DETECTION OF ORTHOPOXVIRUS ANTIBODIES IN SERA OF SOME DOMESTIC SPECIES IN THE SUDAN

BY Siham Tag Elsir Karam Alla Alkidir

(B.V.M.2002, University of Khartoum)

Athesis submitted to the University of Khartoum in

fulfillment of requirement for the degree of Master of Veterinary Medicine by Research

Department of Microbiology Faculty of Veterinary Medicine

University of Khartoum

(June 2008)

DEDICATION

To my mother, father

Husband, daughters,

Sisters, brothers

And friends

With deepest love

Acknowledgments

Praise to Allah who gave me the ability and health to accomplish,

this research work.

My sincere thanks and gratitude to my supervisor Prof.

Abdelmalik Ibrahim Khalafalla for his guidance, support and

encouragement throughout this work.

I am also indebted to Dr.Tag Elsir Mohamed, Central Veterinary

Research Laboratory in Soba for his help and provision of some essential

materials. My deep thanks to my colleague Dr. Afraa Tagelsir for her help

and support. I am extremely grateful to the staff of the Department of

Microbiology, Faculty of Veterinary Medicine, University of Khartoum,

for providing facilities to conduct this work, special thanks to Mr.Sharani

Omer for his technical assistance during the laboratory work, And to Miss.

Mawahib Awad for her assistance during this study. My gratitude is also

extended to all staff of the Department of Preventive Medicine, namely Dr.

Kitma Hassan Elmalek, Dr. Abdelwahid Saeed for their support, and Mr.

Hussein Abdelraheem and Miss. Zainab Altayeb for their assistance. Also I

want to thank Prof. Ahmed Gameel, Department of pathology, for his Help

in histopathology.

TABLE OF CONTENT

DEDECATION .................................................................................... i

ACKNOWLEDGMENT ..................................................................... ii

TABLE OF CONTENT ..................................................................... iv

LIST OF FIGUERS.......................................................................... viii

LIST OF TABLES ............................................................................. ix

LIST OF ABBREVIATIONS ............................................................. x

ARABIC ABESTRACT .................................................................... xi

ENGLISH ABESTRACT ................................................................. xii

INTRODUCTION............................................................................... 1

CHAPTERI: LITERATURE REVIEW .............................................. 3

1.1 Diseases caused by viruses of the genus orthopoxvirus ................ 3

1.2 Camel pox ..................................................................................... 3

1.2.1 Etiology of CP ............................................................................ 3

1.2.2 Importance of the disease ........................................................... 4

1.2.3 Epidemiology of CP ................................................................... 4

1.2.4 Clinical symptoms ...................................................................... 6

1.2.5 Diagnosis of CP.......................................................................... 7

1.3 Cowpox ......................................................................................... 8

1.3.1 Cause of cow pox ....................................................................... 8

1.3.2 Occurrence ................................................................................. 8

1.3.3 Transmission .............................................................................. 8

1.3.4 Clinical & Pathologic Features................................................... 9

1.3.5 Diagnosis.................................................................................... 9

1.3.6 Prevention ................................................................................ 10

1.3.7 Public health significance......................................................... 10

1.4 Poxviridae ................................................................................... 10

1.5 Taxonomic structure of the family .............................................. 10

1.6 Orthopoxvirus ............................................................................ 11

1.7 Classification............................................................................... 12

1.8 Morphology................................................................................. 13

1.9 Physiochemical and physical properties...................................... 14

1.9. 1 Nucleic acid............................................................................. 14

1.9.2 Lipids ....................................................................................... 14

1.10 Diagnosis of Orthopoxvirus ...................................................... 14

1.11 Vaccinia virus............................................................................ 16

1.12 Basic biology............................................................................. 16

1.13 Host resistance........................................................................... 17

1.14 Origin ........................................................................................ 18

1.15 Use as a vaccine ........................................................................ 18

1.16 History....................................................................................... 19

CHAPTER II: MATERIALS AND METHODS .............................. 20

2.1 Virus strain .................................................................................. 20

2.2 Hyper immune serum .................................................................. 20

2.3 Serum samples ............................................................................ 20

2.4 Preparation and sterilization of glassware ................................... 20

2.5 Preparation of solution and cell culture media ............................ 21

2.5.1 Phosphate buffer saline (PBS) and phosphate diluent (PD) ..... 21

2.5.2 Glasgow Minimum Essential Medium (GMEM) ..................... 21

2.5.3 Tryptose phosphate broth (TPB) .............................................. 21

2.5.4 Lactalbumin hydrolysate .......................................................... 21

2.5.5 1%Yeast extract solution.......................................................... 21

2.5.6 7.5%Sodium bicarbonate solution ............................................ 21

2.5.7 Thioglycolate medium.............................................................. 22

2.5.8 Trypsin-Versene solution ......................................................... 22

2.5.9 Penicillin-Streptomycin solution .............................................. 22

2.5.10 Fungizon solution................................................................... 22

2.6 Embryonated Chicken Eggs ........................................................ 22

2.6.1 Chorioallantoic Membrane Inoculation.................................... 23

2.6.2 Harvest of Chorioallantoic Membrane ..................................... 23

2.7 Pock lesion histopathology.......................................................... 24

2.8 Preparation of Cell Culture.......................................................... 24

2.9 Virus propagation........................................................................ 25

2.10 Virus titration ............................................................................ 25

2.11 Agar Gel preparation ................................................................. 25

2.12 Agar gel immunodiffusion test .................................................. 25

2.12.1 Preparation of agar gel ........................................................... 25

2.12.2 Standardization of AGID test ................................................. 25

2.12.3 Examination of the sera for antibodies against Orthopox viruses

using AGID test……………… …. ………………………………...26

CHAPTER III: RESULT .................................................................. 27

3.1Growth of Vaccinia virus on the chorioallantoic membrane ........ 27

3.2 Histopathology of the CAM0 ...................................................... 27

3.3 Growth of vaccinia virus in Cell Culture..................................... 27

3.4 Sero-prevalence of orthopox virus in Sheep, cattle and camel

serum samples ............................................................................. 27

CHAPTER IV: DISCUSSION.......................................................... 35

CONCLUSIONS............................................................................... 40

REFERENCES.................................................................................. 41

APPENDICES................................................................................... 52

LIST OF FIGURES

Figure 1 Pock lesions produced by Vaccinia virus on CAM of

embryonated eggs................................................................................ .31

Figure 2 CAM section showing swollen of the cells with pale cytoplasm

and some inclusions bodies……………………………………………32

Figure 3 CAM section showing foci of epithelial thickening…….…...33

Figure 4 Vero cell culture infected with Vaccinia Virus showing

rounding and plaque formation ………………………………………34

Figure 5 Precipitation line in AGID test for detection of antibodies

against orthopox viruses……….………………………………..…….35

LIST OF TABLES

Table 1 Testing sera collected from cattle, sheep and camels For antibodies against orthopox virus antibodies by AGID……………………………………………………………………29 Table 2 Testing sera collected from different areas in Khartoum for antibodies against orthopoxvirus antibodies by AGID……………………………………………………………………30

ABBREVIATIONS

AGID: Agar gel immuno diffusion test.

CAM: Chorioallantoic membrane.

CPE: Cytopathic effect.

CPV: Camel pox virus.

DNA: Deoxyribonucleic acid.

DDW: Deionized distilled water.

ELISA: Enzyme-linked immunosorbent assay.

GMEM: Glasgow minimum essential medium.

H&E: Haematoxillin and Eosin.

OPV: Orthopoxvirus.

PBS: Phosphate buffer saline.

PC R: Polymerase chain reaction.

PDB: Phosphate diluent buffer.

TPB: Tryptose phosphate broth.

VV: Vaccinia virus.

ملخص االطروحه

هدفت هذه الدراسه لمعرفة بعض الخصائص الحيويه لفيروس الفاآسينيا في جنين

آما هدفت ايضا لدراسة وبائية فيروس الجدري الحقيقي .البيض النامي والزرع الخلوي

.في االبقار والضان والجمال في السودان آأول دراسه من هذا النوع ) اورثوبوآس(

من فيروس الفاآسينيا وتم اآثارها في الغشاء عترهاستعملت في هذه الدراسه

واضحه تاخذ اللون االبيض المعتم فاعطت افات المشيمي اللقانقي لجنين البيض النامي

ايضا اخذت عينات من هذه االفات واجرى عليها اختبار . ملم0.2-0.1ويتراوح حجمها بين

انتفاخ بعض الخاليا ذات سيتوبالزم , واوضحت وجود تكاثر بؤري للخاليا االنسجه المريضه

.وجود اجسام اشتماليه قاعديه وارتشاح خاليا وحيده االنويه,شاحب

واعطت اثرا مرضيا خلويا يتمثل ) آلي القرد االفريقي( فيرو ايضا تم اآثار الفيروس في خاليا

.قنينة الزرع الخلوي الخاليا عن سطح لطعات وانفكاك في استدارة الخاليا وتكوين

استخدم في هذه الدراسه اختبار االنتشار المناعي في الجل لتحديد وجود اجسام مضاده لفيروسات

من السوداناالورثوبوآس في عينات مصل جمعت من ابقار وضان وجمال من مناطق مختلفه

. ل قياسىآمص_ الفاآسينيا حقنت بفيروس _ واستخدم مصل ممنع من ارانب

اوضحت الدراسه وجود اجسام مضاده لفيروسات االورثوبوآس بنسب متفاوته في هذه

وآانت في االبل77.9% وفي الضان 35.7% حيث آانت النسبه في االبقار الحيوانات

بصوره ) اوثوبوآس( وذلك يعني انتشار االجسام المضاده لفيروس الجدري الحقيقي 53.5%

.والضان والجمال في السودان واسعه في االبقار

Abstract

This study aimed to know the biological properties of Vaccinia

virus in the Chorioallantoic membrane of the chick embryo and cell

culture. Also to study the epidemiology of orthopox virus in sheep, cattle

and camels in the Sudan as the first study from this type.

Vaccinia virus strain is used in this study for propagation in

the Chorioallantoic Membrance (CAM) of embryonated eggs. Lesions

were seen as small pock ranging from 0.1-0.2 mm in diameter, round,

opaque and white in colour. The histopathology of these lesions showed

foci of epithelial thickening, swollen cell with pale cytoplasm,

eosinophilic inclusion bodies and infiltration of mononuclear cells.

When propagated in Vero cells, this virus gave clear Cytopathic

effect characterized by rounding of the cells, plaque formation, syncytia

and detachment of cells from flask surface.

Agar Gel Immuno Diffusion Test (AGID) was used in this study

to detect orthopoxviruses antibodies in serum samples collected from

cows, sheep and camels from different areas of Sudan.

The study revealed that orthopoxvirus antibodies are present in

these animals with varying range. The seroprevalence was 35.7% in

cattle, 77.9% in sheep and 53.5 % in camel. That means antibodies

against orthopox viruses are widely distributed in sheep, cattle and camel

in the Sudan.

INTRODUCTION

The orthopoxvirus genus encompasses eight members of the

Poxviridae family of viruses, Orthopoxviruses is characterized by large

brick-shaped virus particles, containing a double-stranded DNA genome of

approximately 200,000 bp, from which vaccinia virus (VV) is the prototypic

virus. Vaccinia virus shares with its closely related virus cowpox virus the

capacity to infect a wide range of hosts, among them humans, cows, rodents

and zoo animals (Moss, 1996).

While humans are the only natural host for variola virus, both vaccinia

virus and cowpox virus have a much broader host spectrum, and the natural

reservoir for cowpox virus is most likely the rodent (Marennikov et al.,

1977).

Orthopoxviruses were pathogenic for animals and man like cowpox

and monkeypox virus (Baxby, 1988). Cowpox viruses are of interest as they

have been identified more frequently during the last three decades (Bennett

et al., 1989). Several outbreaks of severe generalized poxvirus infection in a

variety of zoo animals have been shown to be caused by these agents

(Marennikov et al., 1977). Since cowpox virus has a wide host range,

sporadic cases occur in many other mammals. In particular infection of

domestic cats is an increasingly recognized condition and several reports

documented the transmission of virus from cats to man (Eis-Hubinger et al.,

1990, Egberink et al., 1986, Czerny et al., 1991). From another member of

the genus Orthopoxvirus, camelpox virus, caused severe generalized

infections especially in young camels were reported. However, transmission

to man has not been described so far (Jezek et al., 1983).

The ability of vaccinia virus (VV), the virus used for immunization

against smallpox and the best-studied laboratory model for poxvirus biology

and immunity, to infect almost any cell line in culture has resulted in

descriptions of its broad cellular tropism and presumed widespread

expression of a virus binding receptor (Moss, 2001 and Stuart, 2004).

However, certain biological properties of vaccinia virus are not studied yet.

In this work we studied the histopathology of pock lesion made by vaccinia

virus in the chorioallantoic membrance and beside that the infection by

orthopoxviruses in cattle, sheep and camel as measured by seroconversion in

the Sudan is not determined.

Objectives of the study:

1- To determine biological properties of Vaccinia virus in particular the

growth characteristic in Chorioallantoic membrane (CAM) of embryonated

egg and cell culture.

2- To determine the prevalence of antibodies against orthopoxvirus by Agar

Gel Immunodiffusion Test (AGID) test in cattle, sheep and camel in the

Sudan.

CHAPTER 1

LETERATURE REVIEW

1.1 Diseases caused by viruses of the genus orthopoxvirus

The genus orthopoxvirus within the family Poxviridae consists of

Several species causing diseases in a wide range of animal species and in

humans (Fenner, 1996). Member of the genus Orthopoxvirus such as

camelpox, cowpox and monckeypox are pathogenic for animals and Man

(Baxby, 1988, Fenner et al., 1989).

1. 2 Camel pox

Camel pox (CP) is a systemic disease. Atypical pox exanthema

appears over the entire body and on the head; in particular Camel pox causes

a severe generalized disease in camel, with extensive skin lesion (Murphy,

Gibbs, Marian, Horzinek and Studdert, 1999). Munz, et al., (1990) described

CP as a species-specific highly contagious disease of camel characterized by

pox lesion that causes a severe out break in young animals with mortality

rate up to 10%.

1.2.1 Etiology of CP

Camel pox is caused by a virus which has been classified under the

orthopoxvirus genus of family Poxviridae (Mahnel, 1974, Meyer,

Osterrieder and Pfeffer, 1993 and Wernery and Kaaden, 2002).

The virus has the characteristic and properties of a true poxvirus and

is closely related to the vaccinia-variola group (Mahnel and Bartenbach,

1973). Andrews, Pereira and Wildey (1978) considered the virus to be a

member of the orthopoxvirus group with resemblance to the smallpox virus.

1.2.2 Importance of the disease

Camels are important live stock resource adapted to hot and cold

environment. They have been utilized by man for meat, milk, wool, hides

and transportation. Sudan is the second most density camel populated

country in the world after Somalia (Schwarz and Dioli, 1992).

The mortality in infected herds range from 25% to 100% in young animals,

and from 5% to25% in older animals (Ramyar and Hessami, 1972; Kriz,

1982). The presence of CP in Sudan was first reported in 1953 (Anon,

1954). However, identification of its causative agent has not been made yet

(Shommein and Osman, 1987).

1.2.3 Epidemiology of CP

Camels may become infected with poxvirus through small abrasions

of the skin, by aerosol infection of respiratory tract or by mechanical

transmission through biting arthropods. Several scientists have reported an

increase in CP outbreaks during wet season (Munz, 1992; Wernery, Meyer

and Pfeffer, 1997 and Wernery, Kaaden and Ali, 1997) when the diseases

become more severe. During the dry season, it usually causes a milder

course (Pfahler and Munz, 1989).

Since the CPV has been isolated from the camel tick Hyalomma

dromedarii, it is generally believed that a larger arthropod population builds

up during rainy seasons, forcing a greater virus pressure and virus doses onto

the camel populations (Wernery and Kaaden, 2002). Camels aging 2-4 years

often develop the localized form, with lesion on skin and mucous

membranes of lips and nose. Young camels up to one year old and female

camels in the final month of pregnancy are affected mainly by the

generalized form (Khalafalla and Mohamed, 1998). In two principal camel-

rearing areas of Kenya, the disease was found in Turkana where outbreaks

were detected in two herds of young animals, while in Samburu, outbreaks

were found in two herds of adult animals, as well as in two herds of young

camels. In all cases, there was 100% morbidity in the affected herds. When

the young camels were involved, the main lesions were confind to the

mouth, nose and muzzle as distinct pustular lesions. In adult animals, there

was also extensive odema of the head and neck (Gitao, 1997).

According to Munz (1992) the high prevalence of antibodies against

CPV in camel sera and the occurrence of clinical outbreak in Kenya,

Somalia and Sudan indicate that the disease may be enzootic or sometimes

epizootic in these countries. In four areas of the Sudan Kalafalla, Mohamed

and Agab (1998) found that the prevalence of seropositive animals was

higher in adults more than 4years old (87%) than in calves less than 1 years

old (40%) and than in young animals of 1-4 years old (75%) and the

prevalence rates were higher in female camels (76.5%) than in males

(66.7%).

1.2.4 Clinical symptoms

The incubation period ranges from 9-13 days, pustules develop on the

nostrils and eyelids as well as on the oral and nasal mucosa in mild cases,

presenting with generalized clinical signs such as fever, lassitude, diarrhea,

and anorexia; the eruptions are distributed over the entire body ( Werney and

Kaaden, 2002). Abortion of female camels has been reported (Brisovich and

Orekhov, 1966; Buchnev and Sadykou, 1969; Munz, 1992). Mortality can

reach 28% in generalized forms of the disease (Jezek, Kriz and Rothbauer,

1983). Secondary bacterial and mycotic infections can complicate the course

of the disease (Werney and Kaaden, 2002).

Pox-lesions were also observed in the trachea and lungs of young

dromedaries (Werney and Kaaden, 1995; Kinne, Cooper and Werney, 1998).

Classical lesions in the skin start as erythematous macules, which develop

into papules and vesicles. Vesicles develop into pustules with depressed

centers and raised erythematous borders the so called pock. After the

pustules have ruptured, they become covered by crusts. Healing of pustules

might take 4-6 weeks with or without scars (Werney and Kaaden, 2002).

Associated lymph nodes are often swollen (Munz, 1992). Mammary glands,

genitalia and anal areas are also frequently affected (Kriz, 1982) Lesions

also develop on the mucous membranes of the oral cavity resulting in

difficulty in eating and consequent loss of condition (Munz, 1992).

1.2.5 Diagnosis of CP

Preliminary diagnosis of CP is based on clinical epizootiological and

pathological findings (Buchnev et al, 1987). Confirmatory diagnosis may be

accomplished by electron microscopic detection of orthopoxvirus particles

in pox lesions, cultivation of the virus in tissue culture cells or in the

chorioallantoic membrane (CAM) of embryonated chicken eggs as well as

by serological tests (Munz, 1992).

The systematization and laboratory differentiation is of great

importance in demarcating the orthopoxvirus from the Para poxvirus, as both

viruses can be found in the same camel (Wernery and Kaaden, 1995). Newer

diagnostic methods include the ELISA technique with monoclonal

antibodies, DNA restriction enzyme analysis (Munz et al, 1992) and a dot

blot assay digoxgenin-labeled DNA probes (Meyer et al, 1993). Czerny,

Meyer and Mahnel, 1989; Johann and Czerny, 1993 and Pfeffer, Wernery,

Kaaden and Meyer, 1998) have described various laboratory methods for the

diagnosis of CP. This include electron microscopy, ELISA, immuno-

histochemistry and polymerase chain reaction (PCR). CPV-antigen detection

by immuno-histochemistry is a new method for the diagnosis of CP, which

can easily be performed in laboratories not possessing an electron

microscope. In addition to the diagnosis, immuno-histochemistry is of

particular interest for histopathologists because it facilitates visualization of

the morphological changes induced by the poxvirus (Wernery and Kaaden,

2002). Kalafalla and Mohamed (1998) identified CPV using virus

neutralization, agar gel diffusion, immunofluorecent tests and

histopathological pictures of the skin lesions. Ropp, Jin, Knight, Massung

and Esposito, (1995) developed a PCR strategy to differentiate between

orthopoxvirus species including CPV. They have successfully used this

strategy to identify virus DNA in clinical material, infected cell culture and

CAMs. Meyer, Pfeffer and Rziha, (1994) established PCR and restriction

enzyme protocols for detection and differentiation of species of the genus

orthopoxvirus.

1.3 Cowpox

1.3.1 Cause of cow pox disease

Cowpox is caused by the cowpox virus, a member of the

orthopoxvirus genus of the family poxviridae, which also includes smallpox

and vaccinia.

Cowpox virus is complex double-stranded DNA virus that have

the Potential capacity of encoding more than 200 gene products along

their ~200 kb linear genomes. Their replication cycles occur entirely

within the cytoplasmic compartment of infected host cells (Moss, 1996).

1.3.2 Occurrence

Additional hosts of cowpox virus are human beings and various

animals, including large zoo cats, domestic cats, anteaters, and rodents. The

latter are considered the natural reservoir hosts (Marennikov et al., 1974).

1.3.3 Transmission

Milkers and milking machines are the main means of spread of the

virus. Insects may also serve as mechanical vectors for the virus.

1.3.4 Clinical & Pathologic Features

Cowpox virus produces what is usually a benign infection of the

udder and teats. Papules are first seen, followed by vesicles, which rupture

leading to scab formation. Scabs drop off in about two weeks. Decrease in

milk production result from the soreness of affected teats and also from

secondary bacterial infection, which may complicate the disease and

contribute to development of mastitis (Carter et al., 2005).

1.3.5 Diagnosis

Clinical specimens: Vesicular fluid, scabs, and scrapings from lesions.

It is difficult clinically to distinguish cowpox from pseudo cowpox and other

infections of the teats. Diagnosis is most easily confirmed by the

examination of distilled water lysates of lesion material by electron

microscopy. Orthopoxviruses are "brick-shaped" as opposed to the virions of

pseudo cowpox (a Para poxvirus), which are ovoid in appearance.

Cowpox virus can be grown in cell cultures of bovine and human

origin, and on the chorioallantoic (CAM) membrane of chicken embryos.

The latter method of cultivation also provides a means to differentiate

cowpox virus from pseudo cowpox virus, which does not grow on the CA M

membrane. Vaccinia virus produces smaller pocks on the CAM membrane

than does cow pox. (Carter et al., 2005).

1.3.6 Prevention

Vaccination is not practiced. Prevention is best accomplished by

sound milking practices. Milkers and milking machines can spread the virus.

1.3.7 Public Health Significance

Milkers may contract the infection from cows. The human infection

usually involves a single, benign lesion on the hand or face. Serious

systemic disease has been reported in immunosuppressed individuals.

(Carter et al., 2005)

1.4 Poxviridae

Poxviruses are large double-stranded DNA viruses with genomes

ranging from 130 to 380 kbp (Moss, 2001). Pox viruses are complex viruses

that replicate in the cytoplasm and encode many enzymes and

immunodulatory protein (Moss, 1996). The serological relationship between

several members of the poxvirus group were investigated by a variety of

techniques, including complement fixation ,gel diffusion and ring

precipitation,heamaglutinin inhibition pock and plague, neutralization, and

staining with fluorescent-coupled antibody.(Gwendo et al.,1961) .

1.5 Taxonomic Structure of the Family

Subfamily 00.058.1. Chordopoxvirinae

Genera

00.058.1.01.Orthopoxvirus

00.058.1.02.Parapoxvirus

00.058.1.03.Avipoxvirus

00.058.1.04.Capripoxvirus

00.058.1.05.Leporipoxvirus

00.058.1.06.Suipoxvirus

00.058.1.07.Molluscipoxvirus

00.076.0.01. Trichovirus.

By ICTV –International Committee on Taxonomy of Viruses (2006).

1.6 Orthopoxvirus

Orthopoxviruses are DNA viruses and can be identified by EM. They

are large and brick shaped virions of 220-450 nm long by 140-260 nm wide,

presenting an irregularly structured surface pattern .The orthopoxvirus

genome prototype comprises a160-220 kbp linear duplex DNA with a

variable- sized invated terminal repetition (ITR) that contains distally ,

ahypovariable array of tandemly repeated sequences adjacent to covalently

closed is omerized “ hairpin’’ ends (Hollowczak, 1982, Wittek, 1982,

McFadden and Pales, 1982, Pales and Pogo, 1981, Baroudy et al., 1982a,b

,1983). Previous comparisons of genome endonuclease cleavage

electrophores patterns and maps of different orthopoxvirus indicated that

there was considerable middle region DNA conservation within the genus

and that species ,strains and variant specific differences were represented

mainly as variations in terminal region (conserved and unconserved)

nucleotide sequences and DNA lengths (Wittek et al ., 1977, Muller et al.,

1978,Espoito et al., 1978, 1981b, Mackett and Archard ,1979, Archard and

Machett, 1979, Dumbell and Archard,1980., Moyer et al., 1980b,

Schumperli et al., 1980). Orthopoxviruses exhibit extensive serologic cross-

reactivity and nucleic acid homology (Woodroofe and Fenner 1962, Baxby,

1975, 1977, Esposito et al, Moss, 1978, Mackett and Archard, 1979).

Biological and serological techniques have been used to show that the

orthopox viruses are closely related (Andrewes and Pereira, 1978). Members

of the orthopoxvirus genus share common antigens, although they differ

biologically from one to another.

1.7 Classification

Orthopoxvirus classified by International Committee of Taxonomy of

Viruses (ICTV) in the subfamily Chordoviridae of the poxviridae family.

Orthopoxvirus

Virus classification

Group: Group I (dsDNA)

Family: Poxviridae

Genus: Orthopoxvirus

Species:

Buffalopoxvirus

Camelpoxvirus

Cowpoxvirus

Ectromeliavirus

Monkeypoxvirus

Rabbitpoxvirus

Raccoonpoxvirus

Sealpoxvirus

Skunkpoxvirus

Taterapoxvirus

UasinGishudiseasevirus

Vacciniavirus

Variolavirus

Volepox

1.8 Morphology

Virions consist of an envelope, a surface membrane, a core, and

lateral bodies, or a surface membrane, a core, and lateral bodies. Virions

have a buoyant density in CsCl of 1.23-1.27 g cm-3.During their life cycle,

virions produce extra cellular particles and produce intracellular particles;

can occur in two phenotypes; may be enveloped during their extra cellular

phase. The infection is initiated by extra cellular virions. Virus may be

sequestered within inclusion bodies that are not occluded and typically

contain one nucleocapsid. Virus capsid is enveloped and virions mature

naturally by budding through the membrane of the host cell. Virions are

generally brick-shaped, or pleomorphic and measure 200 nm in diameter;

250-300 nm in length; 250 nm in height displaying tubular units. The core is

biconcave with two lateral bodies nested between the core membranes, or

between the surface membranes.

1.9 Physicochemical and Physical Properties

1.9.1 Nucleic Acid

The genome is not segmented and contains a single molecule of linear

double-stranded DNA. The complete genome is 185000 nucleotides long.

The genome has a guanine + cytosine content of 36 %. The genome

sequence has termini with cross-linked hairpin ends (i.e. single-stranded

loopes thus forming one continuous polynucleotide chain). The genome has

terminally redundant sequences. The terminally redundant sequences have

reiterated inverted terminal sequences which are tandemly repeated. The

genome sequence is repeated at both ends. Double-stranded DNA is

covalently. Double-stranded DNA is linked at both ends.

1.9.2 Lipids

Lipids are present and located in the envelope. Virions are composed

of 4% lipids by weight. The composition of viral lipids and host cell

membranes are similar. The lipids are host derived and synthesized de novo

(during the early phase of virus replication) and are derived from plasma

membranes. Viral membranes include glycolipids. (ICTV management)

1.10 Diagnosis of Orthopoxvirus

Biologic and antigenic properties are often useful for identifying and

differentiating orthopoxviruses (OPV). However, polymerase chain reaction

(PCR) amplification, with either restriction cleavage or sequencing of

amplicons, has been gaining credibility as a more rapid, specific, sensitive,

and often cost-saving technique for research and diagnostic laboratories

( Meyer et al., 2004).

Other diagnostic methods have included differentiation by viral

protein-profile following separation by sodium dodecyl sulfate-

polyacrylamide gel electrophoresis and by various serologic assays ( Arita et

al., 1977, Obijeski et al., 1973). All of these methods required virus isolation

and propagation, and clear-cut differentiation was often difficult. Therefore,

a test that can directly detect OPV in clinical specimens is desirable. More

recently, PCR methods have been developed which eliminate the need to

propagate virus (Knight et al 1995, Meyer et al., 1994, Meyer et al., 1997,

Ropp et al, 1995). But they were not suitable for strain differentiation. OPV

strain differentiation is important for several practical applications including

epidemiological investigations. OPV PCR tests were developed based on the

determination of complete DNA sequences for VAC (Goebel et al., 1990,

Johnson et al, 1993) and VAR (Massung et al., 1993) and the availability of

partial genomic sequences from other OPVs. These sequence data have

shown that a large central genomic region is highly conserved among OPV

isolates, which explains the significant degree of cross-reactivity in various

tests (Fenner, F.1996, Fenner et al., 1988). However, the data also revealed

that genes within the terminal regions have both conserved and variable

segments, and several of these genes have been associated with host range,

tissue tropism, and/or virulence of different OPV species and strains (Goebel

et al., 1990, Massung et al., 1993, Massung et al., 1996). Thus, the species-

specific genes make these terminal variable regions ideal targets for use in

PCR-based diagnostics.

1.11 Vaccinia

Vaccinia virus (VACV or VV) is a large, complex enveloped virus

belonging to the poxvirus family ( Ryan et al (2004). It has a linear double-

stranded DNA genome, which is approximately 190 kbp in length and

encodes for approximately 250 genes. The dimensions of the virion are

roughly 360 × 270 × 250 nm. Vaccinia virus is well-known for its role as a

vaccine that eradicated the smallpox disease, making it the first human

disease to be successfully eradicated by mankind. This endeavour was

carried out by the World Health Organization under the Smallpox

Eradication Program. Post eradication of smallpox, scientists have been

studying vaccinia virus to use as a tool for delivering genes into biological

tissues (gene therapy and genetic engineering). Moreover, due to recent

concerns about smallpox resurfacing as a possible agent for bioterrorism,

scientists have renewed their interests in studying vaccinia virus. .(Smith et

al., 2002).

1.12 Basic biology

Vaccinia virus is unique amongst all DNA viruses because it

replicates only in the cytoplasm of the host cell outside of the nucleus.

(Tolonen et al., 2001). Therefore, the large genome is required for encoding

various enzymes and proteins involved in viral DNA replication and gene

transcription. During its replication cycle, VV produces several infectious

forms which differ in their outer membranes: intracellular mature virion

(IMV), the intracellular enveloped virion (IEV), the cell-associated

enveloped virion (CEV) and the extra cellular enveloped virion

(EEV)(Smith et al., 2002). Although the issue remains contentious, the

prevailing view is that the IMV consists of a single lipoprotein membrane,

while the CEV and EEV are both surrounded by two membrane layers and

the IEV has three envelopes. The IMV is the most abundant infectious form

and is thought to be responsible for spread between hosts. On the other hand,

the CEV is believed to play a role in cell-to-cell spread and the EEV is

thought to be important for long range dissemination within the host

organism (Smith and Law, 2004).

Maps and sequences of vaccinia (WR) virus and cow pox (Brighton)

virus donid DNA Tandem array regions have revealed as similar core

sequence constituling the repeated units in these species (Wittek and Moss,

1980, Baroudy et al., 1982a, b, 1983, Pick up etal, 1982). Sequence analysis

of vaccinia virus DNA tips (telomeres) showed two equirmolor isomeric in

averted complementary (Flip-Flop) forms of hair pin loops (Baroudy et al.,

1982a, 1983, pick up et al., 1983)

1.13 Host resistance

Vaccinia contains within its genome several proteins that give the

virus resistance to interferon. K3L is a protein with homology towards the

protein eIF-2alpha. K3L protein inhibits the action of PKR, an activator of

interferon. E3L is another protein encoded by vaccinia. E3L also inhibits

PKR activation; and is also able to bind to double stranded RNA. (Davies

MV, 1993)

1.14 Origin

Vaccinia virus is closely related to the virus that causes cowpox.

Historically the two were often considered to be one and the same

(Huygelen C, 1996). The precise origin of vaccinia virus is unknown due to

the lack of record-tracking as the virus was repeatedly cultivated and

passaged in research laboratories for many decades (Henderson DA, Moss

B, 1999). The most common notion is that vaccinia virus, cowpox virus and

variola virus, the causative agent for smallpox, were all derived from a

common ancestral virus. There is also speculation that vaccinia virus was

originally isolated from horses (Huygelen C, 1996).

1.15 Use as a vaccine

Avaccinia virus infection is very mild and is typically asymptomatic

in healthy individuals, but it may cause a mild rash and fever. Immune

response generated from a vaccinia virus infection protects the person

against a lethal smallpox infection. For this reason, vaccinia virus was, and

is still being used as a live-virus vaccine against smallpox. Unlike vaccines

that use weakened forms of the virus being vaccinated against, the vaccinia

virus vaccine cannot cause smallpox because it does not contain the

smallpox virus. However, certain complications and/or vaccine adverse

effects occasionally arise. The chance of this happening is significantly

increased in people who are immunocompromised. Approximately one in

one million individuals will develop a fatal response to the vaccination.

Currently, the vaccine is only administered to health care workers or

research personnel who have a high risk of contracting vaccinia virus, and to

the military personnel of the United States of America. Due to the present

threat of smallpox-related bioterrorism, there is a possibility the vaccine may

have to be widely administered again in the future. Therefore, scientists are

currently developing novel vaccine strategies against smallpox which are

safer and much faster to deploy during a bioterrorism event.(from

Wikipedia).

1.16 History

The original vaccine for smallpox, and the origin of the idea of

vaccination, was cowpox, reported on by Edward Jenner in 1798 (Henderson

DA, Moss B, 1999). The Latin term used for cowpox was variolae

vaccinaeX, essentially a direct translation of "cow-related pox". That term

lent its name to the whole idea of vaccination. When it was realized that the

virus used in smallpox vaccination was not, or was no longer, the same as

the cowpox virus, the name 'vaccinia' stayed with the vaccine-related virus.

Chapter II

Materials and Methods

2.1 Virus strain

Vaccinia virus (v.v) (Elstree strain) was obtained from the virus stock

of the Virology Laboratory, Department of Microbiology, Faculty of

Veterinary Medicine, University of Khartoum.

2.2 Hyper immune serum preparations

This was prepared in rabbits using Vaccinia virus. Four rabbits with

the same age inoculated with vaccinia virus. Then we collect the serum after

10 days, 20 days and 30 days.

2.3 Serum samples

305 serum samples were collected randomly from cattle and sheep

from Khartoum state, and 101 samples were collected from camels from

Gadarif and Tambool.

2.4 Preparation and Sterilization of Glassware

Flasks, beakers, bijou, and volumetric bottles, measuring cylinders,

tissue culture bottles, tubes and other glassware were rinsed in running tap

water, brushed with soap and then rinsed several times in tap and distilled

water (D.W).The clean dry glassware were sterilized in the hot –air oven at

160c° for 1hr.Volumetric glass pipettes were soaked overnight in potassium

dichromate, then, they were washed several times in (D.W).

2.5 Preparation of Solution and cell culture Media (see appendix 1).

2.5.1 Preparation:

Solutions a, b and c were autoclaved separately at 121 °c for 10

minutes and left to cool. To prepare working solution of PBS (Ix) solutions a

and b were mixed. Solution c was then added and the final volume was

brought to 2 liters with sterile DDW. To prepare PD (Ix) solution a was

made up to 2 liters with sterile DDW.

2.5.2 Glasgow Minimum Essential Medium (GMEM)

A- Stock Solution (5x)

The entire content (125.7g) of one bottle of powdered media (Sigma)

was dissolved in 2 liter of DDW. The solution was immediately filtered

through a Millipore filter (o.22 u) under positive pressure, tested for sterility

using thioglycolate media and store at 4c°.

B- Outgrowth and Maintenance Media (see appendix 2)

2.5.3 Tryptose Phosphate Broth (TPB)

Three grams of TPB powder were dissolved in 100 ml DDW,

sterilized by autoclaving at 121c° for 10 min and stored at 4c°.

2.5.4 Lactalbumin hydrolysate

Five grams of lactalbumin powder were dissolved in 100 ml of DDW,

sterilized by autoclaving at 121c for 10 min and stored at 4c°.

2.5.5 1% Yeast extracts solution

One gram yeast extract powder was dissolved in 100ml of DDW,

autoclaved at 121c for 10 min .and stored at 4c°.

2.5.6 7. 5% Sodium bicarbonate solution (NaHCO3)

Seven and half grams of NaHCO3 were dissolved in 100ml DDW and

autoclaved at 121c° for 10 min and stored at 4c°.

2.5.7 Thioglycolate medium

Twenty nine and half grams of thioglyconate medium were dissolved

in 100ml of DDW, dispensed in bijou bottles and autoclaved at 121c° for 10

min and stored at 4c°.

2.5.8 Trypsin – Versene solution

Trypsin (2.5 % solution ) was sterilized by filtration (Millipore filter

0.22ul ) versene (5% solution ) sterilized by autoclaving, then 6 ml of trypsin

were added to 4 ml of versene and the mixed solution completed to 100 ml

with sterile phosphate diluent (1x)

2-5-9 Penicillin – Streptomycin Solution

One gram of streptomycin powder and 2 million units of penicillin

were dissolved in 10 ml of sterile DDW so that 1 ml of the prepared solution

contained 100 mg streptomycin and 20.000 units of penicillin. The solution

was kept at -20c°.

2.5.10 fungizon solution

The content of one vial of fungizon (Amphotericin B 50 mg) was

dissolved in 10 ml of sterile DDW and kept at 4c°.

2.6 Embryonated Chicken Eggs

Embryonated chicken eggs were obtained from the poultry unit of

Virology Reseach Laboratory (VRL) Dept. of Microbiology, Faculty of

Veterinary Medicine, University of Khartoum, Shambat.The eggs were

cleaned, disinfected and incubated at 37c° in a humidity range of 60 -65%.

Embryonated chicken eggs were used for production of pocks on the

chorioallantoic membrane (CAM) at the age of 10- 12 days. Embryonated

eggs were candled in dark room to check for embryo viability.

2.6.1 Chorioallantoic Membrane Inoculation

A cross was made in an area over the air sac and another one on the

egg side using a pencil. Eggs were then swabbed with 70% alcohol and a pore

was made at the crosses and drilling was made just deep enough to penetrate

the shell membrane to ensure that each chorioallantoic membrane (CAM)

drops to from a false air sac. To drop the membrane, a rubber bulb was used.

The bulb was placed over the hole in the air cell end of the egg and slowly

aspirated from the cell by releasing pressure on the deflated bulb. Holes were

drilled to the proper depth, to create a false air sac to from in the area of the

second hole. Eggs were inoculated by chorioallantoic membrane (CAM) route

with sterile disposable I ml syringes. Using just the tip of the needle, 0.1-0 2

ml of inoculum was injected from the syringe. The pores were sealed with

melted paraffin wax. Eggs were incubated at 37c° for 5 days with daily

candling to check for embryo death.

Embryos that died during 24 hours of inoculation were considered as

nonspecific and discarded and those that survived thereafter were killed by

chilling at 4c°.

2.6.2 Harvest of Chorioallantoic Membrane

Eggs were removed from the refrigerator, disinfected with 70 %

alcohol and the shell over air sac was removed using sterile forceps. The

embryo and yolk were extracted with forceps. Care was taken not to disturb

the CAM, and the area of inoculation was examined for lesion before

removal from the shell, the CAM was then detached from the shell with

sterile forceps, stripped of excess fluid with another forceps and placed in a

sterile Petri dish, then examination of the pock lesion was performed. CAM

samples showing pock lesion were collected and homogenized using sterile

mortars and pestles with aid of sterile sand. Samples were centrifuged at

1000 rpm for 10 min. Supernatant fluids were collected into sterile bottles

and treated with antibiotics and then stored in sterile bijou bottles at -20c°.

2.7 Pock lesion histopathology

Pock lesion were collected and fixated in 10% neutral formalin for 2

days. First the tissues were cutting into small square pieces about one cubic

cm. Section were put in 60% alcohol for ½ an hour, 70% alcohol during the

day (4_5hrs), 90% alcohol overnight, 100% alcohol 6 hrs (2 hrs each),

chloroform overnight, and melted paraffin wax 3-4 hrs and quickly cooled.

Section were cutted with aid of rotary microtome ,and transferred in a warm

water path containing little amount of gelatin powder , and left to float then

fixed to the slide glass and then incubated for 30 minutes at 60 c° to dry.

We used zylene to remove wax from the section for 10 minutes, and used

100% alcohol to remove Zylene for 10 minutes, and then the section were

rehydrated by rinsing in 90% alcohol for 5 min., 70% alcohol for 5 min and

D.W for 5 min. The section were then stained with Heamatoxyline and

Eosin (H&E) and covered with a cover glass, then dried overnight at room

temperature and examined microscopically.

2.8. Preparation of Cell Culture

A flask containing confluent monolayer culture of Vero cell was

obtained from the Central Veterinary Research Laboratory (Soba), the

growth medium was removed and the cells were briefly washed with PD.

0.5ml of warm trpsin – versene solution was added and the flask incubated

at 37c° until cells flew freely when the flask was tilted. Few drops of bovine

serum were added to stop the action of trypsin and versene .The cells were

diluted in GMEM growth medium and sub cultured in appropriate plastic

tissue culture flasks and incubated at 37c°.

2.9 Virus Propagation

A 24 wells plate containing semi–confluent monolayer was

inoculated with 50 ul volume of inoculum (every 4 wells inoculated with

one sample). The inoculated plate was kept at 37c° for 60 minutes

adsorption time. Incula were then removed and monolayers washed twice

with PD and refed with maintenance medium. The plate and a set of control

wells were examined daily with an inverted microscope and, when

cytopathic effect (CPE) involved 70 % or more of the sheet cover, the whole

cultures were harvested after three repeated cycle of freezing and thawing.

The harvested cell lysate was used as inculum to infect new plastic tissue

culture flasks.

2.10 Agar gel Preparation (see appendix 3).

2.11 Agar gel immunodiffusion test (AGID)

2.11.1 Preparation of agar gel

The agar and other chemical powders were dissolved in it’s specific

buffer , heated in a microwave for 2 minutes, 9 ml of the agar gel was

poured in the Petri dish to give a thickness about 3mm , using a template and

a cutter, a rosette six peripheral wells and a central well were cut in the agar.

Then plugs were carefully removed.

2.11.2 Standardization of AGID test

All the agars that mentioned in the material were prepared and used in

AGID test to detect precipitin lines when a vaccinia virus antiserum was

added to the central well and the vaccinia virus antigen mixed with sodium

deoxycholate were added to the peripheral wells .The plates were incubated

in a humid chamber at 35_37c .The plates were examined daily for

precipitation bands. (Fig: 5)

2.11.3 Examination of the sera for antibodies against orthopoxviruses

using AGID test

Purified agar1 was used to test the sera since it was the best agar tried

.The antigen which was mixed with sodium deoxycholate was added to the

central wells, while the sera were placed in the peripheral wells. Then plates

were incubated at 37c in humidity chamber, Then result were obtained and

reported.

Chapter 111

Results

3.1 Growth of vaccinia virus on the Chorioallantoic Membrane

Vaccinia virus was inoculated onto the CAM of 12 embryonated eggs.

The results showed the production of small pock lesions, round, opaque-

white in color and approximately 0.1-0.2 mm in diameter and without

hemorrhagic or necrotic center (Fig: 1)

3.2 Histopathology of the Chorioallantoic Membrane (CAM)

A number of 2 CAM were collected from inoculated eggs for

histopathology. CAM section showed foci of epithelial thickening .The cells

appear swollen with pale cytoplasm and indistinct cell boundaries. Many

cells showed degenerative changes. Some cell exhibited small round

eosinophilic inclusion bodies in the cytoplasm. Increase in connective

tissues cells at the site of epithelial proliferation showed mononuclear cell

infiltration (lymphocytes, monocytes) (Fig: 2 and 3).

3.3 Growth of vaccinia virus in Cell Culture

Vaccinia virus replicated in Vero cell and produced 100% Cytopathic

effect (CPE) three days post inoculation which was characterized by

rounding, plaque formation and destruction of the cell sheet (Fig: 4).

3.4 Sero-prevalence of orthopox virus in Sheep , Cattle and camel

Serum Samples

Four hundred and sex sera were examined by AGID to investigate

orthopox virus antibodies. The Test showed that 222 samples were positive

(54.7%) and 184 samples were negative (45.3%) (Table1).

The result showed that 109 (77.9%) out of 140 sheep sera were positive, 59 (35.7 %) out of 165 cattle sera were positive and 54 (53.5%) out of 101

camel sera were positive (Table 1).

Table 2 showed that 97 (92.3 %) out of 105 sheep sera collected from Bahry

were positive, 12(34.3%) out of 35 sheep sera collected from Omdorman

were positive, and showed that 14 (28 %) out of 50 cattle sera collected from

Bahry were positive, 45(39.1%) out of 115 cattle sera collected from

Omdorman were positive.

In camel showed that 37 (61.6 %) out of 60 camel sera collected from

Gadarif were positive, 17(41.5%) out of 41 camel sera collected from

Tambool were positive.

Table 1: Testing sera collected from cattle, sheep and camels for antibodies against orthopox virus antibodies by AGID

Species

Positive (%)

Negative (%)

Total

Cattle

59(35.7%)

106(64.2%)

165

Sheep 109(77.9 %) 31(22.1 %) 140

Camel 54(53.5%) 47(46.5%) 101

Total 222(54.7%) 184(45.3%) 406

Table 2: Testing sera collected from different areas in Khartoum for antibodies against orthopox virus antibodies by AGID

Species areas No. samples

+ve (%) -ve (%)

cattle Bahary Omdorman

50 115

14 (28%) 45 (39.1%)

36 (72%) 70 (60.9%)

sheep Bahary Omdorman

105 35

97 (92.3%) 12 (34.3%)

8 (7.6%) 23(65.7%)

camel Gadarif Tambool

60 41

37 (61.6%) 17 (41.5%)

23 (38.4%) 24 (58.5%)

Fig 1: Pock lesions produced by Vaccinia virus on CAM of embryonated eggs

Small pock lesions, round, opaque-white in color and approximately 0.1-0.2 mm in diameter and without hemorrhagic or necrotic center

Fig 2: CAM section showed swollen of the cells with pale cytoplasm and some inclusions bodies

The cells appear swollen with pale cytoplasm and indistinct cell boundaries. Many cells showed degenerative changes

Fig 3: CAM section showed foci of epithelial thickening

CAM section showed foci of epithelial thickening .

Fig 4: Vero cell culture infected with Vaccinia Virus showing rounding and plaque formation

Cytopathic effect (CPE) three days post inoculation which was characterized by rounding, plaque formation and destruction of the cell sheet

Fig 5: Precipitation line in AGID test between Vaccinia virus antigen (control well) and cows sera.

35

Chapter IV

DISSCUSION

Biological methods like growth on the Chorioallantoic

Membrance of embryonated eggs (CAM) and cell cultures were used to

determine the biological properties of Vaccinia virus (VV) and diagnosis.

In this study, VV was able to grow on CAM and produced

small pock lesions, which were round, opaque, white in colour and with a

diameter of about 0.1-0.2mm.The results in this study confirm the

observations of Marennikova et al (1974) who report that when Vaccinia

Virus was propagated on CAM at 370c, monomorphic punctuated, rather

dense white pock lesions small in size were seen.

When Vaccinia virus was grown in Vero cells it produced

clear Cytopathic effect (CPE) consisting of rounding of cells, plaque

formation, syncytia and detachment from the glass in 3 days after

inoculation. These observations agree with Maria-Lucia (2002) who

observed that the CPE in Vero cell culture by VV consist of small

syncytia, or multinucleated giant cells, result from fusion of cell

membranes bearing viral glycoproteins. Also there are inclusion bodies,

which are seen as eosinophilic areas of altered staining in the cytoplasm.

36

We also determined here the histopathology of the pock lesions to

show the VV effect on the CAM. CAM sections showed foci of epithelial

thickening, swelling of the cells, eosinophilic inclusions bodies and

infiltration of mononuclear cells. This result is similar to that of Davies

(1975) in histopathological section of the CAM of chick embryos

inoculated with camelpox virus which gave cellular inclusions and the

eosinophilic intra-cytoplasmic inclusions and Feulgen positive reaction

could be shown in the cells in the pocks. Khanna et al (1996) made

sections through pock lesions formed by camel pox and showed

proliferation of CAM cells and a few intracytoplasmic eosinophilic

inclusions. This is expected since camel pox virus is also a member of

the orthopoxvirus of the family Poxviridae. On the other hand the section

of pock lesions which formed by fowlpox were hyper plastic and

exhibited large easinophillic intra cytoplasmic inclusion bodies (Nyaga

1979). Gaffar et al (1980) described the inclusions bodies in pock lesions

of CAM section formed by Fowl pox virus as eosinophilic bodies in the

cytoplasm. The inclusions bodies were rounded; oval or irregular in

shape, often had granular appearance, and lacked discrete boundaries.

Inclusions bodies frequently appeared occupy the entire cytoplasm

37

except from a thin margin, pushing the nuclei to one side. Some

inclusions bodies appeared vacuolated, containing remnants of the outer

layer. With haematoxylin-shorr S3 stain, these inclusions bodies were

distinguished very clearly, taking the brilliant- red stain.

Various serological tests have been used for demonstration of

antibodies against poxviruses. These include gel diffusion test (Tantawi,

1974, Gwendolyn et al, 1962), neutralization test (Al Falluji et al, 1979;

Davies et al, 1985; Hafez et al, 1992), plaque reduction test (Nguyen and

Richard, 1985) and ELISA test (Munz et al 1986).

In this study, we used AGID test to detect orthopoxviruses in

sera of some domestic animal species in the Sudan. This test is

commonly used for identify and classification of the orthopox virus

(Joseph et al, 1977), and previously used for survey on antibody against

Parapoxvirus among cattle in Japan (Hiroshi et al, 2000). We have

examined 406 sera, 305 sera collected from sheep and cattle from

Khartoum state and 101 sera collected from camel from Gadarif and

Tambool. Our finding showed that 240 (59%) samples out of 406

samples collected from sheep, cattle and camel were positive. 186 sera

out of 305 samples collected from sheep and cattle were positive

38

(55.1%), 109 (77.9%) samples were positive out of 140 samples collected

from sheep, 59 (35.7%) samples were positive out of 165 samples

collected from cattle and 54(53.5%) samples were positive out of 101

sera collected from camel.

These findings demonstrate for the first time a serological

evidence for orthopoxvirus infection in sheep and cattle. Cattle are

known to be infected by cow pox virus which is also a member of the

orthopoxvirus genus. This disease is not yet reported in the Sudan

probably due to its benign course and difficulty in clinically distinguish

cowpox from pseudo cowpox of the genus parapox and other infections

of the teats. Thus our study points out for the existence of this disease.

Cowpox is benign infection of the udder and teats that causes no

mortality in cattle.

Orthopox viruses are not pathogenic in sheep, but seroconversion

in sheep to orthopox virus could be due to exposure of the sampled sheep

to camel pox or cowpox virus. The production system in most areas of

the Sudan is characterized by raising different species of domestic

animals including sheep, goat, cattle and camels. According to Tantawi

39

(1974) sheep experimentally infected with camel pox virus developed

high serum antibodies but they remain susceptible to sheep pox.

Our study showed that 53.5% of camel sera collected from eastern

and central Sudan were positive for camel pox virus antibodies. Camel

pox was already reported in the Sudan (Shommein and Osman, 1987,

Khalafalla 1998). The disease is the most contagious viral disease in

camels characterized by localized and generalized pox lesions that cause

severe out break in young animals with mortality rates of up to 10%.

Khalafalla et al (1998) detected antibodies against camel pox virus

in sera collected from 4 different region of the Sudan using ELISA. The

result showed 72.5% seropositivity. Our findings support the finding of

Khalafalla et al (1998) on the seroprevalence of camel pox virus

antibodies in eastern and central Sudan.

Further studies are needed to determine the economical impact of

orthopox virus infection in the Sudan particularly in cattle and sheep.

40

CONCLUSIONS

From this study, it can be concluded that Vaccinia Virus when

grown on the chorioallantoic membrance of embryonated eggs and Vero

cell gives clear pock lesions and Cytopathic effect (CPE) that can

differentiate this virus from related viruses. Additionally histopathology

of the CAM can give characteristic lesions.

Antibodies against Orthopoxvirus are widely distributed in sheep,

cattle and camel in the Sudan.

41

REFERENCES

Al Falluji, M.M, Tantawi, H.H and Shony, M.O (1979): Isolation, identification and characterization of camel poxvirus in Iraq. J. Hyg. Lond. 83 (2), 276-272. Andrews, C, Pereira, H. G and Wildey, P (1978): Viruses of Vertebrates, 4th. Edit. 356-389. Bailliere. Tindal, London. Anonymous (1954): Annual report of the Sudan Veterinary Services 1953-1954. Archard, L.C., and Mackett, M (1979): Restriction endonuclase analysis of red cowpox virus and its white variant. Gen. Virol. 45, 51-63. Arita, M., and I. Tagaya. (1977): Structural polypeptides of several strains of orthopoxvirus. Microbiol. Immunol. 21, 343-346. Baroudy, B., Venkhtesan, S., and Moss, B (1982): Incompletely base-paired flip-flop terminal loops link the two DNA strands of the vaccinia virus genome intone uninterrupted polynucleotide chain. Cell 28, 315-324. Baroudy, B., Venkhtesan, S., and Moss, B (1983): Structure and replication of vaccinia virus telomers. Cold Spring Harbor Symp-Quant-Bid.4762, 723-730. Baroudy, B.M., and Moss, B (1982 b): Sequence homologous of diverse length tandem repetitions near end of vaccinia virus genome suggests unequal crossing over. Nucleic Acids Res. 10, 5673-5679. Baxby, D. (1975): Identification and interrelationships of the variola /vaccinia subgroup of poxviruses. Prog. Med. virol. 19, 215-246. Baxby, D. (1977): Poxvirus hosts and reservoirs.Arch-virol.55, 169-179.

42

Baxby, D. (1988): Human poxvirus infection after the eradication of smallpox .Epidemiology and infection 100,321-334. Bennett M., Gaskel R.M., Gaskel C.J., Baxby D., Kelly D. F (1989): Studies on pox virus infection in cats. Arch Virol 104, 19-33. Brisovish, Y. F and Orekhov, M. M (1966): Camel pox. Veterinaryia 3, 50. (Abstract: Veterinary Bulletin, 36, 794 (1966). Buchnev, K. N, Tulepbaev, S. Z and Sansyzbaev, A. R (1987): Infectious diseases of camels in the USSR. Revue Sci. tech. Off. Int. Epizoot. 6(2), 487-495. Buchnev, K. N., Sadykov R.G., Tulepbayer S., Roslyakov A.A (1969): Small pox-like disease of camels. Trudy Alma-atinskogo Zootekhnickeskogo Inistituta. 16, 36-47. Carter, G.R., Wise, D.J and Flores, E.F (2005): A concise Review of Veterinary Virology. International Veterinary Information Service, Ithaca NX. (www.ivis.org).

Czerny, C. P., A. M. Eis-Hubinger, A. Mayr, K. E. Schneweis, and B. Pfeiff. 1991. Animal poxviruses transmitted from cat to man: current event with lethal end. Zentbl. Veterinarmed. B 38,421-431.

Czerny, C. P; H. Meryer and H. Mahnel (1989): Establishment of an ELISA for the detection of orthopox viruses based on neutralizing monoclonal and polyclonal antibodies. J. Vet. Med. B. 36,537-546. Davies, F. G., J. N. Mungai and P. O. Kabete (1975): Charactaristics of a Kenyan camelpox virus. J. Hyg. Cqmb. 75, 381. Davis F.G., Mungal H, Atema C and Wilson A (1985): The prevalence of antibodies to camel pox in six different herds in Kenya. Journal of Comparative Pathology 95(4):633-635.

43

Dumbell, K.R., and Archard, L.C. (1980): Comparison of white pock (h) mutants of monkey poxvirus with parental monkey pox and with variola like viruses isolated from animals. Nature (London 286, 29-32. Egberink H.F., Willemse A., Horzinek M.C (1986): Isolation and identification of apox virus from a domestic cat and a human contact case.J Vet Med 33,237-240. Eis-Hubinger A.M., Genitzen A., Schneweis K.E., Pullmann H., Mayr A., Czerny C.P (1990) : Fatal cowpox-like virus infection transmitted by cat-lancet 336,880. Esposito, J.J., Cabradill A, C.D., Nakano, J.H., and Obijeski, J.F. (1981b): Intragenomic sequence transposition in monkey poxvirus. Virology 109, 231-243. Esposito, J.J., Obijeski, J.F., and Nakano, J.K (1978): Orthopoxvirus DNA: strain differentiation by electrophoresis of restriction endonuclase fragmented virion DNA.Virology 89, 53-66. Fenner, F (1996): Poxviruses, p. 2673-2702. In B. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Fields virology, 3rd ed. Raven Press, Inc., New York, N.Y. Fenner, F, Wittek, R. and Dumbell, K. R (1989): The Orthopoxviruses. New York and London: Academic Press. Fenner, F., and J. H. Nakano (1988): Poxviridae: the poxviruses, p. 177-207. In E. H. Lennette, P. Halonen, and F. A. Murphy (ed.), Laboratory diagnosis of infectious diseases, vol. 2. Viral, rickettsial and chlamydial diseases. Springer-Verlag, New York, N.Y. Gaffar E., M.H. Tageldin and S.H. Babiker (1980): Fowl pox virus in the Sudan. Avian Disease. Vol 24. No 3. P763-770. Gitao, C. G (1997): An investigation of camel pox outbreak in two principal camel (camel dromedaries) rearing areas of Kenya. Rev. Sci. Tech. Office Intl. des Epiz.16 (3), 841-847.

44

Goebel, S. J., G. P. Johnson, M. E. Perkus, S. W. Davis, J. P. Winslow, and E. Paoletti. 1990. The complete DNA sequence of vaccinia virus. Virology 179, 247-263. Gwendolyn, M., Woodroofe and Frank, F (1961): Serological relationships within the poxvirus group: on antigen common to all member of the group.Virology.16, 334-341. Hafez, SM, AI-Sakayran, A, Delta Cruz D, Mazloum KS, AL-Bokmy AM, AL-Mukayel A and Amjad AM (1992): Development of live cell culture camelpox vaccine. Vaccine 10(8), 533-539. Henderson DA and Moss B (1999): "Smallpox and Vaccinia", in Plotkin SA, Orenstein WA: Vaccines, 3rd ed, Philadelphia, Pennsylvania: WB Saunders. ISBN 0-7216-7443-7. Hiroshi, S. Yasuo, I. Akihro, M. Yasunori, Y. Kenji, M. and Shinya, S (2000): Survey on Antibody against Parapoxvirus among Cattle in Japan. Microbiol. Immunol. 44(1), 73-76. Holowczak, J. A (1982): Poxvirus DNA. Curr. Top Microbia. Immund. 97, 27-79. Huygelen C (1996): "Jenner's cowpox vaccine in light of current vaccinology" (in Dutch; Flemish). Verh. K. Acad. Geneeskd. Belg. 58 (5),479-536; discussion 537-8. PMID 9027132. ICTVdB Management (2006): 00.058.1.01. Orthopoxvirus. In: ICTVdB - The Universal Virus Database, version 4. Büchen-Osmond, C. (Ed), Columbia University, New York, USA. ICTVdB Management (2006): 00.058.1.01.001. Vaccinia virus. In: ICTVdB - The Universal Virus Database, version 4. Büchen-Osmond, C. (Ed),ColumbiaUniversity,NewYork,USAhttp://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB.

45

Jezek, Z, Kriz, B and Rothbauer, V (1983): Camel pox and its risk to the human population. J. Hyg. Epidem. Microbiol. Immun. 27 (1), 29-42. Johann, S. and Czerny, C.P (1993): A rapid antigen capture ELIZA for the detection of orthopoxviruses. J. V. Med. B. 40,569-81. Johnson, G. P., S. J. Goebel, and E. Paoletti (1993): An update on the vaccinia virus genome. Virology 196, 381-401. Joseph, J. Esposito. John, F. Obijeski and James, H (1977): Serological Relatedness of Monkey pox, Variola and Vaccinia viruses. Journal of Medical Virology 1, 35-47. Khalafalla, A .I and Mohamed, M.E.M (1998) part1: Camel pox in the Sudan. Isolation and identification of the causative virus. J. Camel. Prac and Res. 5 (2), p229-233. Khalafalla, A .I and Mohamed, M.E.M (1998) part2: Some properties of camel pox viruses isolated in the Sudan. J. Camel. Prac and Res. 5 (2), p235-238. Khalafalla, A .I and Mohamed, M.E.M and Agab, H (1998): Serological survey in camels of the Sudan for prevalence of antibodies to camel pox virus using ELISA Technique. J.camel prac and Res.5 (2), 197-200. Khanna, N. D. , P. K. Uppal, N. Sharma and B. N. Tripathi (1996): Occurrence of pox infections in camels. National Research Centre On Camel. Indian Vet. J. 73, 813-817. Kinne, J; Cooper, J. E and Wernery (1998): Pathological studies on camel pox lesions in the respiratory system in the Arab Emirates (U.A.E).J.comp.path.118-25. Knight, J. C., R. F. Massung, and J. J. Esposito (1995): Polymerase chain reaction identification of smallpox virus, p. 297-302. In Y. Becker, and G. Darai (ed.), Diagnosis of human viruses by polymerase chain reaction technology, 2nd ed. Springer-Verlag KG, Berlin, Germany.

46

Kriz, B (1982): A study of camel pox in Somalia .J.comp.path.92, 1-8. Mackett, M., and Archard, L.C (1979): Conservation and variation in orthopoxvirus genome structure. J. Cen. Virol. 45, 683-701. Mahnel, H (1974): Labordifferenzierung der orthopocken viren. J. Vet. Med. B, 21, 242-258.Cited by Wernery, U& Kaaden, O. R (1995). Mahnel, H&Bartenbach, G (1973): Systematisierung des kamel pocken virus (KPV).Zbi.Vet.Med.B20, 572-576. Marennikova, S, Maltseva N.N, Korneeva V.I, Garania N.M (1977):Outbreak of pox disease among carnivora(felidae) and edentata.J Infect Dis 135,358-366. Marennikova, S, Shenkman, L, S, Shelukhina, E. M and Maltseva, N. N (1974): Isolation of camel poxvirus and investigation of its properties . Acta Virologica, Praque, 18, 1-7. Maria, L, Racz (2002): Viral cytopathic effect in cell culture-Vaccinia virus. University of Sao Paulo. Brazil.

Massung, R. F., J. J. Esposito, L. I. Liu, J. Qi, T. R. Utterback, J. C. Knight, L. Aubin, T. E. Yuran, J. M. Parsons, and V. N. Loparev. 1993. Potential virulence determinants in terminal regions of variola smallpox virus genome. Nature 366,748-751.

Massung, R. F., V. N. Loparev, J. C. Knight, A. V. Totmenin, V. E. Chizhikov, J. M. Parsons, P. F. Safronov, V. V. Gutorov, S. N. Shchelkunov, and J. J. Esposito. 1996. Terminal region sequence variations in variola virus DNA. Virology 221,291-300.

Mc Fadden, G., and Dales, S (1982): Organization and replication of Poxvirus DNA .In “Organization and replication of viral DNA” (A.S.Kaplan, ed.), pp.173-190.CR (Press, Boca Raton, Fla. Meyer, H., Damon, I.K, Esposito, J.J (2004): Orthopoxvirus diagnostics methods 269, 119-34.

47

Meyer, H., M. Pfeffer, and H. Rziha (1994): Sequence alterations within and downstream of the A-type inclusion protein genes allow differentiation of orthopoxvirus species by polymerase chain reaction. J. Gen. Virol. 75, 1975-1981. Meyer, H., S. L. Ropp, and J. J. Esposito (1997): Gene for A-type inclusion body protein is useful for a polymerase chain reaction assay to differentiate orthopoxviruses. J. Virol. Methods 64, 217-221. Meyer, H; Martin Pfeffer and Hanns-Joachim Rziha (1994): Sequence alterations within and downstream of the A-type inclusion protein gene allow differentiation of orthopoxvirus species by polymerase chain reaction. J. Viro. 75, 1975-1981. Meyer, H; Osterrieder, N& Pfeffer, M (1993): Differentiation of species of genus orthopoxvirus in adot blot assay using digoxigenin labeled DNA-probes. Vet. Micr. 34, 333-334. Moss, B. (1996) Poxviridae: the viruses and their replication. In field Virology,3rdedu,p26372671.EditedbyB.N.Fields,D.M.Knipe&P.M.Howly. Philadelphia: lippincoH-Raven. Moss,B(2001):Poxviruses,InD.M.Knipe,P.M.Howley,D.E.Griffin,R.A.Lamb,M.A.Martin,B.Roizman,andS.E.Strans(ed.), Fields virology, 4thed.,Vol.2-Lippincott Williams and Wilkins, Philadelphia, Pa. P.2849-2884. Moyer, R.W., Graves, R.L., and Rothe, C.T (1980b): The white pock (m) mutants of rabbit poxvirus. 111. Terminal DNA sequence duplication and transposition in rabbit poxvirus. Cell 22, 545-553. Muller, H.K., Wihek, R., Schaffner, W., Schumperli, D., and Wyler, R.(1978) : Comerison of five poxvirus genomes by analysis with restriction endonucleases Hind III, Baml, and Ecorl .J.Gen.Virol .38,135-147.

48

Munz, E (1992): Pox and pox-like diseases in camels. Proc. 1st int. Camel conference. Eds: W. R. Allen, A. J. Higgins, I. G. Mayhew, D. H. Snow and J. F. Wade: R. and W. publications, New market, UK: 43-46. Munz, E, Kropp, M and Reimann, M (1986): Demonstration of antibodies against Orthopox virus cameli in sera of eastern Africa dromedaries by ELISA. Journal of Veterinary Medicine B 221-230. Munz, E; Moallin, A. S. M; Mahnel, H and Reimann, M (1990): Camel papillomatosis in Somalia. J. Vet. Med. B 37, 191-19. Murphy, F. A; Gibbs, E. P. J; Marian, C; Horzinek and Studdert, M. J (1999): Poxviridae: In Veterinary Virology. Third Edition. Academic press in United State of America. Chapter 16. p283. Nguyen .BA.VY, and Richard D (1985): Titration of camel pox antibodies by plaque reduction on IB-RS 2cells. Revuo. Elev. Med. Vet. Paya. Trop. 38 (3), 223-228. Nyaga, P. O. , J. S. Kaminjolo, E. R. Mutiga and L. C. Bebora (1979): Occurrence of Atypical Fowlpox in Poultry Farm in Kenya. Avian disease 33, 3. Obijeski, J. F., E. L. Palmer, L. G. Gafford, and C. C. Randall (1973): Polyacrylamide gel electrophoresis of fowl pox and vaccinia virus proteins. Virology 51, 512-516. Pales, S., and Pago, B.G.T. (1981): Biology of pox viruses “springer-verlag, New York. Pfahler, W. H. E and Munz, E (1989): Camel pox. Int. J. Anim. Sci. 4,109-114. Pfeffer, M, Wernery. U, Kaaden, O-R and Meyer, H (1998): Diagnostic procedures for poxvirus infections in camelids. J. Camel. Prac. And Res 5(2), 189-195.

49

Pickup, D.J., Bastia, D., and Joklik, W.K (1983): Cloning of terminal loop of vaccinia virus DNA. Viro124, 215-217. Pickup, D.J., Bastia, D., Stone, H.O., and Joklik, W.K (1982): Sequence of terminal regions of cow poxvirus DNA: Arrangement of repeated and unique sequence elements.Proc.Natl.Acad. Sci. USA 79, 7112-7116. Ramyar, H and Hessami, M (1972): Isolation, cultivation and characterization of camel poxvirus. J. Vet. Med. B 19, 182-189. Ropp, S. L., Q. Jin, J. C. Knight, R. F. Massung, and J. J. Esposito. (1995): Polymerase chain reaction strategy for identification and differentiation of smallpox and other orthopoxviruses. J. Clin. Microbiol. 33, 2069-2076M. Ryan KJ, Ray CG (editors) (2004): Sherris Medical Microbiology, 4th ed., McGraw Hill. ISBN 0-8385-8529-9. Schwartz, H. J& Dioli, M (1992): The one-Humped Camel in Eastern Africa. A pictorial guide to disease, health care and management. Verlag Josef Margraf Scientific Books. Shommein, A. M& Osman, A. M (1987): Diseases of camels in the Sudan. Rev. Sci. Tech. Off. Epiz. 6, 481-484. Smith, G.L. and Law, M (2004): The exit of vaccinia virus from infected cells. Virus Res. Dec; 106(2), 189-97. Smith, G.L., Vanderplasschen, A., and Law, M (2002): the formation and function of extracellular enveloped vaccinia virus. Gen Virol. Dec; 83(12), 2915-31. Stuart N. Isaacs (2004): Vaccinia Virus and Poxvirology Methods and Protocols Methods in Molecular Biology.269 , 1-13. Tantawi H. H (1974): Comparative studies of camelpox, sheeppox and vaccinia viruses. Aeta Virologica 18:347-351.

50

Tolonen N, Doglio L, Schleich S, Krijnse Locker J (2001): "Vaccinia virus DNA replication occurs in endoplasmic reticulum-enclosed cytoplasmic mini-nuclei". Mol. Biol. Cell 12 (7),2031-46. PMID 11452001. Wernery, U and Kaaden, O-R (1995): Infectious Disease of camelids. Blackwell Wissenschafts- Verlug, Berlin. Wernery, U. and Kaaden, O-R (2002): Infectious Diseases of camelida 2nd edition. Blackwell Science. Berlin. Vienna. P 177. Wernery, U; Kaaden, O-R and Ali, M (1997b): Orthopoxvirus infection in dromedary camels in United Arab Emirates (U.A.E) during winter season. J. Camel Prac and res. 4(1), 51-55. Wernery, U; Meyer, H and Pfeffer, M (1997a): Camel pox in the United Arab Emirates and its prevention. J. Camel prac and Res. 4(2): 135-139. Wikipedia, The Free Encyclopedia, www.wikipedia.org. Wittek, R (1982): Organization and expression of the Poxvirus genome. Experientia 38, 255 -410. Wittek, R., and Moss, B (1980): Tandem repeats within the invented terminal repetition of vaccinia virusDNA.Cell.21, 277-284. Wittek,R.,Menna,A.J.,Schumperli,D.,Stoffel,S.,Muller,S.,Muller,H.K.,andWyler,R (1977): Hind 111and Ss+I restriction sites mapped on rabbit poxvirus and Vaccinia virus DNA .J.Virol -23,669-678. Woodroofe, G.M., and Fenner, F (1962): Serological relationships within the poxvirus group.Virology16, 334-334.

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Appendices

Appendix 1: Phosphate buffer saline (PBS) and phosphate diluent (PD)

A-Ingredients:

Solution (a)

NaCl 16.0g

KCl 0.4g

Na2 HPO4 2.3g

KH2PO4 0.4g

DDW 1500.0 ml

Solution (b)

MgCI .6H2O 0.426g

DDW 200.0 ml

Solution (c)

CaCI2 2H2O 0.264g

DDW 200.0ml

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Appendix 2: Outgrowth and Maintenance Media Outgrowth and maintenance media were prepared according to Ali

(1971) as shown below:

Medium (liter)

Stock solutions Outgrowth Maintenance

GMEM 200 ml 200 ml

Lactalbumin hydroysate 25 ml 25 ml

Yeast extract (1%) 25 ml 25 ml

Sodium bicarbonate (7.5%) 7.5 ml 10 ml

Penicillin – streptomycin solution 1 ml 1 ml

Fungizon (5000ug / ml) 1ml -

DDW To IL To IL

Tryptose Phosphate broth (TPB) 50 ml 50 ml

Bovine serum 100 ml 20 ml

53

Appendix 3: Purified agar I (Procedure I)

Purified agar 1.60 g

Nacl 8.00 g

Phenol 0.5 ml

Distilled water 100 ml

Purified agar II (Procedure 2)

Purified agar 1.4. g

Nacl 8.00 g

Phenol 0.5 ml

Distilled water 100 ml