Molecular and Biological characterization of Sudan isolates of Newcastle disease virus

Abstract

A field survey for the prevalence detection of Newcastle Disease in Khartoum State was carried out in the period 2008 -2010,and to study the antigenic and pathotype characteristics, which will lead to understanding the types of the disease prevailing and enable to compare them with the current vaccine strains. The virulence of NDV was determined in the laboratory by experimental inoculation of disease- controlled day old chicks with the isolates. The intracerebral pathogenicity index, (ICPI) values were found to be 1.83 and 0.1 for virulent and avirulent isolates respectively. A total of 18 isolates were examined by hemagglutination activity. HA activity was detected in all the 18 samples when the chicken red blood cells (RBCS) were used, while when the horse RBCS were used only 10 samples agglutinated them indicating that they were lentogenic, whereas the other 8 were velogenic and were negative. The hemagglutination inhibition (HI) test results showed that all allantoic fluids were inhibited when tested against positive ND antisera. All viruses revealed clear CPE 24-48 hours p.i. in chick embryo fibroblast cell culture. Plaques were observed only after inoculation of virulent strains and after the addition of bovine serum albumen to the overlay medium. Hence plaque production in cell culture can be used to differentiate between virulent and a virulent pathotypes of the virus. Organs (bursa, spleen, lung, bone marrow, trachea, cecal tonsils, kidney, brain, intestine) were collected and inoculated in 9-11 day old xxiii embryonated chicken eggs. All the embryos died within 48 hours post inoculation and the virus titer ranged from 8-256 for the virulent groups, and no virus titer was recorded for avirulent positive isolates, probably due to the short incubation period. Tissue samples from both virulent and avirulent isolates including spleen, bursa, lung, brain, trachea, and .intestine were collected after intravenous inoculation of 8-weeks old chickens; these were fixed in 10% formalin and examined microscopically for histopathological changes. Allantoic fluids were sent to OIE, FAO and National Reference Laboratory for Newcastle disease and avian influenza in Italy for advance confirmation by using molecular analysis at the cleavage site of the fusion glycoprotein (F protein), all samples which were previously confirmed positive by conventional methods were found positive using one step RT – PCR; nucleotide sequencing were also found positive. This work reported the absence of the circulation of the new genetic lineage, described in the Western and Central parts of Africa; all Sudan NDV isolates belong to genotype 5d, containing the virulent fusion protein cleavage site (FO) motif 112RRQKRF117.