Research On The Molecular Characteristics Of Endogenous Avian Leukosis Viruses In Ten Types Of Chinese High Quality Flocks And Their Roles In The Prevalent Subgroup J Avian Leukosis Viruses Strains In China

Avian leukosis virus (ALV) is a type of retrovirus which results in neoplastic and immunosuppression in virus-infected chickens. ALVs are divided into 10 subgroups(A-J) based on viral envelope. It also can be separated into exogenous virus and endogenous virus according to their transmission ways. Recently, ALV-J has become widespread in China and has resulted in severe economic losses to the poultry industry. Research has indicated that ALV-J is believed to have arisen through a recombination between exogenous ALVs and the ev/J endogenous retrovirus. However, the status of endogenous viruses infections in Chinese chickens and their correlation with ALV-J epidemic strains remains unclear.This study examined embryonated eggs obtained from 10 types of chicken breeds which were flocks of economic importance in China using PCR with the primer sets specific for endogenous ALVs. The PCR assays detected the genomes of EAV, ev, ev/J and ART-CH belonging to the endogenous ALV from Chinese important economic flocks embryos. The results indicated that, four types of endogenous ALVs were all existing in these 10 types of chicken breeds. A comparison of the nt sequence of the EAV detected from all the tested chickens showed a higher sequence identity with identity with EAV-0 but less with E51, and the phylogenetic tree analysis showed that they were in the same evolutionary branch as EAV-0. Paired comparisons of the gag-related nt sequences showed that the ART-CHs detected in the tested chickens had a sequence identity of 91.8%-98.3% to each other but a slightly higher sequence identity with the ART-CH clones 5 (93.5%-99.2%) and 14 (92.1%-99.1%). Phylogenetic tree analysis showed that, in addition to the Ross Brown layer and Lohmann Brown layer are in the evolutionary branch with the two prototype strains, the rest of the chicken species are in a separate branch.In the paired comparisons of the nt sequence of the ev genomes (a part of the env gene), the nt sequence substitutions of the ev genomes indicated the sequence identity between them were as high as 98%. However, phylogenetically, the prototype ev genomes, including ev-1, ev-3, and ev-6 were clustered in a separate branch except Lohmann Brown layers and White Leghorns. A part of the env gene nt and aa sequences from the ev/J genome demonstrated a high level of identity (nt; 95.9%-99.1%) among all of the tested chickens and the other known ev/J sequences. Moreover, the ev/J genomes detected in all of the tested chickens, and the other known ev/J revealed 92.8%-98.1% nt identity to that of ALV-J prototype strain HPRS-103.According to the different deletion of ev/J, they can be divided into four types: Type Ⅰ ev/J to TypeⅣev/J. Primer pair (PrimerF/PrimerR) which located in the U3 region of the LTR of ev/J were set for the amplification of intact ev/J. Products of approximately 3.8 kb were amplified with the genome DNA template from all of the tested chickens. However, additional products of approximately 2.2 kb were amplified with Shouguang chickens,Beijing fatty chickens, Langshan chickens, and three adventitious breeds (Ross Brown layer, White Leghorn, Lohmann Brown layer) The result indicated that ev/J were different between Chinese indigenous chicken breeds and adventitious breeds, and there were also differences existing between the indigenous chicken breeds.The 568 bp product was amplified using primer pair EVJFOR/103ER with all the genome DNA templates from all of the tested chickens in the test for detection of the pol gene in ev/J, with the exception of Taihe and Pudong chickens. These results illustrated that the complete ev/J genome differed between chicken breeds and none of the ev/J pol gene (Intact pol gene is 2618bp) was intact in the tested chickens. Sequence alignment showed that the 568bp fragments belonged to 2.2kb fragments. The reason for the chicken breeds that do not have the 2.2kb fragment but amplified the 568bp fragment was possibility of a large change in the region of the downstream primer 103ER binding to the 2.2kb fragment.Primer pair H83REV/H8, which were located in the gag gene and env gene of ev/J, were used to amplify the deletion part of the ev/J of Chinese high quality chicken breeds. Single fragment was amplified from each of the chicken, and these fragments were of three sizes,638bp,661bp and 728bp, respectively.Analysis showed that the chickens (Shouguang chicken, Langshan Chicken and Pudong chicken) which amplified 638 bp fragment got the same deletion as type Ⅲ ev/J.The chickens(Ross Brown layer, Lohmann Brown layer, Suqin chicken and Taihe chicken) which amplified 728 bp fragment got the same deletion as type Ⅰ ev/J. However, the deletion of ev/J in the chicken breeds (Green eggshell chicken, White Leghorn and Beijing fatty chicken) which amplified 671bp fragment were different from the four types of Prototype strains of ev/J, but the sequences of both sides of the deletion region showed high identity with Type Ⅰ ev/J and Type Ⅲ ev/J, which were 97.0%-97.3% and 94.0%-94.3%, respectively.This new type of deletion in ev/J implied that the gag-env fusion gene of ev/J in Green eggshell chicken,White Leghorn and Beijing fatty chicken got a new connection form.Sequence analysis showed that the 2.2kb fragment of ev/J showed high identity with TypeIV ev/J prototype strain EAV-HP clone4-1, but quite low homology with the other three types ev/J. In addition, the deletion of the 2.2kb fragments were completely the same with EAV-HP clone4-1. This result confirmed that all the 2.2kb fragments of the ev/J in the tested chicken breeds belonged to TypeIV ev/J.Comparing four types of ev/J with the amplified 3.8kb fragments of ev/J showed that there were two significant different deletions in the 3.8kb fragment. The first deletion position was in the region of Matrix of gag gene. In this position, Beijing fatty chicken, Langshan chicken, Lohmann Brown layer, Shouguang chicken and Suqin chicken were consistent with Type I ev/J and Type II ev/J. However, the rest chicken breeds were different from the four types of ev/J. The location of the second deletions was the same as that of the product of PCR using primer pair H83REV/H8. It was also confirmed by that this region of 3.8kb fragments showed high identity with PCR amplification products using primer pairH83REV/H8. However, further study found that the deletion of 3.8kb fragment were not completely consistent with PCR amplification products using primer pair H83REV/H8. Only Langshang chicken and Taihe chicken showed the same. It was indicated that there were several copies of ev/J in the genomic DNA of the same chicken and these copies belonged to different types of ev/J.A sequence comparison of the env gene of ALV-J prototype strain HPRS-103 and ALV-J strains of popularity in different provinces of China with env gene of ev/J (using primer pair F7/R7) showed that the env gene of ev/J showed high homology with the env gene of exogenous ALV-J strains. Among them, the highest homology was 94.1% through the comparison of Taihe chicken and Shouguang chicken with ALV-J prototype strain HPRS-103. The lowest was 86.1% through the comparison of Lohmann Brown layer and GDI 109 strain. Besides, the identity between env gene of ALV-J prototype strain HPRS-103 and env gene of the ev/J of the 10 types of chicken breeds were higher than any other ALV-J strains in this study.Additionally, sequence alignment showed that,the deletions from 67-68bp and 80-81bp in RB7, RB7, PD7 and SG7 (in the region of 5602bp-5633bp and 5645bp-5673bp of ALV-J prototype HPRS-103), the deletions from 609bp-610b in GS7 and 552-553bp in RB7 (in the region of 6144bp-6175bp) didn’t appear in the corresponding region of any other the env genes of the exogenous ALV-J and ev/J of the rest chicken breeds.Moreover, the sequence alignment showed that the env gene of ALV-J IMC10200 strain got a 6bp insertion (423-430bp) compared with the env of ev/J (276bp-277bp of BJ7) of the 10 types of chicken breeds and the other exogenous ALV-J strains. In addition, the sequence analysis showed that the env gene of ALV-J IMC 10200 strain(669bp-679bp) and SCDY1 strain (6042bp-6052bp) got a 9bp insertion compared with the env of ev/J (521bp-522bp of BJ7) of the 10 types of chicken breeds and the other exogenous ALV-J strains.Phylogenetically, the genetic distance between the env gene of ev/J of the 10 types of chicken breeds and the env gene of exogenous ALV-J strains were quite close, while the closest was prototype strain HPRS-103, and the furthest one was IMC 10200 isolated from Inner Mongolia.Sequence analysis showed that the env region of 2.2kb fragment of ev/J of Beijing Fatty chicken, Langshan chicken, Lohmann Brown layer, Ross Brown layer, Shouguang chicken and White Leghorn showed high identity with the env of exogenous ALV-J(Including prototype strain HPRS-103 and the other strains in China).Among them, the highest homology(98.3%) was homology between Ross chicken and prototype strain HPRS 103, and the lowest one(81.4%) was Shouguang chicken and strain HB09-1 isolated from Hubei Province.In this study, endogenous avian leukosis virus:EAV, ART-CH, ev loci, and ev/J in the high quality chicken flocks in China were detected and analyzed. It will be of great significance for understanding the endogenous avian leukosis virus, especially the endogenous retrovirus ev/J, and it will found theoretical significance in further study for the subgroup characteristics and evolution of ALV-J.Besides, it will lay a foundation for further study of the mechanism of the immune tolerance caused by different types of ev/J when chickens are infected with ALV-J.