Jiangsu LUHUA 1,686 KY773921 SDAUAJ-11 2015 Jiangsu LANGYA 1,662 KY773922 SDAUAJ-12 2015 Jiangsu LANGYA 1,692 KY773911 SDAUAK-11 2015 Jiangsu LANGYA 7,814 KY773912 SDAUAK-12 2015 Jiangsu LANGYA 7,811 KY767731 SDAUAK-13 2015 Jiangsu LANGYA 7,813 Accession no. Strain name Year Province Host Length (bp) KY773915 SDAUAB-1 2014 Shandong HY-LINE VARIETY BROWN 1,824 KY773913 SDAUAA-2 2014 Hebei HY-LINE VARIETY BROWN 1,798 KY767730 SDAUAJ-3 2014 Hebei HY-LINE VARIETY BROWN 1,791 KY773914 SDAUAA-4 2014 Hebei HY-LINE VARIETY BROWN 1,788 KY773916 SDAUAJ-5 2015 Jiangsu LANGYA 1,686 KY773917 SDAUAJ-6 2015 Jiangsu LANGYA 1,671 KY773918 SDAUAJ-7 2015 Jiangsu BEIJING YOU 1,689 KY773919 SDAUAJ-8 2015 Jiangsu BEIJING YOU 1,686 KY773920 SDAUAJ-9 2015 Jiangsu LUHUA 1,686 KY773921 SDAUAJ-11 2015 Jiangsu LANGYA 1,662 KY773922 SDAUAJ-12 2015 Jiangsu LANGYA 1,692 KY773911 SDAUAK-11 2015 Jiangsu LANGYA 7,814 KY773912 SDAUAK-12 2015 Jiangsu LANGYA 7,811 KY767731 SDAUAK-13 2015 Jiangsu LANGYA 7,813 View Large Sequencing and Analysis of the Complete Proviral Genome of the Three ALV-K Strains Sequence analysis for the complete proviral genomes of the three ALV-K strains were determined by PCR and sequencing, and were named SDAUAK-11, SDAUAK-12, and SDAUAK-13. The lengths of the genomes were 7,814, 7,813, and 7,811 bp, respectively, with a genetic organization typical of replication-competent type C retroviruses lacking viral oncogenes (5΄-LTR-leader-gag-pol-env-3΄-LTR). The nucleotide and amino acid sequences of different regions were analyzed in comparison to the published reference ALVs from GenBank (Tables 4 to 6). The results showed that the gag and pol genes of the three ALV-K strains were highly conserved, and shared at least 97.0% and 91% nt identity, respectively. Analysis of the env gene and LTR showed that the strains were divided into two different situations, respectively. The env genes of the three ALV-K strains were closely related to those of the ALV-A, ALV-B, ALV-C, ALV-D, and ALV-E subgroups (with ∼90% nt identity), but showed only 50% nt identity to env from ALV-J. The LTR of the three ALV-K strains exhibited high sequence similarity to ALV-A and ALV-D strains, with 75.5 to 91.8% nt identity, but showed relatively low similarity to ALV-B, ALV-C, ALV-E, and ALV-J strains, with 32.5 to 42.1% nt identity. In a nutshell, the strains that were most similar to SDAUAK-11, SDAKAK-12, and SDAUAK-13 are GD14LZ, GDFX0601, and JS11C1, respectively, and the three ALV-K strains showed 90.8 to 97.7%, 90.8 to 97.7%, and 91.2 to 97.3% nt identity, respectively, to other ALV-K reference strains. Sequence of three newly identified ALV-K strains, the homology of their full-length genome, LTR, pol, gag, and env gene were 96.5 to 98.3%, 84.8 to 99.4%, 99.4 to 99.7%, 99.1 to 99.8%, 96.6 to 97.2%, respectively. Table 4. Segmental sequence comparison between isolate SDAUAK-11 and other ALV strains from different subgroups. UTR POL GAG ENV GENOME SDAUAK-11 Na N AAb N AA N AA N A 75.5% to 84.9% 97.2% to 99.3% 98.0% to 99.2% 95.4% to 97.3% 96.7% to 97.4% 87.1% to 90.9% 86.3% to 89.2% 87.4% to 94.4% B 32.5% to 38% 97.1% to 98.2% 98.0% to 98.5% 94.7% to 96.4% 96.6% to 97.9% 88.2% to 88.6% 85.1% to 86.6% 82.2% to 90.8% C 36.8% 98.5% 99.0% 96.3% 96.4% 91.3% 89.3% 88.4% D 76.1% 98.0% 98.7% 95.2% 96.4% 89.2% 87.3% 88.9% E 40.3% to 41.4% 99.2% to 99.5% 98.3% to 99.7% 98.7% to 99.1% 98.2% to 98.7% 93.1% to 93.2% 90.1% to 90.6% 94.9% to 95.8% J 36.0% to 37.3% 97.2% to 98.3% 97.4% to 98.9% 95.4% to 98.5% 96.2% to 97.7% 52.3% to 54.8% 48.5% to 50.3% 78.6% to 82.1% K 74.7% to 85.4% 98.8% to 99.6% 99.0% to 99.7% 96.7% to 99.1% 97.3% to 99.1% 95.9% to 99.3% 95.6% to 98.8% 90.8% to 97.7% UTR POL GAG ENV GENOME SDAUAK-11 Na N AAb N AA N AA N A 75.5% to 84.9% 97.2% to 99.3% 98.0% to 99.2% 95.4% to 97.3% 96.7% to 97.4% 87.1% to 90.9% 86.3% to 89.2% 87.4% to 94.4% B 32.5% to 38% 97.1% to 98.2% 98.0% to 98.5% 94.7% to 96.4% 96.6% to 97.9% 88.2% to 88.6% 85.1% to 86.6% 82.2% to 90.8% C 36.8% 98.5% 99.0% 96.3% 96.4% 91.3% 89.3% 88.4% D 76.1% 98.0% 98.7% 95.2% 96.4% 89.2% 87.3% 88.9% E 40.3% to 41.4% 99.2% to 99.5% 98.3% to 99.7% 98.7% to 99.1% 98.2% to 98.7% 93.1% to 93.2% 90.1% to 90.6% 94.9% to 95.8% J 36.0% to 37.3% 97.2% to 98.3% 97.4% to 98.9% 95.4% to 98.5% 96.2% to 97.7% 52.3% to 54.8% 48.5% to 50.3% 78.6% to 82.1% K 74.7% to 85.4% 98.8% to 99.6% 99.0% to 99.7% 96.7% to 99.1% 97.3% to 99.1% 95.9% to 99.3% 95.6% to 98.8% 90.8% to 97.7% aN represents the nucleotide sequence homology, bAA represent the amino acid sequence homology. View Large Table 4. Segmental sequence comparison between isolate SDAUAK-11 and other ALV strains from different subgroups. UTR POL GAG ENV GENOME SDAUAK-11 Na N AAb N AA N AA N A 75.5% to 84.9% 97.2% to 99.3% 98.0% to 99.2% 95.4% to 97.3% 96.7% to 97.4% 87.1% to 90.9% 86.3% to 89.2% 87.4% to 94.4% B 32.5% to 38% 97.1% to 98.2% 98.0% to 98.5% 94.7% to 96.4% 96.6% to 97.9% 88.2% to 88.6% 85.1% to 86.6% 82.2% to 90.8% C 36.8% 98.5% 99.0% 96.3% 96.4% 91.3% 89.3% 88.4% D 76.1% 98.0% 98.7% 95.2% 96.4% 89.2% 87.3% 88.9% E 40.3% to 41.4% 99.2% to 99.5% 98.3% to 99.7% 98.7% to 99.1% 98.2% to 98.7% 93.1% to 93.2% 90.1% to 90.6% 94.9% to 95.8% J 36.0% to 37.3% 97.2% to 98.3% 97.4% to 98.9% 95.4% to 98.5% 96.2% to 97.7% 52.3% to 54.8% 48.5% to 50.3% 78.6% to 82.1% K 74.7% to 85.4% 98.8% to 99.6% 99.0% to 99.7% 96.7% to 99.1% 97.3% to 99.1% 95.9% to 99.3% 95.6% to 98.8% 90.8% to 97.7% UTR POL GAG ENV GENOME SDAUAK-11 Na N AAb N AA N AA N A 75.5% to 84.9% 97.2% to 99.3% 98.0% to 99.2% 95.4% to 97.3% 96.7% to 97.4% 87.1% to 90.9% 86.3% to 89.2% 87.4% to 94.4% B 32.5% to 38% 97.1% to 98.2% 98.0% to 98.5% 94.7% to 96.4% 96.6% to 97.9% 88.2% to 88.6% 85.1% to 86.6% 82.2% to 90.8% C 36.8% 98.5% 99.0% 96.3% 96.4% 91.3% 89.3% 88.4% D 76.1% 98.0% 98.7% 95.2% 96.4% 89.2% 87.3% 88.9% E 40.3% to 41.4% 99.2% to 99.5% 98.3% to 99.7% 98.7% to 99.1% 98.2% to 98.7% 93.1% to 93.2% 90.1% to 90.6% 94.9% to 95.8% J 36.0% to 37.3% 97.2% to 98.3% 97.4% to 98.9% 95.4% to 98.5% 96.2% to 97.7% 52.3% to 54.8% 48.5% to 50.3% 78.6% to 82.1% K 74.7% to 85.4% 98.8% to 99.6% 99.0% to 99.7% 96.7% to 99.1% 97.3% to 99.1% 95.9% to 99.3% 95.6% to 98.8% 90.8% to 97.7% aN represents the nucleotide sequence homology, bAA represent the amino acid sequence homology. View Large Table 5. Segmental sequence comparison between isolate SDAUAK-12 and other ALV strains from different subgroups. UTR POL GAG ENV GENOME SDAUAK-12 Na N AAb N AA N AA N A 85.0% to 91.3% 97.2% to 98.8% 97.8% to 99.0% 95.5% to 97.3% 96.7% to 97.7% 87.4% to 90.5% 85.2% to 87.5% 87.1% to 95.8% B 33.3% to 36.4% 97.3% to 98.3% 97.9% to 98.3% 94.8% to 96.4% 96.7% to 97.9% 88.0% to 88.8% 84.9% to 86.6% 85.9% to 90.5% C 35.0% 98.6% 98.8% 96.5% 96.6% 91.1% 88.6% 89.3% D 86.1% 98.0% 98.2% 95.4% 96.6% 88.8% 86.6% 88.9% E 40.2% to 41.4% 99.4% to 99.6% 98.1% to 99.6% 98.7% to 99.1% 97.9% to 98.6% 92.6% to 93.0% 89.4% to 90.3% 94.9% to 95.8% J 35.4% to 36.7% 97.5% to 98.5% 97.2% to 98.7% 95.6% to 98.0% 96.3% to 98.0% 51.7% to 54.4% 48.0% to 49.9% 78.6% to 82.1% K 85.8% to 99.7% 98.9% to 99.7% 98.5% to 99.4% 97.0% to 99.8% 97.4% to 99.9% 96.0% to 99.2% 94.5% to 99.0% 90.8% to 97.7% UTR POL GAG ENV GENOME SDAUAK-12 Na N AAb N AA N AA N A 85.0% to 91.3% 97.2% to 98.8% 97.8% to 99.0% 95.5% to 97.3% 96.7% to 97.7% 87.4% to 90.5% 85.2% to 87.5% 87.1% to 95.8% B 33.3% to 36.4% 97.3% to 98.3% 97.9% to 98.3% 94.8% to 96.4% 96.7% to 97.9% 88.0% to 88.8% 84.9% to 86.6% 85.9% to 90.5% C 35.0% 98.6% 98.8% 96.5% 96.6% 91.1% 88.6% 89.3% D 86.1% 98.0% 98.2% 95.4% 96.6% 88.8% 86.6% 88.9% E 40.2% to 41.4% 99.4% to 99.6% 98.1% to 99.6% 98.7% to 99.1% 97.9% to 98.6% 92.6% to 93.0% 89.4% to 90.3% 94.9% to 95.8% J 35.4% to 36.7% 97.5% to 98.5% 97.2% to 98.7% 95.6% to 98.0% 96.3% to 98.0% 51.7% to 54.4% 48.0% to 49.9% 78.6% to 82.1% K 85.8% to 99.7% 98.9% to 99.7% 98.5% to 99.4% 97.0% to 99.8% 97.4% to 99.9% 96.0% to 99.2% 94.5% to 99.0% 90.8% to 97.7% aN represents the nucleotide sequence homology, bAA represent the amino acid sequence homology. View Large Table 5. Segmental sequence comparison between isolate SDAUAK-12 and other ALV strains from different subgroups. UTR POL GAG ENV GENOME SDAUAK-12 Na N AAb N AA N AA N A 85.0% to 91.3% 97.2% to 98.8% 97.8% to 99.0% 95.5% to 97.3% 96.7% to 97.7% 87.4% to 90.5% 85.2% to 87.5% 87.1% to 95.8% B 33.3% to 36.4% 97.3% to 98.3% 97.9% to 98.3% 94.8% to 96.4% 96.7% to 97.9% 88.0% to 88.8% 84.9% to 86.6% 85.9% to 90.5% C 35.0% 98.6% 98.8% 96.5% 96.6% 91.1% 88.6% 89.3% D 86.1% 98.0% 98.2% 95.4% 96.6% 88.8% 86.6% 88.9% E 40.2% to 41.4% 99.4% to 99.6% 98.1% to 99.6% 98.7% to 99.1% 97.9% to 98.6% 92.6% to 93.0% 89.4% to 90.3% 94.9% to 95.8% J 35.4% to 36.7% 97.5% to 98.5% 97.2% to 98.7% 95.6% to 98.0% 96.3% to 98.0% 51.7% to 54.4% 48.0% to 49.9% 78.6% to 82.1% K 85.8% to 99.7% 98.9% to 99.7% 98.5% to 99.4% 97.0% to 99.8% 97.4% to 99.9% 96.0% to 99.2% 94.5% to 99.0% 90.8% to 97.7% UTR POL GAG ENV GENOME SDAUAK-12 Na N AAb N AA N AA N A 85.0% to 91.3% 97.2% to 98.8% 97.8% to 99.0% 95.5% to 97.3% 96.7% to 97.7% 87.4% to 90.5% 85.2% to 87.5% 87.1% to 95.8% B 33.3% to 36.4% 97.3% to 98.3% 97.9% to 98.3% 94.8% to 96.4% 96.7% to 97.9% 88.0% to 88.8% 84.9% to 86.6% 85.9% to 90.5% C 35.0% 98.6% 98.8% 96.5% 96.6% 91.1% 88.6% 89.3% D 86.1% 98.0% 98.2% 95.4% 96.6% 88.8% 86.6% 88.9% E 40.2% to 41.4% 99.4% to 99.6% 98.1% to 99.6% 98.7% to 99.1% 97.9% to 98.6% 92.6% to 93.0% 89.4% to 90.3% 94.9% to 95.8% J 35.4% to 36.7% 97.5% to 98.5% 97.2% to 98.7% 95.6% to 98.0% 96.3% to 98.0% 51.7% to 54.4% 48.0% to 49.9% 78.6% to 82.1% K 85.8% to 99.7% 98.9% to 99.7% 98.5% to 99.4% 97.0% to 99.8% 97.4% to 99.9% 96.0% to 99.2% 94.5% to 99.0% 90.8% to 97.7% aN represents the nucleotide sequence homology, bAA represent the amino acid sequence homology. View Large Table 6. Segmental sequence comparison between isolate SDAUAK-13 and other ALV strains from different subgroups. UTR POL GAG ENV GENOME SDAUAK-13 Na N AAb N AA N AA N A 86.2% to 91.8% 97.4% to 99.7% 97.9% to 99.3% 91.3% to 96.6% 96.9% to 97.9% 88.0% to 90.8% 86.1% to 88.9% 87.2% to 95.8% B 34.4% to 36.4% 97.5% to 98.5% 97.9% to 98.4% 95.0% to 96.4% 96.9% to 98.0% 88.1% to 89.0% 85.6% to 87.4% 85.9% to 90.6% C 35.5% 98.9% 99.1% 96.5% 96.7% 91.1% 89.1% 89.9% D 87.4% 98.2% 98.5% 95.4% 96.7% 88.9% 87.6% 89.3% E 40.6% to 42.1% 99.5% to 99.8% 98.1% to 99.6% 98.7% to 99.1% 98.1% to 98.7% 92.9% to 93.2 90.5% to 91.0% 94.8% to 95.8% J 35.4% to 36.7% 97.6% to 98.6% 97.2% to 98.7% 95.6% to 98.9% 96.4% to 98.1% 52.3% to 54.9% 48.0% to 49.7% 78.7% to 82.0% K 87.7% to 99.4% 99.1% to 99.9% 98.9% to 99.6% 97.0% to 99.9% 97.6% to 99.9% 96.2% to 97.5% 95.8% to 96.8% 91.2% to 97.3% UTR POL GAG ENV GENOME SDAUAK-13 Na N AAb N AA N AA N A 86.2% to 91.8% 97.4% to 99.7% 97.9% to 99.3% 91.3% to 96.6% 96.9% to 97.9% 88.0% to 90.8% 86.1% to 88.9% 87.2% to 95.8% B 34.4% to 36.4% 97.5% to 98.5% 97.9% to 98.4% 95.0% to 96.4% 96.9% to 98.0% 88.1% to 89.0% 85.6% to 87.4% 85.9% to 90.6% C 35.5% 98.9% 99.1% 96.5% 96.7% 91.1% 89.1% 89.9% D 87.4% 98.2% 98.5% 95.4% 96.7% 88.9% 87.6% 89.3% E 40.6% to 42.1% 99.5% to 99.8% 98.1% to 99.6% 98.7% to 99.1% 98.1% to 98.7% 92.9% to 93.2 90.5% to 91.0% 94.8% to 95.8% J 35.4% to 36.7% 97.6% to 98.6% 97.2% to 98.7% 95.6% to 98.9% 96.4% to 98.1% 52.3% to 54.9% 48.0% to 49.7% 78.7% to 82.0% K 87.7% to 99.4% 99.1% to 99.9% 98.9% to 99.6% 97.0% to 99.9% 97.6% to 99.9% 96.2% to 97.5% 95.8% to 96.8% 91.2% to 97.3% aN represents the nucleotide sequence homology, bAA represent the amino acid sequence homology. View Large Table 6. Segmental sequence comparison between isolate SDAUAK-13 and other ALV strains from different subgroups. UTR POL GAG ENV GENOME SDAUAK-13 Na N AAb N AA N AA N A 86.2% to 91.8% 97.4% to 99.7% 97.9% to 99.3% 91.3% to 96.6% 96.9% to 97.9% 88.0% to 90.8% 86.1% to 88.9% 87.2% to 95.8% B 34.4% to 36.4% 97.5% to 98.5% 97.9% to 98.4% 95.0% to 96.4% 96.9% to 98.0% 88.1% to 89.0% 85.6% to 87.4% 85.9% to 90.6% C 35.5% 98.9% 99.1% 96.5% 96.7% 91.1% 89.1% 89.9% D 87.4% 98.2% 98.5% 95.4% 96.7% 88.9% 87.6% 89.3% E 40.6% to 42.1% 99.5% to 99.8% 98.1% to 99.6% 98.7% to 99.1% 98.1% to 98.7% 92.9% to 93.2 90.5% to 91.0% 94.8% to 95.8% J 35.4% to 36.7% 97.6% to 98.6% 97.2% to 98.7% 95.6% to 98.9% 96.4% to 98.1% 52.3% to 54.9% 48.0% to 49.7% 78.7% to 82.0% K 87.7% to 99.4% 99.1% to 99.9% 98.9% to 99.6% 97.0% to 99.9% 97.6% to 99.9% 96.2% to 97.5% 95.8% to 96.8% 91.2% to 97.3% UTR POL GAG ENV GENOME SDAUAK-13 Na N AAb N AA N AA N A 86.2% to 91.8% 97.4% to 99.7% 97.9% to 99.3% 91.3% to 96.6% 96.9% to 97.9% 88.0% to 90.8% 86.1% to 88.9% 87.2% to 95.8% B 34.4% to 36.4% 97.5% to 98.5% 97.9% to 98.4% 95.0% to 96.4% 96.9% to 98.0% 88.1% to 89.0% 85.6% to 87.4% 85.9% to 90.6% C 35.5% 98.9% 99.1% 96.5% 96.7% 91.1% 89.1% 89.9% D 87.4% 98.2% 98.5% 95.4% 96.7% 88.9% 87.6% 89.3% E 40.6% to 42.1% 99.5% to 99.8% 98.1% to 99.6% 98.7% to 99.1% 98.1% to 98.7% 92.9% to 93.2 90.5% to 91.0% 94.8% to 95.8% J 35.4% to 36.7% 97.6% to 98.6% 97.2% to 98.7% 95.6% to 98.9% 96.4% to 98.1% 52.3% to 54.9% 48.0% to 49.7% 78.7% to 82.0% K 87.7% to 99.4% 99.1% to 99.9% 98.9% to 99.6% 97.0% to 99.9% 97.6% to 99.9% 96.2% to 97.5% 95.8% to 96.8% 91.2% to 97.3% aN represents the nucleotide sequence homology, bAA represent the amino acid sequence homology. View Large Molecular Characteristics of the Pol Gene The three ALV-K strains share a common mutation, consisting a single nucleotide deletion at position 24 and an 8-nucleotide deletion at positions 32 to 39 (Figure 2), which caused the replacement of the corresponding amino acid PLKWK with RS, and has not been previously reported. This mutation did not prevent the expression and translation of the pol gene. However, its influence on ALV requires further study. Figure 2. View largeDownload slide Comparison of the amino acid sequences of the pol gene of SDAUAK-11, SDAUAK-12, and SDAUAK-13 to those of other ALVs from different subgroups. Figure 2. View largeDownload slide Comparison of the amino acid sequences of the pol gene of SDAUAK-11, SDAUAK-12, and SDAUAK-13 to those of other ALVs from different subgroups. Recombination Analysis The recombination events in the complete proviral genomes were analyzed by RDP4, and several potential recombination events were detected (Table 7), including four events in SDAUAK-11, two in SDAUAK-12, and three in SDAUAK-13, which were supported by Bootscan (Figure 3) and five other methods. The above results suggest that the three ALV-K strains are recombinant isolates. All these events can be divided into four groups; events a, b, and c occurred in the 5΄ LTR gene of the three ALV-K strains, with GDFX0602 (ALV-K) as the major parent and HPRS-103 (ALV-J) as the minor parent; events d and e mapped to the env gene of SDAUAK-11 and SDAUAK-13, with EV-3 (ALV-E) as the major parent and GDFX0602(ALV-K) as the minor parent; events f, g, and h were located in the 3΄ LTR of the three ALV-K strains, with SD07LK1 (ALV-J) as the major parent and JS11C1 (ALV-K) as the minor parent, and event i occurred in the 3΄ LTR of SDAUAK-11, with GD14LZ (ALV-K) as major parent. Figure 3. View largeDownload slide Bootscan analysis of the potential recombinant and major and minor parent sequences (a, b, c, d, e, f, g, h, and i). The Bootscan was based on the pairwise distance model with a window size of 200, step size of 50, and 1,000 bootstrap replicates generated by the RDP4 program. (j) The positon of the recombination events in the three ALV-K isolates. Figure 3. View largeDownload slide Bootscan analysis of the potential recombinant and major and minor parent sequences (a, b, c, d, e, f, g, h, and i). The Bootscan was based on the pairwise distance model with a window size of 200, step size of 50, and 1,000 bootstrap replicates generated by the RDP4 program. (j) The positon of the recombination events in the three ALV-K isolates. Table 7. Detected potential recombination events. Events Beginning breakpoint—ending breakpoint Recombination position Recombinants Major parent Minor parent a 142–239 5΄UTR SDAUAK-11 GDFX0602 HPRS-103 b 161–279 5΄UTR SDAUAK-12 GDFX0602 HPRS-103 c 142–239 5΄UTR SDAUAK-13 GDFX0602 HPRS-103 d 5,597–6,153 ENV SDAUAK-11 EV-3 GDFX0602 e 5,597–5,920 ENV SDAUAK-13 EV-3 GDFX0602 f 6,119–7,321 ENV, 3΄UTR SDAUAK-11 SD07LK1 JS11C1 g 5,199–7,642 POL, ENV, 3΄UTR SDAUAK-12 SD07LK1 JS11C1 h 6,119–7,163 ENV, 3΄UTR SDAUAK-13 SD07LK1 JS11C1 i 7,118–7,814 3΄UTR SDAUAK-11 GD14LZ SDAUAK-13 Events Beginning breakpoint—ending breakpoint Recombination position Recombinants Major parent Minor parent a 142–239 5΄UTR SDAUAK-11 GDFX0602 HPRS-103 b 161–279 5΄UTR SDAUAK-12 GDFX0602 HPRS-103 c 142–239 5΄UTR SDAUAK-13 GDFX0602 HPRS-103 d 5,597–6,153 ENV SDAUAK-11 EV-3 GDFX0602 e 5,597–5,920 ENV SDAUAK-13 EV-3 GDFX0602 f 6,119–7,321 ENV, 3΄UTR SDAUAK-11 SD07LK1 JS11C1 g 5,199–7,642 POL, ENV, 3΄UTR SDAUAK-12 SD07LK1 JS11C1 h 6,119–7,163 ENV, 3΄UTR SDAUAK-13 SD07LK1 JS11C1 i 7,118–7,814 3΄UTR SDAUAK-11 GD14LZ SDAUAK-13 View Large Table 7. Detected potential recombination events. Events Beginning breakpoint—ending breakpoint Recombination position Recombinants Major parent Minor parent a 142–239 5΄UTR SDAUAK-11 GDFX0602 HPRS-103 b 161–279 5΄UTR SDAUAK-12 GDFX0602 HPRS-103 c 142–239 5΄UTR SDAUAK-13 GDFX0602 HPRS-103 d 5,597–6,153 ENV SDAUAK-11 EV-3 GDFX0602 e 5,597–5,920 ENV SDAUAK-13 EV-3 GDFX0602 f 6,119–7,321 ENV, 3΄UTR SDAUAK-11 SD07LK1 JS11C1 g 5,199–7,642 POL, ENV, 3΄UTR SDAUAK-12 SD07LK1 JS11C1 h 6,119–7,163 ENV, 3΄UTR SDAUAK-13 SD07LK1 JS11C1 i 7,118–7,814 3΄UTR SDAUAK-11 GD14LZ SDAUAK-13 Events Beginning breakpoint—ending breakpoint Recombination position Recombinants Major parent Minor parent a 142–239 5΄UTR SDAUAK-11 GDFX0602 HPRS-103 b 161–279 5΄UTR SDAUAK-12 GDFX0602 HPRS-103 c 142–239 5΄UTR SDAUAK-13 GDFX0602 HPRS-103 d 5,597–6,153 ENV SDAUAK-11 EV-3 GDFX0602 e 5,597–5,920 ENV SDAUAK-13 EV-3 GDFX0602 f 6,119–7,321 ENV, 3΄UTR SDAUAK-11 SD07LK1 JS11C1 g 5,199–7,642 POL, ENV, 3΄UTR SDAUAK-12 SD07LK1 JS11C1 h 6,119–7,163 ENV, 3΄UTR SDAUAK-13 SD07LK1 JS11C1 i 7,118–7,814 3΄UTR SDAUAK-11 GD14LZ SDAUAK-13 View Large ALV-K Growth Kinetics in DF-1 Cells The replication rates of the three ALV-K strains were evaluated in vitro using DF-1 cells, which are commonly used as host cells for ALV. DF-1 cells were infected with the three ALV-K strains along with SDAU09C1 (ALV-A), SDAU09C2 (ALV-B), and NX0101 (ALV-J) as controls. As shown in Figure 4, the replication rates of the three ALV-K strains were markedly lower than those of other ALVs, and NX0101(ALV-J) showed the highest replication rate. Figure 4. View largeDownload slide Comparison of the viral replication dynamics of ALV-K to ALV-A, ALV-B, and ALV-J. Growth curves were generated by measuring the viral titers at various time points. The viral titers were determined using samples harvested every day and were expressed as TCID50 mL–1. The mean ± SD values from three independent experiments are shown. Figure 4. View largeDownload slide Comparison of the viral replication dynamics of ALV-K to ALV-A, ALV-B, and ALV-J. Growth curves were generated by measuring the viral titers at various time points. The viral titers were determined using samples harvested every day and were expressed as TCID50 mL–1. The mean ± SD values from three independent experiments are shown. DISCUSSION ALVs can be classified as endogenous (ALV-E) or exogenous according to their mode of transmission. Exogenous ALVs from chickens are classified into five different subgroups (A, B, C, D, and J) based on their host range and viral interference and cross-neutralization activities (Payne et al., 1991, 1992). Recently, a new subgroup, ALV-K, was identified in the in the indigenous chicken breeds of East Asia (Cui et al., 2014). To assess the status of avian leukosis virus infection in indigenous chicken breeds in China, a total of 121 plasma samples collected from various indigenous chicken breeds from 2015 to 2016 were tested for the presence of ALV. In this study, 14 ALVs were isolated and identified from several Chinese indigenous breeds. The phylogenetic analysis showed that the isolates consisted of two ALV-A strains, one ALV-B strain, eight ALV-J strains, and three ALV-K strains, which demonstrated the exist of different subgroups of ALVs in the indigenous chicken breeds of China. A genome analysis of those three ALV-K was performed. The results, in keeping with previous reports (Cui et al., 2014; Dong et al., 2015b; Li et al., 2016; Shao et al., 2017), revealed that SDAUAK-11, SDAUAK-12, and SDAUAK-13 belong a single clade along with other ALV-K and are distantly phylogenetically related to reference strains of other existing ALV subgroups. The nucleotide and amino acid sequences of different regions of SDAUAK-11, SDAUAK-12, and SDAUAK-13 were analyzed, and the results showed that the pol gene was highly conserved (>97% nt identity) among them; however, compared to other ALVs, the genes contained a single nucleotide deletion at position 24 and an 8-nucleotide deletion at positions 32 to 39, which led to the corresponding amino acid sequence PLKWK being replaced by RS, which has not been previously reported. Pol encodes several enzymatic activities. For example, the reverse transcriptase encoded by the pol gene catalyzes DNA synthesis using DNA or RNA as a template. The pol protein also has nuclease H activity, which degrades the RNA in a DNA: RNA heterodimer. Subunit b of pol has an IN domain, specifically an integrase (p32) domain, which could catalyze integration of the provirus DNA into the genome of host cells. This mutation, and the resulting amino acid sequence changes, did not prevent the transcription and translation of the pol gene; however, its effects on the biology of ALV require further study. As a retrovirus, the replication of ALV requires RNA as intermediate in reverse transcription, which lacks a proofreading function and tends to allow recombination. In recent years, recombination events have been frequently detected among the different subgroups of ALV. An exogenous ALV-A strain was isolated from Malik vaccine by Silva et al. (2007), which was a recombinant virus containing the gp85 gene of ALV-A and a partial gene from an endogenous virus. An ALV strain was isolated by Cai et al. (2013) that was identified as a recombinant virus composed of a gp85 gene from ALV-C, a gp37 gene from ALV-E, and a LTR from ALV-J. SDAU09E1 (ALV-A), which was isolated from a Chinese indigenous breed (Zhang, 2010), also has a partial sequence from ALV-E in the LTR region. Those recombination events lead to variation among ALVs. In particular, ALV isolates carrying the endogenous LTR induce lower level p27 antigen expression, thus reducing the relevance ratio, and easily triggering the wide spread of ALV. In this study, potential recombination events were detected in the three new ALV-K isolates, which showed the complex evolutionary processes among ALVs. More importantly, potential recombination events were detected in the env gene (a major determinant of subgroup and pathogenicity) of all three ALV-K strains, which suggested that recombination might be the primary cause of ALV evolution. On the other hand, the replication rates of the three ALV-K strains were markedly lower than those of the other ALVs in DF-1 cells, which may be the reason for the difficulty in their isolation and identification. Future studies should investigate the large geographic distribution of these novel ALVs, and monitor their variation, rearrangements, host range, and associated diseases. Acknowledgements This work was supported by the National Key Research and Development Program of China (2016YFD0501606) and the Shandong “Double Tops” Program (SYL2017YSTD11). AUTHOR CONTRIBUTIONS QS and YL conceived and performed the experiments, analyzed the data, and drafted the manuscript. PZ and SCu supervised the project and edited the manuscript. ST and SCu conducted part of the experiments. SCh and ZC analyzed part of the data. PZ and ZC provided important suggestions. REFERENCES Bai J. , Howes K. , Payne L. N. , Skinner M. A. . 1995 . Sequence of host-range determinants in the env gene of a full-length, infectious proviral clone of exogenous avian leukosis virus HPRS-103 confirms that it represents a new subgroup (designated J) . J. Gen. Virol. 76 : 181 – 187 . 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Comparison of Genome and Biological Characteristic of Different Subgroup A Avian Leukosis Virus Strains . [Dissertation]. [Tai’an (SD)] . University of ShanDong Agriculture . © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Poultry ScienceOxford University Press

Published: Dec 18, 2017

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