Biological Characterization Of A Subgroup B Avian Leukosis Virus Strain Isolated From Native Breed

Avian leukosis is a variety of tumor diseases caused by avian leukosis virus and avian sarcoma virus. Avian leuksis virus (ALV) is a member of the family Retroviridae. ALV infection of chickens is widespread and known to be capable of inducing a variety of infectious tumor diseases. Avian leukosis viruses, isolated from chickens, are classified based on their host range, cross-neutralization, and viral interference into six groups (A, B, C, D, E, and J). Subgroup A, B, C, D mainly induced classical lymphoid leukosis (LL), while subgroup J has been found to be associated primarily with myeloid leukosis (ML). In the past 10 years, ALV-J has been widely recognized and reported in many regions of China. In the past few decades, since there is no strict nationwide eradication program for ALV infection on chicken farms in China, it is very likely that Chinese chickens, especially the local breeds, may be infected by other subgroups of ALV. However, the related reports are relatively rare.1. Isolation and identification of subgroup B field strain SDAU09C2In this study, by inoculation blood samples in DF-1 (C/E) cell cultlure, an exogenous avian leukosis virus (ALV) strain SDAU09C2 was isolated from a breeder farm of Chinese native breed"Barred Plymouth Rock chicken"in Shandong province. To full proviral genome cloning, seven overlapping primers were designed according to reference strains published in GenBank. Identity of the sequence of SDAU09C2 was compared with other ALV reference strains of different subgroups. The results show that it had 92.5%-95.1% of amino acid identity in gp85 to four ALV-B reference strains, the gp85 identity to subgroups A, B, C, D, E was only in the range of 78.9%-89.9%. The identity to subgroup J was as low as 36.9 %-39.5%. So SDAU09C2 was divided into subgroup B.Comparison of gag,pol between the isolation and other reference strains showed that the sequence identity for gag and pol between SDAU09C2 and other subgroups was more than 95% on average. Actually, these two genes were very conservative between different subgroups.The analysis of amino acid encoding by gp37 showed that gp37 in SDAU09C2 has high identity to gp37 in ALV-A MAV-1 although SDAU09C2 belonged to subgroup B. The identity of gp37 among different subgroups has no clear correlation with its subgroup.The fragment R identity to subgroups A, B, C, D, E, J was in the range of 80.0%-100.0%. The U5 identity to exogenous subgroups A, B, C, D and J was more than 90.0%, while the identity to subgroup E was in the range of 85.9%-87.5%. The results indicated that R and U5 region had no relationship with subgroups.Its U3 had highest identity of 91% to ALV-A strain MQNCSU, much higher than 84.4% and 36.1% to ALV-B reference strains RSR B and MAV-2. It indicated that the identity of U3 among different subgroups has no clear correlation with its subgroup. Analysis of regulatory elements showed that none of the differences noted in the U3 sequences of SDAU09C2 involved any of the known regulatory elements present in the U3 region of MQNCSU. However, an enhancer CAAT box and a CArG box (CC(A/T)6GG) of U3 in SDAU09C2 is not present in the RSR B. In contrast, a PRE box of RSR B is not present in the SDAU09C2.2. Pathogenicity of SDAU09C2 in different chickensPathogenicity of SDAU09C2 was studied in three different breeds by animal experiments. Because the pathogenicity of NX0101 has been studied deeply in our lab, NX0101 was chosen as a control strain. In the experiments, two viruses were both inoculated in ovo in different chickens, including white Leghorn SPF chicken, HN line egg-type chicken and Barred Plymouth Rock chicken. The comparison analysis of animal experiments was the following:2.1 Rates of tumors in different chickensOn the 20th week after hatching, SDAU09C2 and NX0101 started inducing tumors. Up to now, both two viruses can induce tumor in white Leghorn SPF chicken, the rate was 8.7% and 14.3% respectively. While only SDAU09C2 induced tumor in HN egg-type chicken and the rate was 5.6%. Neither of two viruses can induce tumor in Barred Plymouth Rock chicken. Two viruses lead to a variety of tumors in different organs. SDAU09C2 mainly induced hemangioma, in addition to ovarian and mesenteric tumors. NX0101 can induce a variety of tumors in different parts, such as hemangioma, fibrosarcoma, diffuse tumor and so on.2.2 Viremia induced by two ALVs in different chickensIn white Leghorn SPF chicken and HN line egg-type chicken, both SDAU09C2 and NX0101 induced viremia during 1-3 wk of age in the most birds of white Leghorns SPF chickens and HN line egg-type chickens. Especially in white Leghorn SPF chicken, the rate of viremia can reach 9/10 on 3rd week. The SDAU09C2 demonstrated stronger horizontal transmission ability than NX0101. In the local breed Barred Plymouth Rock chicken, SDAU09C2 only induced transient viremia on the 5th week and the rate of viremia was low (2/12). In horizontal infection groups, horizontal infectivity of SDAU09C2 in Barred Plymouth Rock chicken was relative lower than in other 2 chicken breeds.2.3 Comarison of p27 in cloacal swabIn white Leghorn SPF chicken and HN line egg-type chicken, during the 5-21 week of age, SDAU09C2 induced higher positive detection rate of p27 in cloacal swab than NX0101. The SDAU09C2 demonstrated stronger horizontal transmission ability than NX0101. In Barred Plymouth Rock chicken, during the the 5-21 week of age, NX0101 induced higher positive detection rate of p27 in cloacal swab than SDAU09C2. The horizontal infection of two viruses in Barred Plymouth Rock chicken was lower than that in white Leghorn SPF chicken and HN line egg-type chicken.2.4 Inhibition on body weight by two ALVs in different chickensBoth strains induced growth retardation in all 3 chicken breeds, such growth inhibitory effects of SDAU09C2 were stronger than NX0101 in HN egg type chickens but smaller than NX0101 in white Leghorn SPF chickens. In local breed Barred Plymouth Rock chicken, growth inhibitory effects of SDAU09C2 were a little stronger than NX0101, and the inhibitory effects of two viruses were weaker than that of two viruses in other 2 breeds. 2.5 Inhibition on immunity by two ALVs in different chickens35d after vaccine, SDAU09C2 could inhibit antibody reactions to vaccination against Newcastle disease virus in white Leghorn SPF chicken and Barred Plymouth Rock chickens but not in HN egg type chickens. While, SDAU09C2 didn't affect the antibody reactions to vaccination against H5- and H9- avian influenza viruses (AIV-H5 and AIV-H9) in 3 chicken breeds. It indicated that NX0101 did not show significant inhibitory effects on HI antibody titers to NDV, AIV-H5 and AIV-H9 after vaccination in 3 chicken breeds.6 weeks after hatched, chickens were killed randomly and the immune organs were obtained to determine the inhibition on the development of immune organs. In the White Leghorn SPF chickens, both viruses inhibited the development of bursa of fabricius. In HN egg-type chickens, SDAU09C2 inhibited the development of thymus and bursal of fabricius while NX0101 didn't show inhibition on the development of three immune organs. In the Barred Plymouth Rock chickens, NX0101 inhibited the development of the bursa of fabricius, while SDAU09C2 didn't show inhibition on the development of three immune organs.2.6 Other tests in different chickens2.6.1 Analysis of specific antibodyTwo viruses can induce antibodies both in inoculated groups and horizontal transimission groups in the 3 breeds. In white Leghorn SPF chicken and HN line egg-type chicken, antibody level and positive rate were higher than those in Barred Plymouth Rock chicken.2.6.2 Detection of eggsUp to now, only HN egg-type chicken started to lay eggs. The results showed that p27 antigen in albumen and yolk antibody can be detected in SDAU09C2 and NX0101 inoculated HN egg-type chicken groups. And the eggs contained yolk antibody when the hens have developed antibodies. the positive rates of SDAU09C2 were much higher than NX0101.2.6.3 Influences on white blood ball and lymphocytes in the bloodIn the continuous tracking, the two viruses allowed the number of white blood ball and lymphocytes changed a little, while compared with the control group, the difference was not significant. But on the 21wk, the two viruses could make the total number of white blood ball and lymphocytes either increased or decreased significantly.The results suggested that two viruses showed different infectivity and pathogenicity in different chicken breeds. This difference may be caused by the individual difference of each chicken between chickens. In addition, different genetic background of different breeds of chickens can also play roles in it.