Growth of Peste des Petits Ruminants virus(PPRV) on Embryonated Chicken Eggs and Cell Culture

Abstract

The Peste des Petits Ruminants virus (PPRV) strain Nig. 75/1, obtained from the Central Veterinary Research Laboratories Center (Soba), was used to test growth of PPRV in embryonated hen′s egg and to study the effect of different temperature degrees (35°C, 37°C and 39°C) on the growth of Peste des Petits Ruminants on African green monkey kidney cells (Vero). The positive results for PPR growth on embryonated chicken eggs and cell culture were confirmed by use of agar gel immune diffusion test and immunoperoxidase staining technique. The embryonated chicken eggs were inoculated by four different routes (allantoically, amniotically, chorioallantoic membrane and yolk sac). The PPRV was passage twice in embryonated chicken eggs (passage one and passage two). The virus grew on embryonated chicken eggs when inoculated by the four different routes but without any lesions in embryos and chorioallantoic membranes in the two passages, then the growth of the virus was detected by agar gel immune diffusion test. In passage one, allantoic fluid gave negative results in agar gel immune diffusion test when inoculated by allantoic routes. In passage one and passage two, inoculation by amniotic and allantoic routes showed that the virus grew in amniotic and allantoic fluids but not in yolk. In passage one and passage two, inoculation by chorioallantoic membrane route showed thatthe virus grew in amnioticand allantoic fluids and yolk. In passage one and passage two, inoculation by yolk sac route showed that the virus grew in yolk, which became pale and lost its constitution. The amniotic and allantoic fluids gave inaccurate results inagar gel immune diffusion test. In passage one and passage two, the embryos homogenate gave positive results to agar gel immune diffusion test when inoculated by the four different routes. The PPRVwhich was used in this study was passage in cell culture twice; the PPR vaccine used alsowas passage twice. Nine tissue culture tubes containing semiconfluent Vero cells were inoculated with 0.2 ml virus 5

stock containing 10 6.62 CCID50 /ml and incubated in 35°C, 37°C and 39°C; three tissue culture tubes for each temperature degree were used. The tissue culture tubes were incubated for 48 hrs, 72 hrs and 96 hrs, using three tissue culture tubes for each incubation time interval. After incubation periods the tissue culture tubes were harvested and titrated. At 35°C, the titers were 10 2.5, 10 4.27 and 10 5 in 48, 72 and 96 hours post inoculation, respectively. At 37°C, the titers were 10 3.5 , 10 5 and 10 5.55 in 48, 72 and 96 hours post inoculation respectively, while cultivation of the virus at 39°C for 48 hrs gave titer 10 1.8 CCID50/ml, but there was no viral growth detected when the virus was cultivated for 72 and 96 hrs.The highest titer was at 37°C after 96 hours incubation (10 5.5 CCID50 /ml), while the lowest titer was at 39°C after 48 hrs (10 1.8 CCID50/ ml). The results suggested that the best route to propagate the PPR virus in embryonated hen′s egg is the amniotica or allantoic cavities and the best temperature degree to propagate the PPR virus is 37°C