Restriction endonuclease analysis of duck plague viral DNA

Abstract

Differences in the clinical manifestations of infected ducks, pathomorphology in developing duck embryo (DDE), and developing chicken embryo (DCE) and cytopathic effects in duck embryo fibroblast culture (DEFC) and chicken embryo fibroblast culture (CEFC) caused by two virulent strains of duck plague virus – DPV – 1 (procured from I.V.R.I., Izatnager) and DPV – A (isolated from Alleppey) and a vaccine strain – DPV – V (obtained from V.B.I., Palode) were investigated in this study. Restriction endonuclease analysis (REA) was done to assess molecular differences between the strains. Both the virulent strains produced typical symptoms and lesions of duck plague (DP). However, the level of mortality and severity of certain lesions like gizzard muscle necrosis and haemorrhagic bands in the small intestine were more pronounced in DPV – A infection. DPV – V did not produce any symptoms or lesions on experimental inoculation into ducklings. Embryonated duck eggs were used for passage of DPV – 1 and isolation of DPV – A, and DCE was used for propagation of DPV – V. Mortality of embryos with congestive lesions on the CAM and body of the embryo were observed for all the three strains. DPV – A produced more severe congestion on the extremities of inoculated embryos. Both the virulent strains (1 and A) failed to produce lesions in DCE. Both the virulent strains were cultured in DEFC and DPV – V in CEFC. All three strains produced CPE, characteristic of herpes viruses, with rounding and clumping of cells, syncytium formation, vacuolation of cytoplasm and formation of eosinophilic intranuclear inclusion bodies. DPV – A and DPV – V took more time for production of CPE in the first passage with the time taken for CPE production decreasing with successive passages. The three strains of DPV were titrated in embryonated eggs (ELD50 ) and cell cultures (TCID50). DPV – 1 and A had an ELD50 of 105.27 per ml and 104.86 per ml respectively in DDE. The ELD50 of DPV – V was 104 per ml in DCE. TCID50 of DPV – 1and A in DEFC were 105.75 per ml and 105.25 per ml respectively and that of DPV – V in CEFC was 105 per ml. Virus particles ranging in size from 170 – 190 nm with a core size of 70 – 90 nm were observed on electron microscopic examination of processed and concentrated DPV – A material collected from infected DDE. Strains of DPV cultured in DEFC/CEFC were used as virus source for DNA extraction. DPV – 1, V and A had DNA concentrations of 1830 ug per ml, 1470 ug per ml and 1595 ug per ml respectively. Restriction enzyme analysis was done using Eco RI, Bam HI, Xho 1, Pst 1 and Hind 111. Eco RI cleaved all the three strains of DPV into 17 fragments. The restriction profile of DPV – V and A were nearly identical to each other with slight variation from DPV – 1 in fragment size. The RE Bam HI digested both the virulent strains of DPV (1 and A) into 14 fragments which were similar to each other except for the size of two fragments. DPV – V was cleaved into 16 fragments with differences in the size of six to seven fragments on comparison with DPV – 1 and A. The RE Xho 1 cleaved DPV – 1 and V into21 fragments and DPV – A into 23 fragments. All three strains differed from each other in the size of several fragments. Both the virulent strains (DPV – 1 and A) yielded 21 fragments and DPV – V 22 fragments on digestion with Pst 1. Majority of the fragments were below 10 kbp in size and there was variation in the size of 11 – 17 fragments between the strains. Restriction enzyme Hind 111 cleaved DPV – 1, V and A into 23, 22 and 21 fragments respectively. Apart from the difference in fragment number, all the three strains differed from each other in the size of 4 of their fragments. The average molecular size of the genome of DPV – 1, V and A estimated by REA with five REs were 181.23 kbp (Molecular weight 115.067 Megadalton), 184.218 kbp (116.964 Megadalton) and 181.582 Kbp (115.290 Megadalton) respectively. Of the five REs used in this investigation Bam HI, Xho 1, Pst 1 and Hind 111 were found to be more useful in differentiation of the three strains of DPV.