Studies on Newcastle Disease Virus Thermostablity and Molecular Diagnostics

Abstract

In the first series of experiments the Komarov strain of Newcastle disease (ND) virus was subjected to selection for heat resistance. Four cycles of heat selection at 56 o C were carried out. Residual virus obtained after the last cycle was lyophilized. The stability of haemagglutinin and infectivity at room and at refrigeration temperatures was assessed. Effect of some stabilizers (lactalbumen hydrolysate, lactose, sucrose and skimmed milk powder) on stability of virus haemagglutinin activity at both temperatures was also studied. On heat selection the haemagglutination titre of surviving virus raised from 7log2 after the first cycle to10log2 after the fourth cycle. Results showed that haemagglutinin activity of non-stabilizeradded product maintained a titre of ≥8log2 up to 20 days under refrigeration and for 7 days at room temperature (4 days and ≤1 day for parent virus respectively) while activity in lactalbumen hydrolysate, lactose and sucroseadded products were 9, 9 and 7 weeks and 6, 7 and 8 at both temperatures, respectively. For infectivity ≥8log2 titre was maintained up to 50 days under refrigeration and for 12 days at room temperature (2-5 days and ≤2 days for parent virus respectively). The haemagglutination test could not be accurately performed on the skimmed milk-added product. The results showed that Komarov strain of ND virus could be successfully selected for heat resistance and its stability may be enhanced by addition of suitable stabilizers. In the second series of experiments, a reverse transcriptase (RT) polymerase chain reaction (RT-PCR) was developed to detect field isolates of Newcastle disease virus (NDV) grown in vero cell culture or embryonated chicken eggs (ECE). Five Sudanese isolates of NDV designated (OB, KU, GR, A12, and A105) and five vaccine strains including Komarov, B1, LaSota, Clone30 and Clone79 were used in this study. A pair of primers (nd1 and nd2), targeting a fragmentin the F gene of NDV, was designed for PCR amplification. The RT-PCR assay resulted in amplification of a 356 bp PCR product from RNAs of Sudanese and vaccine strains of NDV. However, nucleic acid extracts of infectious bursal disease (IBD) virus, non-infected Vero cells or ECE failed to produce the specific 356 bp PCR product. The described RT-PCR assay was a simple procedure that involved a single amplification step. In addition, the developed RT-PCR assay provides a rapid, sensitive, and specific method for detection of an outbreak of the disease in susceptible birds. Finally, a nested RT-PCR assay for detection of Newcastle disease virus in clinical samples was evaluated. Clinicalsamples include blood, tracheal swabs and cloacal swabs, liver, spleen, heart, lung, kidney, bursa and brain. Tissue homogenates were made from all samples in sterile phosphate buffered saline containing antibiotics. After centrifugation of homogenates the supernatant fluids were used for virus isolation in embryonated chicken eggs and for viral RNA extraction as well. Two steps of nested RT-PCR were performed. In the first step a pair of primers (nd1 and nd2) flanking a 356 bp long were used to amplify specific region inthe F gene of the virus. In the second step, another (nested) pair of primers (nd3 and nd4) was employed to produce 216 bp amplification products for confirmation the authenticity of the first primers to the Newcastle disease virus. For diagnosis the nested RT-PCR assay was proved to be accurate, sensitive, rapid and less expensive whencompared with virus isolation.