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RT-PCR DETECTION OF AFRICAN HORSE SICKNESS
VIRUS SEROGROUPS IN CELL CULTURE
By
SAMAH ABDELRAHMAN AHMED ELGENAY
(B.V.M, 2003)
University of Khartoum
Supervisor
PROFESSOR: IMADELDIN ELAMIN ELTAHIR ARADAIB
A Thesis submitted to the University of Khartoum in Fulfilment of
the Requirements for M.Sc. Degree in Veterinary Medicine
Department of Medicine, Pharmacology and Toxicology
Faculty of Veterinary Medicine
University of Khartoum
September , 2007
II
To: My great mother My father.
My sisters ,and daughter.
FOR THEIR PATIENCE, CONSTANT ENCOURAGEMENT
AND SUPPORT.
III
Acknowledgements
Several people made critical contributions towards the
success of this study. First and foremost, my heartfelt
thanks to Almighty Allah for giving me the strength,
patience and willpower to complete this challenging task.
I would like to express my gratitude and appreciation to my
supervisor Professor Imadeldin Elamin Eltahir Aradaib, Dean
graduate college and director of scientific research
directorate of The National Ribat University, for valuable
guidance, constructive comments, advices and suggestion
which led to completion of my M.Sc. study.
My sincere thanks and appreciation are extended to my
colleague Dr. Khairalla Mohamed Saeed, and Dr.Tamador
My special thanks extended also to my great friend and
father Abdurrahman Ahmed Elgeny who stood beside me all
the time with kind and unlimited support provided to me. I
would also like to express my deepest thanks to Dr.Afraa
TagAlsir for being helpful.
IV
I am also grateful to Dr. Mohamed Abdullah for being so
willing to render assistance throughout the practical work
of my study. My thanks also go to all members of the
Department of Molecular Biology Laboratory, Faculty of
Veterinary Medicine, University of Khartoum, who
contributed in one way or another toward the completion of
my study.
Last, my great appreciation to my mother and sisters for
their understanding, support and encouragement during the
period of my study.
V
LIST OF CONTENTS
Page
Dedication……………………………………. II
Acknowledgements…………………………...
III
List of contents………………………………..
V
List of Figures………………………………...
VIII
Abstract……………………………………….
IX
Arabic abstract……………………………….
XI
CHAPTER ONE: INTRODUCTION AND GENERAL LITERATURE REVIEW
1
1. The virus…………………………………
1
2. Etiology…………………………………..
2
3. Host Range………………………………
3
4. Geographic Distribution………………..
5
5. Transmission……………………………
5
6. Diagnosis…………………………………
7
6.1. Clinical signs…………………………….
7
6. 1. 1. The peracute or pulmonary form……...
7
6. 1. 2. The subacute form………………………
8
6. 1. 3. The acute or mixed form……………….. 9
6. 1. 4. The febrile form…………………………
9
7. Gross Lesions…………………………….
9
VI
8. Morbidity and Mortality………………
11
9. Laboratory Diagnosis…………………...
11
9. 1. Virus isolation…………………………...
11
9. 1. 1. Samples for virus isolation……………
11
9. 1. 2. Tissue culture……………………………
12
9. 1. 3. Chicken embryos………………………..
12
9. 1. 4. Newborn Mice…………………………
13
9. 2. Serological tests………………………….
13
9. 2. 1. Agar Gel Precipitation test (AGPT)…...
13
9. 2. 2. Serum Neutralization Test (SNT)……...
14
9. 2. 3. Fluorescent Antibody Technique (FAT).
14
9. 2. 4. Haemagglutination test (HT)…………...
14
9. 2. 5. Passive Haemagglutination (PHA)……..
15
9. 2. 6. Immunoblotting…………………………
15
9. 2. 7. Enzyme Linked Immunosorbent Assay
( ELISA)…………………………………
15
9. 2. 8.
Complement fixation test……………….
16
9. 3.
Molecular diagnosis……………………..
17
9. 3. 1.
Nucleic acid hybridization……………...
17
9. 3. 2.
Polymerase Chain Reaction (PCR)…….
17
VII
9. 3. 3. Revers transcriptase polymerase chain reaction (RT-PCR)……………………...
19
9. 3. 4 The process of RT-PCR reaction ............ 20
10.
Differential Diagnosis…………………...
21
11.
Control…………………………………...
22
11. 1.
Preventive measures……………………
22
11. 2.
Vaccines…………………………………
23
11. 2. 1.
Live vaccines…………………………….
23
11. 2. 2.
Inactivated vaccine……………………
24
11. 2. 3.
Natural Immunity………………………
25
CHAPTER TWO: MATERIALS AND METHODS……………………………...
27
1.
Virus and cells…………………………
27
2.
Extraction of viral nucleic acid from infected ell culture and clinical sample……...
27
3.
Design of primers……………………………..
28
4.
RT-Polymerase Chain Reaction (RT-PCR)...
29
5.
Agarose gel electrophoresis………………….
30
CHAPTER THREE: RESULTS…………….
38
CHAPTER FOUR: DISCUSSION………….
42
REFRANCES…………………………………
48
VIII
LIST OF FIGURES
Figure 2.1: Confluent monolayer of vero cells……………………………… 33
Figure 2.2: 80% cytopathic effect (CPE)……………………………………. 34
Figure 2.3: Thermal cycler TECHNE, TC-41……………………………… 35
Figure 2.4: Electrophoresis apparatus……………………………………… 36
Figure 2.5: Gel documentation apparatus………………………………….. 37
Figure 3.1: Detection of all the nine AHSV vaccine strains in infected cell
culture…………………………………………………………………………
40
Figure 3.2: Specificity of the Polymerase Chain Reaction for AHSV RNA. 41
Figure 3.3: Sensitivity of the Polymerase Chain Reaction for detection of
AHSV ………………………………................................................................
42
IX
ABASTRACT
In the present work, a reverse transcriptase polymerase chain
reaction (RT-PCR) protocol was carried out to detect African horse
sickness virus (AHSV) RNA in vero cell culture.
The nine serotypes of African horse sickness virus were used in
this study . All these serotypes were propagated in vero cell culture.
RNAs were extracted from these serotypes and then, they were
detected by the described RT-PCR assay, using primers derived from
segment 3 of AHSV serotype 6.
The specific 240 bp PCR products were amplified from all
AHSV serotypes used in the study, and they were visualized on
ethidium bromide-stained agarose gel.
The Amplification product was not detected when the RT-PCR
assay was applied to RNA from bluetongue virus (BTV) or palyam
virus.
This specific 240 bp PCR product was obtained from an mount of 10
pg ,1.0 pg, 100fg, RNA from AHSV serotype (6).
The results of this study showed that the described RT-PCR
assay, using the mentioned primers, could be applied for detection of
AHSV serotypes.
X
In conclusion, the AHSV RT- PCR can solve the problems
which are found in virus isolation procedures. The rapidity, sensitivity
and specificity of the RT- PCR assay would greatly facilitate detection
of AHSV infection in an out break among susceptible equine.
XI
ةخالصه األطروح
فيروس مرض ( فيروس طاعون الخيل االفريقىأجريت هذة الدراسه لكشف مورثات
.باستخدام تفاعل البلمرة المتسلسل العكسى .وذلك ، الخاليا المستزرعة المصابةفي) النجمة
خاليا الزرع في واستزراعه أجرى الكشف على تسعة انماط مصلية للفيروس حيث
.ا استخالص الحمض النووى الريبى منه. )فيرو(الخلوى
من جينوم النمط 3 رقم هذة التجربة زوج من المبادئات مشتق من الفص في استخدم
د قليل من الخاليا المصابة والذى يتطلب عد، لفيروس طاعون الخيل االفريقى6المصلى
في ى التى استخدمتحماض النووية االخر االارنة مع بوادىء هجين موجبة مقإشارة لإلنتاج
.الكشف عن فيروس طاعون الخيل االفريقى
زوج قاعدى من جميع االنماط 240ناتج تفاعل البلمرة المتسلسل البالغ طوله تم إآثار
وتم إظهاره فى هالم ، لدراسة المصلية لفيروس طاعون الخيل االفريقى المستخدمة فى ا
. المصبوغ ببروميد االيثيديوم االغاروز
هذا الزوج من البوادىء خصوصية لهذة الفيروسا ت حيث لم يتم إآثار الناتج عند أظهر
إجراء تفاعل البلمرة المتسلسل العكسى على الحمض النووى الريبى المستخلص من
. اللسان االزرق أو من فيروس باليام فيروس ذاتفيروسات أخرى من نفس العائلة مثل
هذا الناتج فائق الخصوصية تم إآثاره من آميات مختلفة من الحمض النووى الريبى
، بيكوجرام 10 لفيروس طاعون الخيل االفريقى وهى 6المستخلص من النمط المصلى
من جينوم الفيروس أى ما يعادل أو يفوق حساسية عزل . فمتوجرام100 بيكوجرام و 1.0
ذا ما يؤآد حساسية تفاعل البلمرة المتسلسل العكسى فى وه. الفيروس بالطرق التقليدية
.الكشف عن فيروس طاعون الخيل االفريقى
XII
فان نتائج هذة الدراسة توضح أن تفاعل البلمرة المتسلسل العكسى باستخدام البادئات ،ختاما
ذو خصوصية و حساسية ،سريع ،يمكن إستخدامة آاختبار تشخيصى بسيط ، المذآورة سابقا
صعوبات الموجودة فى الطرق التقليدية المستخدمة فى تشخيص فيروس طاعون لحل ال
.الخيل االفريقى
تفاعل البلمرة المتسلسل العكسى للكشف عن استخدام دراسات الحقه،في يمكن
مثل الدم وأنسجة الحيوانات الفريقى مباشرة فى عينات اآلنيكيه فيروس طاعون الخيل ا
)2 وب1ب(للبادئات الخارجية ) 4 وب3ب(باستخدام بادئات داخلية المصابة طبيعيا وذلك
1
CHAPTER ONE
INTRODUCTION AND GENERAL LITERATURE REVIEW
1. The virus:
African horse sickness (AHS) (Peste equina africana, Peste equine)
is an infectious, noncontagious arthopod-borne disease of equidae, caused
by a double-stranded RNA orbivirus belonging to the family Reoviridae
(Roy P.,et .,al 1991). The genus Orbivirus also includes bluetongue virus,
epizootic hemorrhagic disease virus, and palyam virus which have similar
morphological and biochemical properties with distinctive pathological
and antigenic properties as well as host ranges. The genome of AHS virus
(AHSV) is composed of ten double-stranded RNA segments, which
encode seven structural proteins (VP1-7),) most of which have been
completely sequenced for AHSV serotypes 4, 6 and 9 (Roy P.,et .,al
1991), and four nonstructural proteins (NS1, NS2, NS3 and NS3A)
(Grubman M. & Lewis S. 1992). Proteins VP2 and VP5 form the outer
capsid of the virion, and proteins VP3 and VP7 are the major inner capsid
proteins. Proteins VP1, VP4 and VP6 constitute minor inner capsid
proteins. Recently it was indicated that NS3 proteins are the second most
variable AHSV proteins (Van Niekert et al., 2001). The most variable
being the major outer capsid protein, VP2. This protein, VP2, is also the
2
principal protein responsible for distinguishing AHSV serotypes and,
together with VP5 are respensible for virus neutralisation activity
(Martinez-Torrecuadrada et al., 1999). Nine antigenically distinct
serotypes of AHSV have been identified by virus neutralisation, but no
cross-reactions with other known orbiviruses have been,reported.
2. Etiology:
The etiological agent of AHS is a typical orbivirus measuring 68-
70 nm in diameter, and the virion is composed of a double-layered
protein shell.
The virus is present in the blood and certain organs such as spleen,
lung, and lymph nodes in reasonably high concentration, whereas only
traces are found in serum, tissue fluids, excretions, and secretions
(Erasmus, B.J.1972). Viremia generally lasts for about 4-8 days and
roughly parallels the febrile reaction. In exceptional cases, viremia may
last as long as 17 days in the horse and 28 days in zebra and donkeys.
The AHS virus is relatively heat stable, particularly in the
presence of protein. It can be stored for at least 6 months at 4° C in saline
containing 10 percent serum. Blood in OCG preservative (500 ml
glycerin, 500 ml distilled water, 5 g sodium oxalate, and 5 g carbolic
acid) can remain infective for more than 20 years; lyophilization may
3
preserve infectivity for as long as 40 years. The virus is readily
inactivated at pH values lower than 6.3, but it is relatively stable between
values ranging from 6.5 to 8.5.
Nine distinct serotypes of AHSV are known, the last of which was
isolated in 1960. This suggests that, despite its segmented genome, the
virus can be regarded as genetically stable and those new serotypes do not
readily develop. The present nine serotypes probably evolved over many
centuries.
3. Host Range:
Horses, mules, and donkeys have historically been known as hosts
for AHS virus, as reflected in the name of the disease. In view of the high
mortality rate suffered by horses and mules, these species should be
regarded as accidental or indicator hosts. That AHS failed to establish
itself outside tropical regions of Africa tends to indicate that neither
horses nor mules or donkeys remain long-term carriers of AHS virus and
are therefore not essential for the permanent persistence of the infection
in a particular region. Zebra may fulfill this role, but no irrevocable proof
has been found to substantiate this view.
The dog has long been known to be susceptible to experimental
infection (Theiler, A.1906) Infection of dogs also readily occurs
4
following ingestion of infected horse meat (Bevan, L.E.W.1911).
However, it is extremely unlikely that this species becomes infected by
insect bites, and it is generally accepted that dogs play no role in the
spread or maintenance of AHS (Mcintosh B.M. 1955).
Camels can ostensibly become apparently infected with AHS virus, but
few details are available as to the level and duration of viremia in this
species and its role, if any, in the epizootiology of the disease. A high
percentage of African elephant serum samples reacted positively against
AHS virus in complement fixation tests (Davies, F.G., and Otieno, S.
1977), but no neutralizing antibodies could be demonstrated in such
samples. No evidence of virus replication could be found in elephants
artificially infected with AHS virus (Erasmus et. al, 1976). It can
therefore be concluded that the African elephant is not susceptible to
infection and that the putative serological evidence resulted from
abnormal reactions of elephant sera in a complement fixation test.
There is no evidence that man can become infected with field strains of
AHS virus, either through contact with infected animals or from working
in laboratories. However, it has been shown that certain neurotropic
vaccine strains may cause encephalitis and retinitis in humans following
transnasal infection (Swanepoe et. al 1992.).
5
4. Geographic Distribution:
African horse sickness appears to be endemic in tropical regions of
central Africa from where it regularly spreads southwards to southern
Africa. The Sahara Desert forms a formidable barrier against northward
spread. Occasionally the infection does reach northern African countries,
either by spread along the Nile valley or along the West Coast of Africa.
The disease has also occurred outside Africa on a few occasions, the most
notable of which was the major outbreak in the Near and Middle East
from 1959 through 63 and in Spain (1966 and 1987-1990) (Lubroth, J.
1988).
In temperate regions such as South Africa, AHS has a definite
seasonal occurrence. The first cases are usually noticed towards
midsummer, and the disease disappears abruptly after the onset of cold
weather in autumn. The disease is most prevalent in warm, low-lying
moist areas such as valleys and marshes.
5. Transmission:
African horse sickness is a non contagious disease, and the virus
was the first shown to be transmitted by midges (Culicoides spp.) (Dutoit,
R. M.,1944). The most significant vector seems to be Culicoides imicola,
but other species, such as C. variipennis, which is common in many parts
6
of the United States, should also be considered as potential vectors
(Boorman et al.1975).
Although other insects such as mosquitoes have been implicated as
biological vectors, and large biting flies (e.g., Stomoxys, Tabanus) may
transmit AHS virus mechanically, the role of these insects in the
epizootiology of the disease is regarded as absolutely minimal compared
with that played by the Culicoides species
The virus is transmitted biologically by midges, and these insects
are most active just after sunset and at about sunrise.
Generally, midges disperse only a few kilometers from their
breeding sites, but it has been postulated that they can be borne for longer
distances on air currents (Sellers et al., 1977). Analysis of field
observations on the progression of outbreaks indicates that wind-borne
spread of midges may assist the short-distance spread of the disease but
that long-distance jumps of the infection are invariably the result of
movement of infected Equidae.
7
6. Diagnosis:
6.1. Clinical signs:
There are four classical forms of AHS:
1. The peracute or Pulmonary form
2. The subacute edematous or Cardiac form
3. The acute or mixed form
4. Horse sickness fever (Erasmus B.J., 1973).
6. 1. 1. The peracute or pulmonary form:
This has a short incubation period (3–5 days), is characterized by
very marked severe dyspnoea and progressive respiratory involvement.
An acute febrile reaction, lasting 1–2 days and reaching a maximum of
approximately 40–41°C, may be the only sign. This is followed by
various degrees of respiratory distress – respiratory rate may increase to
60 or even 75 breaths/minute. The animal may be observed to stand with
its forelegs spread apart, its head extended and its nostrils fully dilated.
Common and spasmodic coughing may be observed terminally,
with frothy fluid exuding from the nostrils. Death usually occurs within a
few hours after the first clinical signs are observed, the animal having
literally drowned in its own serous fluid. The pulmonary form is usually
observed in completely susceptible animals, animals infected with a
highly virulent strain of virus, or animals that are worked during the
8
febrile stage of the disease. Recovery from this form is very rare,
occurring in <5% of cases.
6.1.2. The sub acute form:
The incubation period of the sub acute, edematous or cardiac
form varies from about 7 to 14 days, and the onset of clinical disease is
marked by a febrile reaction (39–41°C) that lasts for 3–6 days. Shortly
before the decline of the fever, characteristic edematous swellings may
appear. These initially involve the temporal or supraorbital fossae and the
eyelids, and later extend to the lips, cheeks, tongue, intermandibular
space and laryngeal region. Subcutaneous edema sometimes extends a
variable distance down the neck towards the chest and, in severe cases,
may involve the chest and shoulders, but generally not the lower limbs.
Terminally, petechial hemorrhages may be observed in the conjunctivae
and under the ventral surface of the tongue. The animal finally becomes
restless and may show signs of colic before death from cardiac failure.
Difficulty in swallowing due to paralysis of the esophagus is also seen.
The mortality rate is about 50% and death usually occurs within 4–8 days
after the onset of the febrile reaction. In recovering cases, swelling
gradually subsides within a period of 3–8 days. This clinical form of AHS
is usually associated with infection by virus strains of low virulence or is
9
encountered in immune animals infected by heterologous virus strains, or
may be a function of biological variation in the infected animal.
6.1.3The acute or mixed form:
This form represents a mixture of the pulmonary and cardiac forms.
Although seldom diagnosed clinically, it is the most common form, and it
seen at post-mortem examination in most fatal cases of AHS in horses
andmules.
6.1.4. The febrile form:
Horse sickness fever is the mildest form and is frequently
overlooked in natural outbreaks. The incubation period varies from 5 to
14 days, and is followed by a febrile reaction (39–40°C) of the remittent
type, with morning remissions and afternoon exacerbations, lasting for 5–
8 days. Apart from the febrile reaction, other clinical signs are rare.
7. Gross Lesions:
The lesions observed at necropsy examination depend largely on
the clinical form of disease manifested by the animal before death (.
Erasmus, B.J. 1972). In the peracute form the most characteristic changes
are edema of the lungs or hydrothorax. In very per acute cases, extensive
alveolar edema and mottled hyperemia of the lungs are seen, whereas in
cases with a somewhat more protracted course extensive interstitial and
sub pleural edema is also present, but hyperemia is less evident.
10
Occasionally the lungs may appear reasonably normal, but the thoracic
cavity may contain as much as 8 L of fluid. Other less commonly
observed lesions are per aortic and per tracheal edematous infiltration,
diffuse or patchy hyperemia of the glandular fundus of the stomach,
hyperemia and petechial hemorrhages in the mucosa and serosa of the
small and large intestines, sub capsular hemorrhages in the spleen, and
congestion of the renal cortex. Most of the lymph nodes are enlarged and
edematous, especially those in the thoracic and abdominal cavities.
Cardiac lesions are usually not conspicuous, but epicardial and
endocardial petechial hemorrhages are sometimes evident.
In the cardiac form the prominent lesion is a yellow gelatinous
infiltration in the subcutaneous and intramuscular fascia primarily of the
head, neck, and shoulders. Occasionally the lesion may also involve the
brisket, ventral abdomen and rump. Hydropericardium is a common
feature, and there are extensive petechial and ecchymotic hemorrhages on
the epicardium and endocardium, particularly of the left ventricle. The
lungs are usually normal or only slightly engorged, and the thoracic
cavity rarely contains excess fluid. The lesions in the gastrointestinal tract
are generally similar to those found in the pulmonary form, except that
sub mucosal edema of the cecum, large colon, and rectum tends to be far
more pronounced.
11
In the mixed form the lesions seem to represent a combination of
those found in the pulmonary and cardiac forms.
8. Morbidity and Mortality
In susceptible horse populations, the fatalities range between 70
and 95 percent, and the prognosis is extremely poor. In mules, the
mortality rate is about 50 percent and in the European and Asian donkey
about 5-10 percent. No mortality is observed among African donkeys and
zebra.
In enzootic regions, the mortality rate is modified in proportion to
the immunity acquired by the equine population as a result of previous
vaccination or exposure to natural infection. (Erasmus B.J., 1973).
9. Laboratory Diagnosis:
9.1. Virus isolation:
9.1.1. Samples for virus isolation:
Heparinised blood samples taken in acute phase from sick animals
,and organs such as spleen ,lungs, lymph nodes and heart from a dead
animals were suitable for virus isolation (Blackburn and
Swanepoel,1988a).
12
9.1.2. Tissue culture:
Cultivation of AHSV in sheet of Monkey stable (MS), baby
hamster kidney (BHK-21) and African green monkey kidney (Vero) cell
lines were, all used for virus isolation. (Hazrati and Ozawa, 1965)
The virus showed cytopathic effect in 2-3 days, when both virulent
and attenuated neurotropic strains of AHSV were inoculated in monkey
kidney stable (MS) cell line. Following the virus inoculation, the
incubation period become shorter. There was an eight hours latent period
before the virus production was demonstrated. After 10 days from
inoculation, plaques are formed, their diameters ranged in size from 1-3
mm (Ozawa and Hazrati, 1964). The cytopathic effect (CPE) appeared in
the cell cultures 2-5 days after inoculation, in form of cell rounding and
detaching from the surface. Three serial passages were completed on any
simple specimen before the isolation attempt was considered negative
(Carol House, 1992).
9.1.3. Chicken embryos
Inoculation of fertile eggs in yolk sac, is also possible for
propagation of neurotropic and viscerotropic AHSV strains. In the first
passage the embryos mortality was low and it's increased to 100% by the
sixth passage (Goldsmith, 1967).
13
9.1.4. Newborn Mice:
AHS virus isolation can be done by intracerebral inoculation into
suckling mice (1-3 day – old-BALB/C, CD-1Swiss). After 2-3 days mice
developed nervous signs if the specimen is positive. The brains of the
mice are collected and homogenized as suspension to make two or three
more passages by reinculation intercerebrally into new born mice before
the specimen is considered to be positive. (Alexander, 1935b).
9.2. Serological tests:
9.2.1. Agar Gel Precipitation test (AGPT)
The success of AGPT in the diagnosis of AHS viral infection was
established by Hug and An sari (1961).
The AGPT was described as group specific test for AHS when the
virus was isolated in suckling mice, (Hajer et al., 1980). When AHSV
antigen and positive serum diffused towards each other through an agar
medium, two precipitin lines were found (Ozawa et al., 1968).The agar
gel immunodiffusion (AGID) test, currently used as standard serological
test, is complicated by cross-reactions between related orbiviruses
(Aradaib et al., 1995)
14
9.2.2. Serum Neutralization Test (SNT):
Serum neutralization test using the tube technique was established
in (1966) by Hazarati and Ozawa.They used this technique in the
diagnosis of AHS virus using cell culture (MS) as host system.
The serum neutralization test were also carried out to detect
antibodies in sera of equidae using vero cell culture in 96 well flat tissue
culture microtitre plates (Binepal et al., 1992) . The test can also be used
for serotyping the virus in tissue culture (Hazrati &Ozawa, 1965b,
Blackburn &Swanepoel, 1988b).
9.2.3 Fluorescent Antibody Technique (FAT):
Direct and indirect flurescent antibody techniques were
successfully applied to the identification of virus of AHS isolates in tissue
culture and for the screening of sera for antibody detection . The test was
proved to be group specific test. (Davies &Lund, 1974 )
9.2.4. Haemagglutination test (HA):
Tokuhisa et al., (1982) proved that erythrocytes from horse, sheep,
cattle, goat, rabbits, guinea pig and poultry were agglutinated by
concentrated culture fluid of AHSV at 4C°, room temperature and 37C°.
Optimal titers were obtained when the virus was diluted in normal saline
15
of PH ranging between 6.0 and 7.5. HA test was also used as a specific
test for diagnosis of AHSV. (Tewari et al., 1972).
9.2.5. Passive Haemagglutination (PHA)
(Soliman &Salama ,1979 ) Passive haemagglutination test for
detection of AHS antibodies was carrier out by using partially purified
AHS virus soluble antigen, it gave a positive reaction with tanned
sensitized horse RBCs .
9.2.6. Immunoblotting:
For the determination of anti- AHSV antibodies, antibodies to
viral proteins which separated by electrophoresis and transferred to
nitrocellulose paper, were useful. Small numbers of sera is especially
suitable for this test (OIE, 2000).
Immunoblotting procedure had been developed for the detection
of (AHSV) and identification of antibodies specific for viral proteins
(VP) and non-structural proteins (NS) (Bougrine et al., 1998).
9.2.7. Enzyme Linked Immunosorbent Assay (ELISA):
ELISA was proved to have a great potential as quantitative
serological tool in the detection of antibodies for several viral infection
(Williams 1987). The sensitivity and specificity of ELISA are superior to
16
the complement fixation test (CFT), which is commonly used as routine
quantitative serological test for AHSV diagnosis.
Indirect sandwich ELISA was used for detection of AHSV by
Plessis et al. (1990). Asero group specific, indirect sandwich ELISA was
developed for rapid detection of AHSV (Hamblin et al., 1991).
The problems associated with the use of the (AGID) test have been
solved by using monoclonal antibodies (MAbs) in competitive ELISA
(cELISA) technique (Laviada et al., 1992) . To confirm the orbivirus
identity, agroup –specific sandwich ELISA (S –ELISA) was also
developed recently (Crafford et al., 2003).
9.2.8. Complement fixation test:
Macintosh (1958) reported that complement fixation test (CFT)
with Guinea pig RBCs and acetone ether extracted mouse brain antigen
was a group specific test and was suitable for the detection of all the
established antigenic types.
CFT can be used for typing the AHSV, there were no serological
cross reactions between AHSV and other orbivirus ,(Ozawa &Bahrami
1968) .For demonstration of group –specific antibodies against AHSV,
CFT is frequently used , a sucrose –acetone .mouse brain extract is
commonly used as antigen (Paweska et al., 2000) .
17
9.3 Molecualr Diagnosis
9.3.1 Nucleic acid hybridization:
Serogroup-specific and serotype-specific complementary DNA
(cDNA) probes derived from different AHSV genome segment have been
developed to address virus isolation problems (Zientara et al., 1998).
However, the minimum amount of AHSV RNA these cDNA probes
could detect was found to be in the nanogram range with dot-blot
hybridization (Zientara et al., 1998).
9.3.2. Polymerase chain reaction (PCR):
The polymerase chain reaction (PCR) is a molecular biological
technique (Smithsonian Institution, 2006) for isolating and exponentially
amplifying a fragment of DNA, via enzymatic replication, without using
a living organism. As PCR is an in vitro technique, it can be performed
without restrictions on the form of DNA, and it can be extensively
modified to perform a wide array of genetic manipulations.
Invented in 1983 by Kary Mullis, PCR is now a common technique
used in medical and biological research labs for a variety of tasks, such as
the sequencing of genes and the diagnosis of hereditary diseases, the
identification of genetic fingerprints (used in forensics and paternity
18
testing), the detection and diagnosis of infectious diseases, and the
creation of transgenic organisms.
PCR is used to amplify specific regions of a DNA strand. This can be
a single gene, just part of a gene, or a non-coding sequence. Most PCR
methods typically amplify DNA fragments of up to 10 kilo base pairs
(kb), although some techniques allow for amplification of fragments up to
40 kb in size. (Cheng S, et.,al 1994).
PCR, as currently practiced, requires several basic components (Joseph
Sambrook et.,al 2001). These components are:
• DNA template that contains the region of the DNA fragment to be
amplified
• One or more primers, which are complementary to the DNA
regions at the 5' and 3' ends of the DNA region that is to be
amplified.
• a DNA polymerase (e.g. Taq polymerase or another DNA
polymerase with a temperature optimum at around 70°C), used to
synthesize a DNA copy of the region to be amplified
• Deoxynucleotide triphosphates, (dNTPs) from which the DNA
polymerase builds the new DNA
19
• Buffer solution, which provides a suitable chemical environment
for optimum activity and stability of the DNA polymerase
• Divalent cation, magnesium or manganese ions; generally Mg2+ is
used, but Mn2+ can be utilized for PCR-mediated DNA
mutagenesis, as higher Mn2+ concentration increases the error rate
during DNA synthesis ( Pavlov AR, et.,al 2004)
• Monovalent cation potassium ions
The PCR is carried out in small reaction tubes (0.2-0.5 ml volumes),
containing a reaction volume typically of 15-100 µl, that are inserted into
a thermal cycler. This is a machine that heats and cools the reaction tubes
within it to the precise temperature required for each step of the reaction.
Most thermal cyclers have heated lids to prevent condensation on the
inside of the reaction tube caps. Alternatively, a layer of oil may be
placed on the reaction mixture to prevent evaporation.
9.3.3 Reverse transcriptase polymerase chain reaction (RT-PCR):
Reverse transcriptase polymerase chain reaction (RT-PCR) is based on
the polymerase chain reaction (PCR). More importantly it is based on the
process of reverse transcription, which reversely transcribes RNA into
cDNA. RT initially isolated from retroviruses.
20
The techniques of RT-PCR allows the formation of cDNA
(complementary or copy DNA) from RNA, which stores the sequence of
RNA (such as messenger RNA, mRNA) in the more stable form of
nucleic acid, DNA. This reverse transcription from RNA into its reverse
complement DNA (cDNA) is the first step of a usually two-step process
of RT-PCR. Furthermore, by copying the RNA into cDNA, it will be
possible to amplify the cDNA sequence by using primers specific for the
DNA sequence. This amplification is the final second major step of the
two-step process of RT-PCR.
9.3.4. The Process of RT-PCR:
The First step of RT-PCR is referred to as the "first strand reaction". In
the first-strand reaction, complementary DNA also termed cDNA, is
made from the messenger RNA template of interest using oligo dT
(oligonucleotide poly-dTs act similar to primers and bind to the 3' polyA
sequence located at the 3' UTR - untranslated region, which are present in
most mRNAs), dNTPs, and an RNA-dependent DNA polymerase
(reverse transcriptase) through the process of reverse transcription.
These factors are combined in a reverse transcriptase buffer for 1 hour at
37°C. After reverse transcriptase reaction is complete, and the cDNA has
been synthesized, RNaseH is added (an RNA digestion enzyme) which
21
digests the RNA away from the RNA-cDNA hybrid. After incubation
with RNaseH, standard PCR or polymerase chain reaction is conducted
using DNA oligo primers specific for the sequence of interest. This
second step is referred to as the "second strand reaction".
Thus by adding the thermostable taq DNA polymerase, upstream and
downstream DNA primers, the single stranded DNA becomes double
stranded and is amplified, allowing the detection of even rare or low copy
mRNA sequences by amplifying its complementary DNA (PCR station,
2006).
10. Differential Diagnosis:
Erasmus (2003) reported the clinical signs of AHS particularly
when not fully developed, to be confused with other infections notably
Equine Encephalosis and Equine Viral Arthritis (EVA). Under the same
epizootiological conditions as AHS, those diseases occur. Usually horses
suffering from Equine Encephalitis do not have characteristic lung edema
or subcutaneous edema, and the mortality rate are considerably lower
than in AHS. Sever cases of EVA may readily be confused with AHS ,
the present of ventral edema in EVA of lower limbs ,and much lower
mortality rate should allow differentiation .
22
The early stage of piroplasmosis disease before blood parasites
demonstrated and anemia develops, may be confused with AHS, also
trypanosomasis shares this sign with the disease .(OIE ,2002).
Erasmus (2003), found the necropsy lesions of AHS can be
confused with those of Purpura Hemorrhagica ,in which the hemorrhages
and edema seem to be more sever and widely spread than in AHS and
usually involve the limbs and lower abdomen .
Hemorrhages and sudden death in Anthrax can be confused with
AHS. (OIE, 2002).
11. Control:
11.1. Preventive measures:
Several preventive measures should be taken, when the disease is
introduced into a country, to prevent further spread and eventually to
eradicate the virus in the shortest possible time .Equid animals imported
from infected countries should be quarantined in insect-proof facilities at
the point of entry. (Erasmus, 2003)
Affected equidae should be eliminated immediately and non-
infected one vaccinated and rested for 2 weeks and should be kept in
insect-proof housing because vaccine failure may occur .Vector control
23
should also be adopted by using insecticides repellent .Aircrafts flying
from endemic areas to countries free of the disease should be sprayed
with insecticides on arrival.
An international plan to prevent the spread of AHS involved the
establishment of immune buffer zones around infected areas; extensive
production of vaccines and internationally coordinated vaccination
campaigns was developed. (Losos, 1986).
11.2. Vaccines:
11.2.1. Live vaccines:
In India adverse reactions to the polyvalent AHS mouse adapted
vaccine occurred. (Parvi & Anderson.1963).
Losos, (1986) showed that ,the adaptation of the virus to the brain
of adult mice resulted in neurotropic vaccine that occasionally caused
encephalitis in horses ,mules and particularly in donkeys .The mouse
brain polyvalent vaccine was produced by using up to eight strains of
neurotropic virus propagated intracerebrally in mice . After 5 to 13 days a
mild fever appeared in vaccinated horse .The vaccine provides protection
in 21 days. After 6 months the full immunity is reached and lasts for up to
5-8 years. Foals from immune mothers are effectively vaccinated only
24
after 7month of age. Strains attenuated in mice are potentially neurotropic
for horse.
Blackburn and Swanepoel, (1988a) said that, immunization with
polyvalent attenuated vaccines of AHSV ,leads to broader response to
various serotypes and to higher titre, but some horses failed to respond to
one more serotypes .
Monovalent mouse brain and tissue culture vaccine of AHSV
serotype (9) which contained approximately 6.6 log 10TCID 50 per dose
was prepared and evaluted. No adverse effect due to vaccination was
observed and vaccines were safe in horse, mules and donkeys .The
vaccine titer decreased (0.5 log 10 TCID 50), when it was kept at 37C°
for 7 days. (Hazarti & Ozawa 1965b).
A polyvalent attenuated tissue culture AHS vaccine consisted of 7
neurotropic viruses was prepared, and was used in the Middle East. It
elicited a satisfactory immune response in vaccinated horses. (Mirchamsy
& Taslimi, 1964).
11.2.2. Inactivated vaccines:
The virus was inactivated with either B-propilactone at 0.2
percent, or with formaldehyde in concentration of 1:8000. In order to
enhance the immunogenicity, an aluminum hydroxide was added to the
25
vaccine as an adjuvant .Neutralizing antibodies developed within 4
weeks, when a single dose of either vaccine was used. Much higher
neutralizing antibodies were present in sera 6 month later. When two
injections of inactivated vaccine were administered at interval of 4 weeks.
The animals were resistant to a challenge dose of virulent virus.
(Mirchamsy & Taslimi, 1967).
11.2.3. Natural Immunity:
Alexander ,(1936) ,found that recovery from infection lead to the
development of complement fixing ,neutralizing and haemagglutination
inhibiting antibodies. Complement fixing antibodies are detectable in the
circulation as early as five days after infection. The formation of
neutralizing antibodies however, is somewhat slower and although they
may be detected as early as five days after the cessation of the febrile
reaction, they reach a peak much later and persist for an undetermined
period at lower titers.
Generally the level of antibody in sera of dams correlated with
the level of antibody acquired from colostrums by their foals, and the rate
of decline was proportional to the initial titre. Early immunization was
recommended so as to control the losses of foals because maternal
26
antibody titre decline in 2-4 months after birth (Blackburn
&Swanepoel,1988b) .
27
CHAPTER TOW
MATERIALS AND METHODS
2.1. Virus and cells:
The nine prototype vaccine strains of AHSV were obtained from
(Institut Pasteur Dakar, Senegal). The viruses were processed as
described previously (Aradaib et al., 1995). The virus vaccine strains
were propagated on confluent monolayers of Vero cells (Figure 2.1).
Vero cells were sub cultured in 25 ml Falcon tissue culture flasks.
Glasgow minimal essential medium (GIBCO)
was used for growth and maintenance of cells. All viruses were adapted
to this cell line by 2-3 successive passages. The infectious materials were
harvested when 80 % cytopathic effect (CPE) was observed (Usually 3-5
days after infection) (Figure 2.2), and centrifuged at 1500Xg for 30 min
and the cell-free supernatant was used for RNA extraction using QIAmp
extraction kit (QIAmp, Hamburg, Germany)
2.2. Extraction of viral nucleic acid from infected
Cell culture and clinical sample:
AHSV dsRNAs were extracted from the infected cell culture
supernatant fluid and from infected tissues using QIAmp viral RNA kit as
28
per manufacturers instructions. Briefly, 140 ul of virus suspention were
added to 560 ul AVL buffer containing carrier RNA into a 1.5 ml
microcentrifuge tube and mixed by pulse-vortexing for 15 sec. The
mixture was incubated at room temperature for 10 min. An amount of
560 ul of absolute ethanol was added and mixed by pulse-vortexing for
15 sec .An amount of 630 ul of the mixture were transferred to QIAmp
spin column mounted on 2ml collection tube and centrifuged at 8000 rpm
for min .The column was then transferred to another collection tube and
the remaining 630ul of the mixture was again spined at the same speed.
The column was then washed twice by 500 ul of washing buffers WB1
and WB2, respectivly . finally , dsRNAs were carefully eluted by 60ul
of buffer AVE equilibrated to room temperature .Total nucleic acid was
quantified using aspectrophotometer at 260nm wavelength .
2.3. Design of primers:
Primers were selected from the published sequence of genome
segment 3 of AHSV serotype 6 and used in these PCR assays (Williams
et al .,1998) .Primers 1 and 2(P1 and P2) were selected for the synthesis
of specific AHSV RT –PCR product . P1 included bases 281 -300 bp of
the positive –sense strand of the genome segment 3: 5'-GAT AAA CGC
GCC ACC GAA TT-3'. .P2 included bases 501 -520 bp of the
complementary strand: 5'-TCT GGA TCA GCA ATC TCT GC-3'. AHSV
29
RT-PCR using primer P1 and P2 would result in a product of 240 base
pair (bp). All primers were synthesized on a DNA synthesizer and
purified using oligo-pak oligonucleotide purification columns as per
manufacturer's instructions (Milligen/Biosearch, Millipore, Burlington,
MA, USA ) .
2.4. RT-Polymerase Chain Reaction (RT-PCR):
Amplification of the target sequence was preformed by using
double reaction RT-PCR. 1.0 µl methyl mercuric hydroxide of 80 mM
concentration was used to denaturent a mixture of 5 µl of the target RNA
and 2 µl of primer .The primers were used at a concentration of 20µġ/µl.
The mixture was incubated at 25°c for 10 min. To neutralize the mixture
by, 1.0 µl of βeta mercapto ethanol, 1.0 µl of RNAase inhibitor, 8.0 µl of
dNTPs ( 2.0µl of each dNTTP,dNATP,dNCTP,dNGTP ) .
Reverse transcription step used to synthesize complementary DNA from
RNA template by reverse transcriptase mixture which contained 2.7 µl of
10ҳ PCR buffer , 5.0 µl of Mgcl2 of 1.5 mM concentration and 1.1 µl of
the enzyme reverse transcriptase , . 8.8 µl of reverse transcriptase mixture
was added to each PCR tube which was placed in the thermal cycler at
42ºc for 30 minutes.
Amplification mixture composed 7.3 µl of 10ҳPCR buffer, 8.o µl
of Mgcl2, 57 µl of ddH2o, and 1.0 µl Tag DNA polymerase at
30
concentration of 5units\µl. 73.3µl of the mixture added to each PCR tubes
.
The RT-PCR was performed in TECHNE, (TC-412, USA)
thermal cycler (Figure 2.3). Initial reverse transcription step was done at
94ºc for 2 minutes followed by 30 cycles of amplification .Each cycle
include denaturation at 94ºc for1 minute, annealing at 57ºc for 30 seconds
and extension at 72ºc for 45 seconds .Final extention at 72ºc for 5
minutes .
2.5. Agarose gel electrophoresis:
The amplified products of cDNA transcribted from viral RNA
molecules by RT-PCR were analyzed in agarose gel. The agarose gel
preparation, running buffer preparation and electrophresis were carrid out
as follows:
a) Preparation of 1 percent Aagarose gel:
One gram of agarose powder (molecular biology grade) was suspended in
100 ml of Tris-boric acid-EDTA (TBE) burffer. The suspension was
heated in microwave oven for few minutes until the agarose was
completely melted .When cooled down 30-40 ml of melted agarose was
poured in the gel try loaded with a comb.
b)Preparation of Tris-boric acid-EDTA:
107.81 gm 0.89 M Tris-(hydroxymethyl)-aminomethane .
55.03 gm 0.89 M Boric acid
31
7.44 gm 0.20 M EDTA 2H2O.
Up to 1 litre distilled water.
This mixture (10× TBE) was dissolved on magnetic stirrer then
another litre of distilled water was added tomprepare 5×buffer
concentration. The buffer was then kept at room temperature and used at
1× concentration for gel preparation and electrophoresis.
c) The molecular weight marker:
In each electrophoresis run, two microliters of a one hundred
base-pair ladder molecular weight marker (MW marker ) was placed in
the first lane.
d) Electophoresis:
The agarose gel was submerged in the buffer basin of the
electrophoresis apparatus (Figure 2.4) filled with 1×TBE containing
0.001% Ethidium bromide(50mg/ml). RT-PCR products with the MW
marker were lodded in the gel after being stained with an indicator dye
(Bromophenol blue). Constant electric current of 88 mV was then
switched on for about 45 minutes to allow the migration of the amplified
PCR products. The standard molecular weight marker (1Kb) was
incorporated in each reaction for determination of the size of the
amplified product by comparing this size with the separate
distinguishable bands of the MW marker which were seen when the gel
was stained with ethidium bromide.
32
The gel was then visualized under UV light using gel documentation
apparatus (Figure 2.5). Separate distinguishable bands were seen when
the gel was stained with ethidium bromide .
33
Figure 2.1: Confluent monolayer of vero cells
34
Figure 2.2: 80% cytopathic effect (CPE)
35
Figure 2.3: Thermal cycler (TECHNE, TC-412, USA)
36
Figure 2.4: Electrophoresis apparatus
37
Figure 2.5: Gel documentation apparatus
38
CHAPTER THREE
RESULTS
3.1. Detection of AHSV vaccine strains:
The RT-PCR-based assay described offered sensitive and specific
detection of all the nine AHSV vaccine strains used in this study.
The specific 240 bp PCR products was visualized on ethidium bromide-
stained agarose gel from 1pg RNAof AHSV vaccine strains (Figure 3.1).
3.2 Specificity of AHSV RT-PCR :
The specificity studies indicated that the specific 240 bp PCR product
was not detected from1ng RNA of bluetongue virus (BTV) and 1ng RNA
of Palyam virus (Figure 3.2).
3.3 Sensetivity of AHSV RT-PCR :
The sensitivity studies indicated that the specific 240 bp PCR product
was obtained from an amount of 10pg ,1.0pg ,100fg ,RNA from AHSV
serotype(6) (Figure 3.3).
39
Figure3.1: Detection of all the nine AHSV vaccine strains in
infected cell culture. Visualization of the 240 bp specific AHSV
PCR product on ethidium bromide-stained agarose gel from 1.0 pg
RNA of nine different AHSV vaccine strains. Lane MW:
molecular weight marker; Lanes 1-9: AHSV serotypes 1-9 of the
vaccine strains.
MW 1 2 3 4 5 6 7 8 9
500 bp
1000 bp
40
Figure 3.2: Specificity of the Polymerase Chain Reaction for AHSV
RNA. Amplification product was not detected from 1 ng of BTV RNA
and Palyam virus. Lane MW: molecular weight marker; lane 1: 1 pg
AHSV; lane 2: BTV and lane 3: Palyam virus.
MW 1 2 3
500 bp
1000 bp
41
Figure 3.3: Sensitivity of the Polymerase Chain Reaction for detection of
AHSV. Visualization of the 240 bp specific-AHSV PCR product on
ethedium bromide-stained agarose gel from 100 fg AHSV RNA. Lane
MW: molecular weight marker; lanes 1-6: (AHSV-6) 10 pg, 1.0 pg, 100
fg, 10 fg, 1.0 fg and 0.1 fg, respectively.
MW 1 2 3 4 5 6
500 bp
1000 bp
42
CHAPTER FOUR
DISCUSSION
Several serological methods to characterize AHSV such as serum
neutralization, haemagglutination inhibition, complement fixation, and
agar diffusion were used.Among these methods the neutralization test
was most type specific and thus as as most common used to determine
antibody level as well as serotyping (Hazrati et .al, 1965 b).
Aradaib et al., 1995 stated that serology is useful in
epidemiological studies to identify previous infection with AHSV. The
agar gel immunodiffusion (AGID) test which was used as a standard
serological test, but there were some draw- backs such as the cross-
reaction between related orbiviruses.
Monoclonal antibodies (MAbs) in competitive ELISA (cELISA)
technique were used to solve the problems associated with the use of the
AGID test (Laviada et .al, 1992), but this technique is suitable only to
blood and it takes at least 14 days for the production of anti-AHSV
antibodies by the susceptible host. (Fassi-Fihri et. al, 1998).
Virus isolation is time consuming and expensive.
Serogroup-specific and serotype-specific complementary DNA
(cDNA) probes derived from different AHSV genome segments have
43
been developed to solve those problems mentioned above. (Zientara et.
al, 1998).
With dot-blot hybridization, the minimum amount of AHSV
RNA these cDNA probes could detect was found to be in the nanogram
range.(Zientara et . al, 1998) .
This low sensitivity of recombinant DNA probes restricts their
application for direct detection of virus nucleic acid sequence present at
very low amounts in biological specimens. Now, for the detection and
differentiation of related orbiviruses including BTV and AHSV, the
polymerase chain reaction (PCR) has high sensitivity (Aradaib et .al,
2003).
In the present study, a reproducible, sensitive, and specific RT-
PCR technology was described for detection of nine AHSV serotypes
genomes in cell culture using primers derived from segment 3 of AHSV
serotype 6.
The described assay reproducibly and specifically detected AHSV RNA
in infected cell cultures. Selection of the primers was based on
observation that the genome segment 3-specific probe requires small
numbers of infected cells to produce appositive hybridization signal
compared to other nucleic acid probes used for detection of AHSV. The
specific 240 bp PCR products, visualized on ethidium bromide –stained
agarose gels, were obtained from all AHSV RNA samples tested. The
44
AHSV PCR assay was a simple procedure that efficiently detected all
AHSV strains under the stringent condition used in this study.
This assay is easier than other molecular biological techniques,
many of which are lengthy and cumbersome (Zientara et. al, 1995a.b).
The sensitivity studies indicated that this PCR protocol was
capable of detecting 100fg of total AHSV genomic dsRNA.
The specific 240 bp PCR product was not amplified from high
concentration of 1.0 ng of RNA from BTV serotype 10 under the same
strigency condition described in this study.
The primer annealing and extension, the enzyme and MgCl2
concentrations, the temperature and time for denaturation, and number of
cycles of three temperatures in each step were very important for keeping
sensitivity and specify of the PCR reaction. The time needed for
amplification was approximately 4 hours.
For detection of African horse sickness virus (AHSV) in splenic tissues
from infected horses, a single tube reverse transcription-polymerase chain
reaction (RT-PCR) method was described to amplify the 1179 bp
amplification product (the segment 7 which encodes for VP 7) (Zientara
et al., 2004).
The reverse transcription followed by the polymerase chain
reaction (RT-PCR) technique using specific primers for attenuated AHSV
45
serotype 4 segment 5 (NS1 gene) was used for detection of African horse
sickness virus (AHSV). Using this primer pair, no RT-PCR product was
detected from the RNA samples extracted from ten other orbiviruses
infected cells and their virions. . (Mizukoshi et al, 1994).
A single primer pair which amplified a 423-bp fragment of the
segment 8 gene which encodes the NS2 protein of AHSV, was used for
reverse transcription-PCR (RT-PCR) to detect African horse sickness
virus (AHSV) (Stone-Marschat et al, 1994).
(Koakemoer, et al., 2004) Describes the first one-day serotyping
procedure that requires only a single RT-PCR and hybridization and
which can identify multiple serotypes in mixed infections in one assay.
The same region of genome segment 2 of all nine AHSV serotypes was
amplified in a single RT-PCR. A universal primer set, designed to
amplify the 5'-terminal 521-553bp of genome segment 2 of all of the
1179 bp amplified product nine AHSV serotypes with one reaction, was
used to generate serotype-specific probes from dsRNA prepared from
infected tissue cultures or organ samples.
A coupled reverse transcriptase-polymerase chain reaction assay
(RT-PCR) was developed for the detection of African horse sickness
46
virus (AHSV). The primers which was selected used genome segment 7
and 10 as target templates for RT-PCR. Zientara et al,.1995 )
Sailleau C et al., 1997 used the reverse transcription-polymerase
chain reaction (RT-PCR) assay which was followed by dot-blot
hybridization for detection of African horse sickness virus (AHSV); the
primers employed amplified the S7 gene that encodes the VP7 protein.
The RT-PCR assay was compared with virus isolation for detecting
AHSV in blood samples form horses experimentally infected with
AHSV-4 and AHSV 9.
The AHSV RT-PCR assay described using primers derived from
segment 3 of AHSV serotype 6 to detect AHSV RNA in infected cell
culture .The specific 890 bp PCR product was visualized on ethidium
bromide-stained agarose gel .(Aradaib et al ., 2006).
In conclusion, the described AHSV RT-PCR assay, using
primers derived from genome segment 3 of AHSV serotype 6, provides a
simple, rapid, sensitive and diagnostic method for detection of AHSV.
47
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