| Newcastle disease is one of the most serious infectious diseases caused by Newcastle disease virus (NDV) in poultry. The virus is classified as a member of the Rubulavirus genus sub-family Paramyxovirinae in the family Paramyxoviridae, belonging to Mononegavirales and is designated avian paramyxovirus-1(APMV-1). It causes high morbidity and mortality in non- immunized chickens even in some immunized chickens population with respiratory tract infection as well as egg drop. ND has been regarded as the most devastating disease in poultry industry for more than 70 years. Many scholars tried to discover the molecular variation trend of NDV, and a lot of data on molecular epidemiology have been reported in the past ten years, but the relation between the biological properties and genetic variations, especially the relationship between the genetic and antigenic evolution of the fusion protein (F) and hemagglutinin–neuraminidase protein (HN) is still not clear. In this study, we firstly tried to reveal the influence of the antibody immune selective pressure on the mutations of Newcastle disease virus (NDV) HN and F genes in cell cultures.Eight 21-day-old SPF chickens were inoculated in muscle with 0.5 ml of oil emulsion inactivated vaccine prepared by NDV field strain TZ060107. SPF chickens were raised in an isolator with filtered air of positive pressure. Chickens were vaccinated again two weeks later. Ten days after the second vaccination, chickens were bled and serum samples were collected as antiserum against NDV strain TZ060107. The primary virus neutralization test was conducted to determine the right dilution of the antiserum for the next serial passages. The concentration of 1︰50 was determined to add into medium, as the immune selective pressure of antibody. The field strain TZ060107 of NDV was inoculated into chicken embryo fibroblast (CEF) cells with (group A) or without (group B) anti-NDV monospecific serum and passed continuously. Each group contained 3 independent passage serials. HN gene and F gene were amplified and sequenced for the 10th, 20th, 30th, 40th and 50th generations of each passage serial, and compared with the original strain. From sequences comparison result, the influence of the immune pressure of antibody on HN gene and F gene were observed.The results demonstrated that HN gene mutations in group A with the antibody were apparently more than in group B without the antibody. In group A, the ratio of nonsynonymous (NS) mutations to synonymous (S) mutations was 6, significantly higher than 3.4 in group B. In group A with the antibody, there were 5 stable NS mutations in HN gene and 3 of them (related to aa#353, 521 and 568) were related to the known epitopes. There were two stable NS mutations in F gene in group A, but no stable NS mutation was found in group B. The NS/S ratios of base mutations in F gene were less than 2.5 no matter in group A or B. This suggests that the antibody strongly influenced mutations in HN gene, while F gene was less influenced by the same antibody. |
