| Avian leukosis virus(ALV) was discovered over 100 years ago, which is the first proposed virus that can cause tumors. ALVs are divided into ten subgroups(A-J) based on the virus and host range associated antigenicity of envelope proteins and subgroup K which was identified recently. Avian leukosis subgroup J is a kind of tumorigenic infectious disease with myelocytoma which is caused by avian leukosis virus subgroup J(ALV-J). In 1988, ALV-J was first detected in UK and has become widespread around the world. In the mainland of China, ALV-J was firstly isolated from broiler chicken in 1999 and the pathogenicity was gradually evolved. In the first decade, avian leukosis subgroup J only happened in imports of meat-type chickens. However, ALV-J infection has emerged in layer chickens and yellow broilers around 2004. The rate of infection and tumor reach up to 30%. Especially, ALV-J infection in layer flocks had high percentage of hemangioma and mortality. Chinese layer flocks have experienced outbreaks of ALV-J during 2007 and 2009. In addition to a longer incubation period(3-6 months) of myelocytoma or hemangioma, acute fibrosarcomas caused by avian leukosis virus subgroup J associated with v-src and v-fps had happened in Chinese flocks in recent years. In the last five years, field cases of ALV-J infection and tumors were found in local breeds of chickens. In this study, we systematically studied the epidemiology and pathogenicity of ALV-J in local breeds, and researched on ALV-J quasispecies diversity under host immune selection pressures by using high-throughput sequencing.1. Epidemiological investigation of ALV in indigenous chicken breeds, ChinaA total of 270 clinical samples were collected from chickens in three provinces in China(Shandong, Zhejiang and Jiangsu) between 2011 and 2014, those chickens originated from 22 indigenous species. Of which a total of 47 ALV strains were isolated and identified, 4(8.5%)were identified as ALV-A, 2(4.3%) as ALV-C, 18(38.3%) as ALV-J, and 23(48.9%) as ALV-K. Phylogenetic tree for gp85 amino acid sequences showed that subgroup J was in one clade, while non-subgroup J isolates were in the other clade. Most of viruses isolated from diseased chickens with tumors were ALV-J. However, ALV-A, ALV-C, and viruses belonging to a new subgroup K were isolated from healthy chickens. On the other hand, with contrast to no isolation of ALV-K from the imported white broilers and layer flocks, such a high separation rate was found with the infection of ALV-K in the local breeds of chickens inChina. The epidemiology phenomenon shows that the novel isolated subgroup K by our laboratory may be an unique and long existing subgroup in chicken flocks in China and east Asia.2. The pathogenicity research of co-infection with ALV-J and REVTo study the co-infection of ALV-J and REV in broiler chickens and layer chickens,one-day-old commercial broilers and SPF chickens were single infected or co-infected with ALV-J and REV. The mortality, body weight, immune organs ratios and antibody responses to Newcastle disease virus(NDV) and H9-Avian influenza virus(H9-AIV) were studied at different times. The results indicated that no matter in broilers or in SPF chickens, the co-infection of ALV-J and REV induced more growth retardation and higher mortality rate than ALV-J or REV single infection(P < 0.05). Chickens co-infected with ALV-J and REV also showed more severe immunosuppression which was manifested by significantly lower immune organ to body weight ratios and lower antibody responses to Newcastle disease virus and H9-avian influenza virus(P < 0.05). These results showed that synergistic pathogenic effects of co-infection of ALV-J and REV are in both broiler chickens and SPF chickens.3. The pathogenicity research of acute fibrosarcoma from chickens infected withsubgroup J avian leukosis virusTo understand the biological relationship between each specific modal karyotype and the acute fibrosarcoma and the characteristics of its pathogenicity, we produced the tumor cells from the sarcoma and performed a karyotype analysis of the fifth generation fibrosarcoma cell cultures. The pathogenicity test was also carried out with the 26 th cell supernatants. The tumor cells were prepared successful and had subcultured to 42 generations, which were in good shape but the growth starting to slow down. The karyotype analysis of the 5th generation cell showed that 29 of 50 qualified spreads of the fibrosarcoma cell culture demonstrated polyploidy of some macrochromosomes, 21 of which were trisomic on chromosome 7, and others were trisomic on chromosomes 3, 4, 5(sex chromosome w) and 10. In addition, one of them was trisomic on both chromosome 7 and the sex chromosome 5(w). The animal experiment showed that the 26 thcell supernatants could also induce fibrosarcoma. In this study, we described cytogenetic mosaicism or polymorphism of the aneuploidy in the acute fibrosarcoma described in this study, which suggests a polyclonal characteristic of the analyzed cells. Also, construction of tumor cell lines provided in vitro platform to study such acute fibrosarcomas caused by avian leukosis virus subgroup J associated with v-src.4. ALV-J quasispecies diversity under host immune selection pressures by usinghigh-throughput sequencingTo study dynamic evolution of ALV-J quasispecies under host immune selective pressures,we inoculated specific-pathogen-free(SPF) chickens with the same founder ALV-J stock. Six(three antibody positive and three antibody negative) chickens with persistent viremia were selected after 36 weeks animal experimental observation. The viral RNA was extracted from plasmas at different time points(2 w, 4 w, 8 w, 12 w, 16 w, 20 w, 24 w, 28 w, 32 w and 36 w)and then sequenced using a high-throughput sequencing platform. This produced the largest ALV-J dataset to date, composed of more than three million clean reads. Our results showed that host immune responses could greatly enhance the genetic diversity of ALV-J quasispecies.In particular, selection pressures also promoted a dynamic change of the top ten variants.According to the former and later dominnat variants from the antibody-positive chicken, we constructed two infectious clones and cross-neutralization experiment was performed with the serum from the corresponding period. The results indicated that the antibodies from the former and later periods from the same chicken showed significantly different antigen specificity, which displayed the co-evolution of virus and host immune responces.Accordingly, more amino acid substitutions were observed to be fixed under positive selection in antibody-positive chickens. In contrast, the antibody-negative chickens provided a weak driving force, and no shift of dominant variant was observed. Moreover, we identified a novel hypervariable region in the gp85(61-68aa). To our knowledge, this is the first report studying the diversity and dynamics of ALV quasispecies in vivo via high-throughput sequencing. Our study sheds light on the interaction between ALV-J and its host immune system. |
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