Molecular Evolution Analysis For Gp85 Gene Of ALV-J Strains In Chicken Flocks From Different Genetic Backgrounds

Avian leukosis virus Subgroup J was first detected and identified from white feather broilers in UK in 1988, which mainly caused myeloid leukosis. In the early 1990 s, ALV-J occurs only in the white feather broilers, not found in layer chickens. The oncogenicity and infectivity of layer chickens was significantly lower than white feather broiler via inoculation at that time. In the 1990’s, ALV-J was imported with white feather broiler into China, only occurring on white feather broiler initially. From the beginning of the 2000’s, ALV-J infection gradually emerged in egg-type chickens, yellow broilers and Chinese indigenous chicken Breeds, and the incidences are increasing. In some chicken flocks, the severity and diversity of tumor developed more than the pathogenicity on white feather broilers. Obviously, the epidemiological characteristic has changed significantly on ALV-J infection in chicken flocks with different genetic backgrounds. Whether this change related to the evolution of ALV-J in the past twenty years? What relationship between these significant changes and the evolution of ALV-J infection in the past twenty years? What evolution caused the ALV-J infection to the chicken flocks with diffident genetic backgrounds? In this study, we attempts to explore the correlation on evolution of gp 85 which likely happen variants with LTR of ALV-J and prone to the different type of chickens by using the molecular virology methods.1 Homologous comparisons of gp85 of ALV-J isolates from different genetic background lines of chicken We compared the gp85 homology in amino acid level of 150 strains from ALV-J infection with different genetic background chickens. These stains including original ALV-J stain HPRS-103 isolated from white feather broilers in UK in 1988; 8 strains from broilers in USA in 1990’s, 22 strains from broilers in China during 1999-2008; 42 strains from yellow feather broilers in China since 2005; 51 strains from layer chickens in China since 2007; 26 strains from Chinese Indigenous Chicken Breeds et al.1.1 Homologous comparisons of ALV-J from chickens with different genetic background and original HPRS-103The results showed that the identities of gp85 amino acid sequence between ALV-J of American broilers and the original virus was 86.6%-95.5%, the homology between the ranges of 85.6%-94.8%; the identities of gp85 amino acid sequence between ALV-J of Chinese broilers and the original virus was 88.9%-95.8%, the homology between the ranges of86.9%-100%; the identities of gp85 amino acid sequence between ALV-J of yellow feather broilers and the original virus was 86.9%-93.8%, the homology between the ranges of87.0%-100%; as well, the identities of gp85 amino acid sequence between ALV-J of layer chickens and the original virus was 87.0%-96.7%, the homology between the ranges of86.6%-99.3%, however, the identities of gp85 amino acid sequence between ALV-J of Chinese Indigenous Chicken Breeds and the original virus was 87.6%-92.4%, the homology between the ranges of 85.9%-100%. From the above results demonstrated that gp85 of ALV-J from chicken flocks with differ greatly genetic background on sequence homology has not showed significantly difference compare with the original strain HPRS-103.1.2 Homologous comparisons of ALV-J among the chickens with different genetic background and original HPRS-103The results showed that the identities of gp85 amino acid sequence between white feather broiler from American and Chinese Chinese white feather broilers, yellow feather broilers, Chinese layer chickens, Chinese Indigenous Chicken Breeds were 83.7%-94.8%,84.9%-94.8%, 84.6%-97.1%, 84.6%-93.1% respectively; the identities of gp85 amino acid sequence between Chinese white feather broilers and Chinese yellow feather broilers, Chinese layer chickens, Chinese Indigenous Chicken Breeds were 84.0%-95.4%, 84.0%-99.7%,84.3%-93.7% respectively; the identities of gp85 amino acid sequence between Chinese yellow feather broilers and Chinese layer chickens, Chinese Indigenous Chicken Breeds were85.5%-95.7%、85.3%-94.4%. As well, the identities of gp85 amino acid sequence between layer chickens and Chinese Indigenous Chicken Breeds was 85%-94.1%.In the comparison among all the 150 strains, the identities of gp85 amino acid sequence of 6827 strain isolated from American white feather broilers in 1997 and SD0102 strain isolated from Shandong in 2001 or NM2002-1strain isolated from Inner Mongolia in 2002 is the lowest, with the homology about 83.7%. From the above results demonstrated that the homology of gp85 seems has no relationship with different genetic background of chicken lines, and has great randomness on variation.1.3 Comparison of genetic lineages of gp85 ALV-J isolates from different genetic background lines of chickenWe analyzed the genetic lineages of gp85 ALV-J isolates from different genetic background lines of chickens, the distribution of different strains in the genetic linages seems to have shown correlation to the infection of chickens with genetic background.The gp85 of ALV-J from the chicken with same genetic background was close to each other in the genetic linages, which are distributed in the same region. It is indicated that sequence genetic lineage analysis is more representative correlation between ALV-J and host genetic background of lines chicken than the analysis of sequence homology. What is the main sequence of distribution in analysis of genetic lineage of gp85 need be studied in the future.2 Variation trend of ALV-J gp85 with yearsWe could analyze the variation trend of ALV-J gp85 due to the amount of strains were collected near 14 years in our lab. The results showed that the identities of gp85 amino acid sequence between prevalent isolates in 1990’s、2000、2001、2002、2003、2005、2006, 2007,2008, 2009, 2010, 2012, 2013 and the original strain HPRS-103 were 86.6%-95.5%,89.9%-92.5%, 90.3%-92.2%, 89.9%-92.2%, 90.6%-95.8%, 89.9%-91.9%, 89.2%-91.2%,88.3%-92.9%, 89.9%-96.7%, 87.9%-96.4%, 87.9%-93.8%, 87.0%-92.5% and 86.8%-92.1%,respectively. Among these newest strains, 2-LH5-15 stain and JS13-LY1-5 strain have the lowest or highest homology with HPRS-103 strian respectively. The identities of gp85 amino acid sequence between prevalent isolates in different years and 2-LH5-15 strain were87.7%-91.4%, 87.4%-89.4%, 86.4%- 91.4%, 87.1%-88.1%, 86.8%-83.1%, 90.4%-91.7%,89.7%-93%, 88%-92.7%, 87%-92.4%, 87.4%-93%, 88.7%-92.1%, 87.7%-91.4% and85.7%-94.4%, respectively. The identities of gp85 amino acid sequence between prevalent isolates in different years and JS13-LY1-5 strain were 87.1%-93.7%, 91.7%-93.1%, 89.8%-92.4%, 91.1%-91.7%, 89.4%-92.4%, 90.4%-92.4%, 91.1%-92.7%, 90%-94.1%, 91.4%-93.4%, 90.4%-94.4%, 89.7%-91.4%, 88.2%-93.4% and 88.4%-100%, respectively. It is indicated that ALV-J gp85 of prevalent strains did not deviate from the original strain HPRS-103, also illustrates that there is irrelevant between the strain from the different years and deviation of prevalent strain and HPRS-103 or strains isolated recently. According to the analysis of homology, in some extent, it will happen to random variation compare the new strain to the original strain HPRS-103 with the sitting years increasing, the lowest homology of the variant strain 6827 from white feather broilers in American in 1997 was 86.6%compare with the HPRS-103.3 The variant trend of the virus quasispecies in the serial passaging between white leghorn chickens and white feather broilers of different ALV-J isolatesTo explore the variation of molecular genetic mechanism on ALV-J in the process of the pathogenicity of the different genetic background lines of chicken, we conducted on there is variation in different genetic background chickens with different strains, inoculated white leghorn chickens and white feather broilers with ALV-J NX0101 which isolated fron Chinese white feather broiler in 2001 and ALV-J 733 strain from layer chickens in 2012. In order to find small variation, in this study we sequenced the gp85 gene from ALV-J RNA genome in the plasma and LTR products by RT-PCR from different passaged chickens using high-throughput sequencing, it can obtain more than 10000 effective sequences from each plasma for each chicken.3.1 Comparisons of quasispecies variation between white leghorn chickens and white feather chickens with ALV-J 733 strain from layer chickensThe statistical data showed that molecular variants and quasispecies composition did not shown the significantly difference either in the serial passaging, on white leghorn chickens with ALV-J 733 strain from layer chickens. When we inoculated white feather broilers with the same strain, even though there are no changes in gp85 quasispecies after third and fifth generation, but, we have found 28 common molecular variantsin rising in the first 60 ones in the three chickens. However, only 4 molecular variants were found in the passaging three white leghorn chickens. All results suggested that 733 strains from layer chickens in the passaging of white feather broilers, there is no selective variation on quasispecies. But, the quasispecies become mutating under selection pressures in the replication environment from chickens with different genetic background.3.2 Comparisons of quasispecies variation between white leghorn chickens and white feather chickens with ALV-J NX0101 strain from white feather chickensThe results showed that more than half groups of gp85 molecular variants have replacement when in the passaging on white leghorn chickens with ALV-J NX0101 from broilers. It includes that three of the gp85-A group, two of the gp85-B groups have significantly difference, but the new quasispecies was found in the gp85-B group is only relative to the original virusBut the molecular variants gp85-B group of emerging dominant quasispecies is only relative to the first five quasispecies of the original virus. The dominant quasispecies have significant difference in the passaging on white feather broilers with ALV-J NX0101,128 thmolecular variants from the original virus become the dominant quasispecies when passaging the third generation; even the dominant quasispecies from gp85-B group has changed, but the quasispecies of emerging dominant quasispecies is only relative to the first third quasispecies of the original virus. The dominant quasispecies have significant difference in the passaging on white leghorn chickens and white feather broilers with ALV-J NX0101,but the dominant quasispecies from gp85-B group are relatively stable. In the first 60 th quasispecies, the rising numbers of quasispecies have not shown significant difference from layer chicken and broilers groups, but the selective pressures from ecosystems on the broilers greater than layer chickens for effects on NX0101 quasispecies.4 Pathogenicity of ALV-J on different species birds In this study, we inoculated birds’ embryos with NX0101 and 733 via yolk sac, while could not isolate any ALV-J from hatched birds after several detection in the first Six weeks.But at the age of 7 months, the pheasant inoculated by ALV-J 733 via embryo showed symptoms of tumor. ALV-J isolated from tumor tissue is high homology with ALV-J 733 strain. The results showed that the ALV-J could infect pheasant and induce tumors, but need a long time. The results showed that quail cann’t be infected with ALV-J.