The characterisation of turkey rhinotracheitis virus from chickens and the development of a suitable vaccine.
Maharaj, Sanjay Balkishore.
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Three turkey rhinotracheitis virus-like (TRTV-like) isolates were isolated from chickens with swollen heads. All grew well via the yolk sac (y/s) , chorioallantoic membrane (CAM), tracheal organ culture (TOC) , chicken embryo fibroblast (CEF) , and Vero cell routes. Affected embryos were stunted and severely congested. No pathological alterations were detected in allantoic sac (a/s) inoculated embryos. The CEF and Vero cells required trypsinisation for five consecutive passages before any visible cytopathic effects (CPE) were observed. Intra-cytoplasmic eosinophilic inclusions were observed in Vero and CEF monolayers . Only isolate 652/93 caused 100% ciliostasis in TOC. The other two isolates were able to cause a maximum of only 70-80% ciliostasis. The isolates were inactivated by chloroform treatment and exposure to 56°C for 1 h. Long term storage could be achieved at -70°C or in liquid nitrogen but not at 4°C or at -20°C. The isolates did not agglutinate chicken red blood cells and were found to contain genomes of RNA. They were able to elicit TRTV antibodies in specific pathogen free (SPF) birds as measured with the Pathasure enzyme linked immunosorbent assay (ELISA) kit. They could also be neutralised by TRTV-specific antisera. Electron microscopy of infective allantoic fluid (A/F) revealed particles of 100-300 nm in diameter with a helical nucleocapsid component approximately 15 nm in diameter and a fringe of approximately 12 nm long spikes. The processes of VLP development and maturation in TOC's and Vero cells were similar with accumulations of virus-like nucleoprotein close to the plasma membrane, forming the nucleocapsid. Virus-specified spikes were then inserted into the plasma membrane after which the VLP budded from the plasma membrane, incorporating this membrane with spikes as its own. Nine viral polypeptides with molecular weights of 200kDa, 83kDa, 53kDa, 40kDa, 37kDa, 28kDa, 19kDa and 15kDa were detected by SDS-PAGE in samples of the three isolates. The 83kDa and 53kDa polypeptides were also detected by western blotting using TRTV specific antisera. Both, a TRTV and a 652/93 isolate non-radioactive DNA probe, appeared specific for TRTV and TRTV-like isolates. The 652/93 probe detected 652/93 virus in SPF chickens for 19 days post-inoculation. A vaccine produced in SPF eggs using the attenuated 652/93 isolate, was able to protect vaccinates against virulent virus in laboratory challenge studies. In field trials, birds vaccinated at day-old or at day-old and again at 14 days, showed slightly improved performance compared to non-vaccinated birds. However, this benefit was not statistically significant. It is suggested that other environmental and disease factors mask the benefit provided by the vaccine in field trials. The three TRTV-like isolates appear to be chicken strains of TRTV and vaccination with an autogenous vaccine appears to afford some benefit to vaccinates.