Isolation and Characterization of PI-3 Virus from Sheep and Goats
Jehan A.M. Gafer,
The present study was directed to isolate the PI-3 virus from sheep and goats which naturally affected with respiratory manifestation and to identify the isolated virus using several assays. Two hundred and five nasal swab samples were collected from diseased sheep and goats for the isolation of PI-3 virus. The samples were taken from diseased animal at 7 different Governorates Kaffer el sheikh, Alexandria, EL Behaira, Port Said, Demiatta El-Qalubia and Giza through the winter seasons of years 1999 to 2003. The virus was successfully isolated from three ovine samples after three successive passages on MDBK cells. The isolated viruses were then titrated and identified using different biological, serological and molecular assay.
Parainfluenza-3 (PI-3) is the most common virus infection of the respiratory
tract of sheep and goats throughout the world. The virus belongs to family Paramyxoviridae
of order Mononegaviralis, the non segmented negative single stranded RNA viruses
(Pringle, 1991). The virus is generally spherical and
150 to 350 nm in diameter but can be pleomorphic in shape. The genomic RNA contained
within a helical nucleocapsid that is enclosed within the lipid bilayer. Inserted
into the envelope are glycoprotein spikes that extended approximately 8 to 12
nm from the surface of the membrane (Kingsbury, 1991;
Field et al., 1996).
Parainfluenza-3 virus was first isolated from sheep in 1966 (Hore,1966)
since then serological surveys have shown that this infection is widely spread
in sheep population in many countries (Fenner et al.,
1996). In Egypt, the first isolation of PI-3 virus from lambs was carried
out in 1971 (Baz, 1971). The virus revealed symptoms like
mild pyrexia, coughing and nasal discharge for several days and necropsy has
shown area of pneumonia, particularly in ventral parts of the apical lobe (Hore
and Stevenson, 1967). The present study was directed to isolate the PI-3
virus from sheep and goats which naturally affected with respiratory manifestation
and to identify the isolated virus using several assays.
MATERIALS AND METHODS
Two hundred and twenty five nasal swabs were collected from sheep and goats
suffering from respiratory manifestation at 7 Govenorates through the winter
seasons of years 1999 to 2003; the dilated data are shown in Table
The samples were collected in sterile tubes containing 2 mL of sterile PBS
and 200 μL of stock antibiotic solution per each sample. The samples were
taken on ice bucket. Clarification was done by centrifugation at 5000 rpm for
5 min at 4°C in cooling centrifuge. The supernatant was taken and kept at
-70°C till used.
||Number of nasal swab samples collected from sheep and goats
in different localities in Egypt
||Normal non infected MDBK cells
First local bovine Egyptian PI-3 virus strain 45 was kindly supplied from
Rinder Pest like disease Department at Veterinary Serum and Vaccine production
Institute Abbassia, Cairo. Second bovine PI-3 virus strain SF4 was
kindly supplied from Rabies Department in VACSERA.
MDBK cells were kindly supplied from Department of Virology, Faculty of
Veterinary Medicine Cairo University (Fig. 1).
Samples were subjected to heamagglutination test to exclude the non-heamaglutinatinating
samples from the isolation work. The test was conducted according to Singh
and Baz (1966) using 1% G. pig RBCs.
Isolation of PI-3 Virus from HA Positive Samples
The isolation was carried out according to Rulka (1987)
using MDBK cells. Three successive passages were done for this isolation.
Titeration of Isolates
The harvest of the inoculated MDBK cells after three passages of the isolates
was titrated. The test was performed according to Frey and
Liess (1971). Briefly, in a microliter plate 90 μL of MEM free serum
were dispensed into each well then 10 μL of the suspected isolate suspension
were added to the wells of the 1st column and serial ten fold dilutions were
made 50.000 cells/well were added while, being in suspension. The plate was
incubated at 37°C in CO2 incubator with daily microscopic examination.
The TCID50 was calculated according to Reed and
Identification by Heamadsorption Technique
The test was conducted according to Elizabeth et
al. (1997). After 16 h of inoculation of cells with isolates, the attached
cells were fixed for 30 min with formaldehyde in PBS (pH 7.2) then 1% suspension
of G. pig RBCs was added. The cells were examined under inverted
Identification of PI-3 Virus by Detecting Inclusion Bodies
The test was made using Zinker Solution as a fixative according to Clayden
Identification of the Isolates Using Immunofluorescence
The test was performed according to Vander Heide (1971)
using Cell Culture Staining Chamber (CCSC). Each cup was inoculated with 100
μL of the isolate and uninfected cups were included as control. The cups
were overlaid with 100 uL of anti PI-3 serum conjugated with FITC (supplied
from Department of Virology, Faculty of Veterinary Medicine Cairo University)
then examined under fluorescent microscope.
Identification of PI-3 Suspected Isolates by Virus Neutralization Test
The test was performed according to Murakami et al.
(1983). Beta neutralization procedure using antibovine PI-3 hyperimmune
serum (supplied from Serum and Vaccine Research Institute Abbassia, Cairo).
The serum was originally supplied from Imes, Iowa, USA.
Detection of PI-3 Virus by Polymerase Chain Reaction
The genomic RNA was extracted from inoculated tissue culture by means of
TRIZOL reagent according to their manufacture instructions (commercially supplied
by life technologies CO.) The TRIZOL reagent is improvement to single step RNA
isolation method was declared by Chomczynski and Sacchi (1987).
RT-PCR was employed using oligonucleotide primer pair designed from conserved
site of fusion protein gene (F gene ) (Table 2) as
previously described by Lyon et al. (1997). The
sequences of utilized primers as follow:
Analysis of the PCR Product Using Agar Gel Electrophoresis
The analysis was carried out according to Sambrook et
al. (1989) using ethidium bromide stained agarose gel 1.5%.
||Cycling protocol for amplification of F gene of PI-3
RESULTS AND DISCUSSION
Results of heamagglutination test are presented in (Table 3).
Five HA-positive samples were cultivated and propagated on MDBK cells. Three
passages in cell culture were applied and resulted three isolates which were
designated as Oseem56/1999, Oseem184/2003 and Nahia70/2000 according to their
localities and the year at which the samples were collected (Table
4) present the designation and passages results of the cell culture work.
The three isolates as well as local bovine strain 45 were titrated according
to infectivity the results presents in (Table 5). It seem
to be titers ranged from 105 to 106.6 TCID50/mL
with the highest test titer was given by the isolate Oseem56/1999 followed by
isolate Oseem 184/2003 and finally the isolate Nahia 70/2000. The isolated viruses
were identified using hemadsorption, inclusion body detection, immunofluorescence,
SNT as well as RT-PCR (Fig. 2).
Parainfluenza-3 (PI-3) virus causes, different clinical syndromes in sheep
and goat specially respiratory infections (Ackermann and
Brodgen, 2000). Serological surveys have shown that infection is widely
spread among sheep population in many countries (Fishman,
1965; Pop et al., 1979; Saddour,
1987; Riedmann et al., 1991). Many of serological
data indicated that the ovine PI-3 virus is antigenically related to bovine
and human strains of PI-3 (Wood et al., 1975).
In the present study, we tried to isolate PI-3 virus from naturally infected
sheep and goats and RT-PCR was used as a recent tool for detection and identification
of the isolated virus. The detection of PI-3 virus by heamagglutination, revealed
five positive samples (all of them were from sheep samples representing 2.2%
of the 225 collected samples. This low percentage of HA positive may be due
to the high amount of virions (at least 105.5-106.5 virions)
required to give one HA unit. The isolation was achieved through three passage
applied on MDBK cells giving rise to three isolates developed a clear CPE (Table
6).The identification by detecting inclusion bodies revealed large eosinophilic
intracytoplasmic (Fig. 3) with no evidence to nuclear inclusions
in contrast to bovine PI-3 virus that produced both cytoplasmic and nuclear
inclusion similar results are previously reported by Howard
et al. (1982). Intracytoplasmic perinuclear florescence in the positive
FA-isolates on MDBK cells shown in Fig. 4. It was noticed
the three isolates were from lambs which may be indicate the prevalence of Parainfluenza-3
(PI-3) virus that among sheep than in goats.
||Haemagglutination titer of the five HA positive nasal swab
samples and their localities
||Results of cultivation of the HA positive (ovine) nasal swab
|CPE = cytopathic effect
||Results of titration of PI-3 isolates on MDBK cells using
||Infected MDBK cells with the isolates after fixation and the
||Large eosinophilic intracytoplasmic inclusion body
||Intracytoplasmic perinuclear fluorescence in the positive
FA-isolates on MDBK cells
||Results of virus neutralization test for PI-3 isolated viruses
against antiparainfluenza-3 reference serum
||The ethidium bromide stained 1.5% agarose gel electrophoresis
of the PCR products. Lane M: The molecular DNA marker. Lane A: The amplified
PCR product of approximately 400 bp, the conserved portion of F gene
of PI-3 virus strain A45 (positive control). Lane 1- 3: The PCR
products of the isolated viruses. Only Oseem56/1999 revealed the expected
band (lane 1)
However, the failure to isolate the virus from goat may be due to the lower
number of collected samples from goats compared with those from sheep and the
fact that the animals were kept under relatively isolated conditions (Erasmus
et al., 1967). The low titer of Oseem184/ 2003 isolate may be taken
as criterion for heterogeneity (Gaul et al., 1982).
This finding agreed with many of the earlier studies, which have shown that
at least three ovine PI-3 strains of different serotypes namely (G2
strain, CsL6 strain and DH-1 strain), do exist (Hore, 1966;
St-George, 1969; Howard et al.,
1982). The heamadsorption used as an indicator system for the presence of
PI-3 virus proved to be more rapid and sensitive than waiting until the cytopathic
changes appear. This finding is similar to those described previously by St-George
(1969) and Toth and Jankura (1990). The RT-PCR technique
waw performed in this study using two primers chosen from conserved site of
fusion protein gene (F gene of whate PI-3 viruses). The primers were
chosen in this site of genome as it is conserved among PI-3 viruses not specific
for ovine PI-3 virus (Fig. 5) Also, both sheep and goats can
catch bovine PI-3 virus. Moreover, these primers could help us in identification
of the standard bovine PI-3 virus strain and the standardization of the technique.
Also, the same primers were developed by Lyon et al.
(1997) and Maria et al. (1998). The detection
and differentiation of PI-3 virus could be studied by amplification and sequencing
of another part of the genome for example HN gene (Ruth
et al., 1994; Echevarria et al., 2000;
Vecherov et al., 2003). Indeed, This study reports
the isolation and characterization of parainfluenza-3 virus from sheep and goats
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