| Over the past 10 years, spreading of subgroup J ALV in poultry industry has resulted in great economic losses. The last two years, leukemia / hemangioma cases increased significantly in layers breeder industry again. avian leukemia become a serious disease,which does severely Harm to layer industry in China. Isolation and identification of virus showed that the vascular tumors was caused by the subgroup J avian leukemia virus or the subgroup A and B. In order to survey epidemic status and pathogenicity of exogenous ALV in Chinese breeder industry, we collected samples from imported white meat-type chickens, layers, a Chinese local breed and inoculated DF-1 cell. Finally, the three strains of exogenous ALV, SDAU09C1, SDAU09C3 and SDAU09E1, were isolated. Additionally, exogenous ALV, SDAU09E2, was isolated from another local breed by my colleague. Genome of isolates were cloned. SDAU09C1 from broilers and SDAU09C3 from layers were inoculated 5-day-old chicken embryos of white broilers, HN layers, SPF chicken separately. At the same time, J subgroup avian leukemia virus NX0101 was used as control, and pathogenicity of the 3 strains were compared.1 Isolation and Identification of Subgroup A Avian Leukosis Virus240 1-day-old white meat-type grand-parent chicks, which were imported from abroad, were kept in an SPF room (with filtered positive air, 10 000 particles per cubic liter). Plasma samples were collected from each chickens at the age of 7d. 30 anti-coagulation samples were obtained from A Hy-line brown field, and 30 eggs were obtained from a Chinese local breed. Samples were were transported to The laboratory under Sterile conditions. After being processed properly, all sample were inoculated into DF-1monolayers respectively. Inoculated DF-1 cultures were incubated for 9 days of 14 days. then the supernatant of each well was tested for ALV group-specific p27 antigen with an ALV p27 antigen detection kit. Supernatant samples which were positive to p27 were inoculated into another fresh DF-1 cell culture at large scale to amplify virus stocks. Positive supernatant samples were used as exogenous ALV and kept.2 Sequences comparison of 4 ALV-A strainsDesign seven pairs of overlapping primers, and amplify corresponding gene fragment from proviral DNA of the four exogenous ALV with genomic DNA of infected DF-1 cells as templates. PCR products were cloned and sequenced. Splicing overlapping nucleotide fragments with DNAStar software to obtain proviral genome sequence of the four isolates. Then each gene of 4 isolates proviral was compared with those in reference strains of the subgroups.2.1 Sequences comparison of the isolates gp85gp85 genes of the isolates was of different sizes, whose amino acid sequence had over 87.7% of identity to 10 reference strains of subgroup A. However, The homology to subgroups B, C, D, E was in the range of 76.9% - 85.5%, and the homology to reference strains of subgroup J was as low as 38%. The phylogenic tree indicates that the 4 isolates definitely fell into subgroup A.2.2 Sequences comparison of the gag and pol from isolatesgag and pol of 4 isolates were consistent with reference strains of subgroups in sizes, and the encoded amino acid sequence were relatively conservative. The homology to reference strains of subgroups were over 95.4% and 97.3% respectively.2.3 Sequences comparison of the isolates gp37gp37 genes of the isolates was of different sizes, whose amino acid sequence displayed high identity with endogenous virus ev-6(92.1%-95.9% ).2.4 LTR sequences comparison of the isolatesLTR of four isolates are of different sizes. SDAU09C1, SDAU09C3 and SDAU09E2 show 85% to 90.7% identity with exogenous virus for LTR sequences and only 70% with Endogenous virus, LTR sequences of SDAU09E1 display high homology(91.3-93.5%) with endogenous virus and vaccine contaminating strains, PDRC-1039, PDRC-3246, and PDRC-3249 reported by American, and only 66% with the exogenous virus.2.5 U3 region comparison of the isolatesWithin the LTR, the U3 region in the SDAU09C1,SDAU09C3,SDAU09E2 isolates exhibited less than 51% identity to U3 in endogenous viruses, but showed 82.4–92.5% identity to the U3 of exogenous viruses. The U3 sequence in the SDAU09E1 isolate exhibited less than 50% identity to U3 of exogenous viruses. But showed 82.7–97.5% identity to the U3 in endogenous viruses and the vaccine-contaminated strains. Sequence analysis demonstrated that the important transcription regulatory elements contained in the U3 region of SDAU09E1 were consistent with those in endogenous viruses. In contrast, the important transcription regulatory elements contained in the U3 region of the other isolates were consistent with exogenous viruses. These results suggested that the SDAU09E1 isolate contained the LTR sequence of endogenous viruses, and SDAU09C1,SDAU09C3,SDAU09E2 isolates contained the LTR sequence of exogenous viruses3 Comparation of replication levels of 4 subgroup A avian leukemia viruses in DF-1 cellsInfecting DF-1 cells with the four isolates equivalently. Detection from ELISA for p27 antigen indicated, SDAU09E2 replicated the fastest, SDAU09C1 second, SDAU09C3 was lower than SDAU09C1, SDAU09E1 the weakest. the results from virus titer shew, the TCID50 of SDAU09C1 isolate was highest, SDAU09E2 second, SDAU09C3 was lower than SDAU09E2, the TCID50 of SDAU09E1 isolate was Significantly lower than the other isolates. replication difference of the four isolates in DF-1 cells closely related with LTR promoter activity.4 pathogenicity Comparison of different strains4.1 pathogenicity Comparison between the SDAU09C1 and SDAU09E11-day-old SPF chickens were inoculated with either the strain SDAU09E1 or the strain SDAU09C1 intraperitoneally and equivally. In SDAU09C1 infected group, all inoculated chickens demonstrated persistant viremia and p27-shedding, but no antibody response. The strain SDAU09C1 induced higher levels of horizontal infection. In SDAU09E1 infection group, the strain established transient viremia in SPF chickens and elicited a weak antibody response. There was no horizontal infection. This indicates that ability of the strain SDAU09E1 with endogenous virus LTR, especially U3, infecting SPF chickens is significantly lower than strain SDAU09C1.4.2 Pathogenicity of the strain SDAU09C1 and SDAU09C3 on the white broilers, HN layers and SPF chickenThe strain SDAU09C1 and SDAU09C3 were selected to inoculate 5-day-old embryos from White Boilers, HN layers and SPF chickens, Simultaneously, with ALV-J strain NX0101 as control. Pathogenicity of 3 strain ALV were compared. 4.2.1 Induced tumor of 3 strains ALV to different chicken strainsFrom 20w to 27w, white broilers challenged with SDAU09C1 and SDAU09C3 appeared 1 case of tumors, respectively, and white broilers challenged with NX0101, 2 cases. There was one case of tumor in HN layer group challenged with SDAU09C1, and no tumor was observed in HN layer group challenged with NX0101 and SDAU09C3. There are 5 cases of tumor with bone sclerosis, enlarged kidney or liver atrophy in SPF chickens group challenged with SDAU09C1. There are 1 cases of tumor with bone sclerosis in SPF chickens group challenged with SDAU09C3. 3 cases of tumor appear in SPF chickens challenged with NX0101. And type of tumor performed more complex. Diversification in tumor types and difference in the number of induced tumors may be associated with viral characteristics, genetic background of host and individual differences.4.2.2 Effect of 3 strains ALV on immunity level of different chicken strainsAt 6 weeks post hatching, For three chickens strains infected, Spleen/body weight, thymus/body weight, and bursa/body weight were not statistically significant(p>0.05). And three strains ALV induced different degrees of immunesuppression against NDV, AIV-H9 and AIV-H5 vaccine.4.2.3 Infectious comparation of 3 strains ALV on different chicken strainsViremia, cloaca p27 and serum antibody were detected by ELISA. Strain NX0101 caused severer infection to white broilers and SPF chickens than to HN layers. But the level of horizontal transmission in White Boilers was higher than in the HN layers and SPF chickens. SDAU09C1 induced immune tolerance and high levels of horizontal transmission in SPF chicken, and established transient viremia and weaker horizontal infection in white broiler. Infection levels of SDAU09C1 in HN layers was between the both, but it could cause a higher level of horizontal transmission. SDAU09C3 induced persistant viremia and a higher level of horizontal transmission in SPF chickens, and Its infection levels in HN layers is lower than in SPF chickens and is higher than in white broiler. It indicates that infection of ALV-J strain NX0101 on white broiler was supreme, infection of ALV-A strain SDAU09C3 on SPF chicken and HN layers was higher, infection of ALV-A strain SDAU09C1 on SPF chicken and HN layers was higher than on white broilers. And in SPF chickens, infection degree produced by three strains of ALV show SDAU09C3> SDAU09C1>NX0101; in HN layers, SDAU09C1 > SDAU09C3 > NX0101; in white broiler, NX0101 > SDAU09C1 > SDAU09C3.4.2.4 comparation of other pathogenic types of 3 strains ALV on different chicken strainsOn the weight growth, white broiler infected with ALV-J strain NX0101 grew slowliest; HN layer infected with ALV-A strain SDAU09C3 grew slowliest; body weight of SPF chickens infected with SDAU09C1 and NX0101 was lower than the growth of SPF chicken infected with SDAU09C3.In blood of three chicken strains inoculated with ALV, counts of WBC and LYM were not statistically significant from 1w to 15w. But at 21w, counts of WBC and LYM of white broiler inoculated with SDAU09C1 and SDAU09C3 were significantly higher than the control (P<0.05), counts of WBC and LYM of SPF chicken inoculated with SDAU09C3 and NX0101 were significantly higher than the control (P<0.05), and counts of WBC and LYM of HN layers inoculated with NX0101 were significantly lower than the control (P<0.05).This study provide a new basis for understanding the prevalence of classical exogenous ALV in Chinese flocks and genetic evolution of ALV through virus isolation and genome sequencing. ALV mainly cause tumor and immune suppression in infected chicken, this study compared pathogenicity of different ALV isolates on different chicken strains and provided an important reference value for the effective control of avian leukemia. |
