Growth of Peste des Petits Ruminants virus(PPRV) on Embryonated Chicken Eggs and Cell Culture
Growth of Peste des Petits Ruminants virus(PPRV) on Embryonated Chicken Eggs and Cell Culture
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Date
2015-04-18
Authors
Elrayah, Thoyba
Journal Title
Journal ISSN
Volume Title
Publisher
UOFK
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
Description
Keywords
Peste des ,Petits Ruminants,virus,Embryonated, Chicken Eggs,Cell Cultur