Study On The Bionmics Of Marek’s Disease Virus Predominant Strains In China

Marek’s disease(MD) is a lymphoproliferative and demyelinating disorder of chickens caused by Marek’s disease virus(MDV).Clinical signs of MD showed chronic polyneuritis, immunosuppression, lymphomas in visceral organs and other tissues, transient paralysis and acute brain oedema. MD was prevalence disease in the world, and causing losses to the industry of $1~2 billion annually. Live vaccines have been widely used to control MD since their development in 1970. MD vaccines are non-sterilizing and, thus, do not prevent vaccinated birds from becoming infected with virulent field strains that collectively replicate and evolve within the same bird. MDV has been increasing in virulence since the middle of the twentieth century.Vaccines with better efficacy have been introduced to combat field strains, and the evolution of MDV appear to continue periodically in successive waves resulting in the emergence of more virulent strains capable of breaking through the immune responses induced by each generations of vaccines.In China, some flocks co-infection of MDV with other immunosuppressive virus including Reticuloendotheliosis(REV), Avian leukosis virus(ALV) or Chicken infectious anemia virus(CIAV). It has made huge economic losses for poultry industries. Therefore,to investigate the pathogenicity, gene variety and the effectiveness of the existing vaccines of epidemic field strains of isolated MDV and co-infection of MDV with REV, ALV and CIAV in China in recent years, which are of great significance for controling MD in the future.To investigate the prevalence of MDV in China,in the course of our continuous surveillance activities for MDV during 2011 to 2013, 522 samples suspected MDV infected were collected from 136 layer or breed farms which were located in Heilongjiang, Jilin, Liaoning, Shandong, Hebei, Jiangsu, Mongolia, Hunan, Henan and Guangdong provinces of China. Three hundred and thirty-six samples contained two or three copies of the MDV genomic 132 bpr as determined by PCR amplification, and those samples, which were obtained from 95 farms in nine provinces, were found to be positive for MD. The positive sample detection rate was 64.4%. These data showed that MD infrequently, but persistently,occurred in vaccinated chicken flocks in many provinces of China. Meanwhile, all MD positive samples were tested by PCR, IFA or/and ELISA for REV, ALV and CIAV, the results showed that polyinfection of MDV with REV、ALV or CAV accounted for 11.7%, 21.0%, 21.3%, respectively. Double, even triple or quar infection was detected. These results indicated that there was serious MDV infection in chicken flocks of China. Forty-three MDV isolates were obtained from 60 samples of MDV positive and ALV, REV and CIAV negative chickens and were shown to be well-adapted to DEF and CEF. PCR detection showed that the 41 MDV isolates contained two copiesand 2MDV isolates contained three copies of the 132 bpr of the MDV genome, which is characteristic of MDV field strains.The cultures of the 43 isolates were confirmed to be negative for ALV, REV and CIAV by PCR, ELISA and/or IFA. Meanwhile, MDV and REV coinfection CEF were obtained and detected by PCR, MDV plaque, IFA and Electron microscope.The meq genes of 43 isolated and reference strains contained the substitutions 77 E, 80 Y, 115 A, 139 A, 176 R and 217 A which are the main characteristics of Chinese pandemic strains. Phylogenetic analysis showed that different groups of MDV were co-circulating in chicken flocks in China and there were new strains circulating in the field.To study of pathogenic charateristics of MDV prevalent field strains, the pathogenic characteristics of three isolates were monitored and they exhibited different characteristics. The LCC is fundamentally characterized by neurological signs, marked thymic and bursal atrophy, and chronic wasting. None of the birds inoculated with the LCC presented visible tumors in any visceral organs during the course of the experiment. The histopathological observation showed abnormal mitotic figures definitive of neoplastic transformation in various types of organs in the LCC-challenged chickens at the end of the experimental period. These results suggest that LCC would later induce the development of lymphoproliferation in chickens. The LLY and LTS are predominantly characterized by visceral lymphomas. These strains induced gross MD lesions in all(100%) of SPF chickens, and the mortality rates of the unvaccinated chickens were 42.9%, 46.7%, and 23.1% by 60 days post challenge(dpc), respectively. MDV and REV coinfection CEF strains CYM+R caused SPF chickens 100% tumors and 93.3% mortality. Histopathologic observations of liver, spleen, kidney, and proventriculus of diseased chickens showed different types of lymphocyte. These results showed that MDV and REV co-infection caused more serious disease.The effectiveness of the existing vaccines, the CVI988 vaccine induced protective indices(PIs) of 85.7, 92.3, and 66.7, respectively. These results showed that the pathogenic characteristics of the Chinese isolates were diverse and that vaccine CVI988 provided different levels of protection against them. These data indicated that the existence of variant MDV strains was a possible reason of immunity failure in China. MDV genome load in the feathers of chickens, the viral genome load of the unvaccinated LCC- or LTS-challenged chickens peaked at 108 copies per million host cells at 14 and 17 dpc, and that of the unvaccinated LLY-challenged chickens peaked at 17 dpc. The viral genome loads of the unvaccinated challenged chickens were more than 106 copies per million host cells between 21 and 60 dpc. These results showed that MDV genome load maybe account for immunity failure. Finally, the charateristics of MDV strains co-infected with REV, constuction the MDV genome as bacterial artifical chromosome(BAC) and recombinant viruses were reconstituted and detected, the genome sequence were determined and analysis showed that shared the high nucleotide similarities with other Chineseprevalentstrains, and did not show obvious evolution.But above all, we investigated and analyzed extensively the prevalence of MDV strains in China in this study. The molecular characteristics, genetic evolutionary, pathogenic characteristic replication features and effectiveness of the existing vaccines against field strains were systematically studied. Meanwhile, co-infection of MDV with other immunosuppressive disease virus wasinvestigated, and analyzed MDV co-infection with REV. This research provides a good material and base information for the fundamental study pathogenic mechanism, gene function and vaccine development of MDV.