Characterization of camelpoxvirus isolates from Africa and Asia

veterinary microbiology ELSEVIER

Veterinary

Microbiology

45 ( 1995) 371-381

Characterization of camelpoxvirus isolates from Africa and Asia I.C.E. Renner-Miiller ’ Institute

uf Microbiology,

a.*, H. Meyer b, E. Munz a

Federal Armed Forces Medical Academy. Munich, Germarly

Received

I2 July 1994: accepted 22 December 1994

Abstracl

Five orthopoxvirus isolates of camels from different geographic regions of Africa and Asia were analysed with respect to their biological and genomic attributes. The behaviour of the isolates in various cell cultures, the type of pock lesions on the chorioallantoic membrane of embryonated chicken eggs, and the reSpective ceiling temperatures were determined. Additionally, physical maps for restriction endonucleases Hind111 and XIzol were established. The data obtained from biological assays and DNA analyses demonstrated minor differences between the five isolates. However, these findings confirm previous reports suggesting that orthopoxviruses of camels constitute a separate species within the genus Orthopoxvirus. Kquvords:

Camels: Camelpoxvirus;

Orthopoxvims

1. Introduction

Camelpoxvirus

(CPV).

the causative

agent of a pox disease of old world camelids is known in almost all came1 raising countries (Wilson et al., 1990). Camelpox can either occur as a localized benign infection or as a generalized malignant disease affecting especially younger animals (Hafez et al., 1992). Similar clinical symptoms can be caused by infections with parapoxvirus (Ecthyrna contagiosum) (Munz et al., 1986) and simultaneous infections with both, paraand orthopoxvirus have been observed ( H. Mahnel, persona1 communication, 1993 ) Infection with papillomavirus can also lead to localized pox-like lesions (Munz et al., 1990). (Catnelus dromedarius and Camelus bactrianus, respectively),

* InstitJt fiir Vergleichende Tropenmedizin und Parasitologie. Ludwig-Maximilians-Universitat, 5,80802 Miinchen. Deutschland. Tel. 089/2180-3516. Fax 0891336112. 0378-l 135/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved .%SDIO3;‘8-1135(94)00143-X

Leopoldstr.

The pathogenicity of CPV for man is extremely low (Jezek et al., 1983), but in camels infections are of great economic importance. CPV was tirst isolated in 1969 ( Buchnev and Sadykov. 1969 ) Since then, several reports about the properties of isolates originated from Africa and Asia have been published (Ramyar and Hessami. 1972: Baxby, 1974: Marennikova et al.. 1974; Tantawi. 1974; Tantawi et al., 1978; Davies et al.. 1975: Al-Falluji et al.. 1979: Chauhan and Kaushih. 1987; El-Kenawy et al., 1989; Nguyen-ba-vy et al., 1989). CPV is a typical orthopoxvirus (OPV) in its morphology and serological cross-reactivity with other OPV. It appears to be enzootic in camels but natural infections in other animals are not known. CPV attracted some attention during the smallpox eradication campaign as it was described as a smallpoxlike virus (Baxby, 1972) sharing many biological properties with variolavirus, i.e. the narrow host range in experimental animals and the production of small white pocks on the CAM ( Baxby. 1975 ) Therefore, experimental work was carried out to differentiate CPV-isolates from variolavirus. It could be demonstrated that infection of HeLa cells with CPV led to multinucleate giant cells whereas variolavirus caused a rounding up of these cells CBaxby et al.. 1975 ) Based on detailed investigations including the CP- I strain from Iran (Ramyar and Hessami, 1972). it has been suggested that OPV-isolates from camels belong to a distinct OPVspecies: Orthopoxvirus cameli ( Mahnel and Bartenbach. 1973: Francki et al., 199 1). Restriction enzyme analysis is a useful tool for the classification of OPV species. Although different species share a large conserved central region of their genomes, this technique proved to be capable of differentiating between members of the OPV-group, e.g. monkeypox-from variolavirus isolates (Mackett and Archard, 1979; Wittek, 1982). Variola major and minor strains could also be distinguished (Esposito and Knight, 1985 ). The most commonly used endonucleases are HirrdIII and XhoI. Physical maps have been constructed for most OPV-species. However. only two reports are dealing with the genome of CPV. Esposito and Knight ( 1985 ) published the HindIll-map of an isolate from Somalia and the HindITI-patterns of four CPV-isolates from Iran and Somalia have been compared by Fenner et al. ( 1989). In these reports only limited data concerning biological attributes of CPV were shown comparatively. In the present study biological properties of live CPV-isolates of different geographic origin have been evaluated and HiFzdIII and X/z01 maps were constructed.

2. Material and methods 2. I. Viruses and cells The following orthopoxviruses isolated from camels ( Canrelus drumedclrius) were investigated: reference strain CP- I isolated in Iran ( Ramyar and Hessami, 1972; Mahnel and Bartenbach, 1973), CP-SA from Saudi-Arabia (obtained from Dr. S.M. Hafez, Riyadh. Saudi Arabia, 1986). CP-MAU from Mauritania, CP-NIG from Niger (both obtained from Dr. Nguyen-ba-vy, Maison-Alfort Cedex, France, 1988), and CP-5 from Dubai (kindly provided by M. Pfeffer, Munchen, 1992). Additionally, vacciniavirus strain Elstree (Lister

I. Remer-Miiller

et al. / Veterinqv

Microbiolqg_v 45 (1995) 371-381

373

institute), cowpoxvirus strain KR-2 Brighton (Downie, 1939), and the cowpoxvirus isolate OPV 85 (~Nasemann et al., 1987) were used. Viruses were propagated on African green monkey kidney cell line (MA 104) as described by Czerny and Mahnel ( 1990). Infected cells were freeze-thawed ( - 20/2O”C) to release virus particles and cellular debris was removed by low speed centrifugation ( 1500 g/ 10 min.). The susceptibility of various cell lines was tested: BHK 21 (baby hamster kidney cells). E-Derm (equine dermal fibroblasts. Bayer. Leverkusen). HEP 2 (human epidennoid carcinoma cells, Boehringer. Mannheim). L929 (mouse fibroblasts), MDBK (Madin Darby bovine kidney cells), MDCK ( Madin Darby canine kidney cells),RK 13 (rabbit kidney cells), and Vero (green monkey kidney cells). Coverslip preparations were prepared from cells infected with a multiplicity of infection of at least 5. At 24 and 48 hours post infection, coverslip were fixed and stained with haemator.ylin and eosin (Mayr et al.. 1974). 2.2. Infection of the chorioallantoic

membrane

Virus-mfected tissue culture supernatants were titrated in log ,,-steps. The chorioallantoic membranes (CAM) from eggs of lo-12 days old chicken embryos were inoculated with 0.1 ml of the corresponding dilutions. The following criteria were determined: the morphology and number of primary pocks after 3 and 5 days. the presence of secondary pocks as a sign of a generalized infection, the ceiling temperatures, and the lethality for the embryo. 2.3. Preparation

and clo?ling of DNA

Viral DNA was isolated from purified virions (Meyer et al.. 199 I ) and cleaved with restriction enzymes according to the manufacturer’s instructions (Boehringer, Mannheim) Cloning of DNA restriction fragments of strain Elstree and KR-2 Brighton was performed by standard procedures (Sambrook et al., 1989). OPV-specific inserts were gel-isolated, DNA was extracted by freeze-squeezing (Sambrook et al., 1989) and cloned into appropriately digested plasmid p2TZl9R (Pharmacia/LKB, Freiburg) using T4 DNA ligase (Boehringer). Transformation of competent E. coli K12 strain DHSaF’ (Gibco/BRL, Eggenstein) was done according to Hanahan ( 1983). Plasmid DNA was isolated by the procedure of Hattori and Sakaki ( 1986) and tested for correct insertion of fragments by restriction enzyme analysis and Southern blot hybridization (data not shown). Isolation of recombinant clones phr and pHA has been described recently ( Meyer et al., 199 1) Table 1 summarizes the designation of the recombinant plasmids and the respective sizes of the inserted poxvirus DNA. The location of cloned sequences in the genomes of vacciniavirus strain Elstree and cowpoxvirus strain KR-2 Brighton is shown in Fig. 1. 2.4. Sowhern blot hybridization DNA of camelpoxvirus isolates CP-1, CP-SA, CP-MAU, CP-NIG, and CP-5 as well as vacciniavirus Elstree and cowpoxvirus Brighton were digested with HirzdIII or XhoI, respectively. DNA-fragments were separated on agarose gels, transferred to nylon membranes (Hybond N + , Amersham-Buchler, Braunschweig) by capillary blotting ( Southern, 1975),

374

I. Rmner-Miiller

Table 1 Designation

et al. / Veterinary Microbiology 45 (1995) 371-381

and origin of DNA-probes

used for the construction

No.

Designation

Origin

1

pA7 pXho J pXho I

vv COWPV COWPV vv vv vv vv vv CPV vv vv COWPV COWPV COWPV

2 3 4 5 6 7 8 9 10 11 I? 13 14

phr pXho B pHind M pB2 plHD pAT1 pXho E PHA pXho L pXho 0 pHind L

of physical maps

WR KR-2 KR-2 CVAh KR-2 Elstree Elstree Elstree Id Elstree CVAh KR-2 KR-2

Sire (kbp)

vector

7.3 6.0 6.6 5.7 28.0 4.0 14.1 11.1 4.8

lambda WESB” pTZI9R pTZ19R pTZ19R _L

7.8 4.8 3.8

pTZ19R pTZ 19R pTZl9R pTZ 19R pTZ 19R pTZ 19R pTZl9R pTZ 19R

7.4

pTZ19R

Il.2

KR-7

W=Vacciniavirus. CowPV= Cowpoxvirus. “kindly provided by R. Wittek (Lausanne). ‘isolated from vacciniavirus strain CVA (Meyer et al.. 19911. ‘DNA was labeled after gel-isolation. “isolated from camelpoxvims strain CP- 1 ( Meyer and Rziha. 1993

).

and fixed by drying at 80°C. OPV-specific DNA isolated from recombinant plasmids (see above) was labeled by random primed incorporation of Digoxigenin-1 I-dUTP using a commercially available kit (DNA labeling kit. non-radioactive, Boehringer). Membranes were preannealed for 3-5 hours at 42°C in 50% (v/v) formamide; 5 X SSC (20 X SSC = 3M NaCI, 0.3 M Nacitrate, pH 7.0) : 0.1% (w/v) N-lauroylsarcosine; 0.02% (w/ v) SDS; and 5% (w/v) blocking reagent (Boehringer). The mixture was changed and hybridization proceeded overnight in the presence of Digoxigenin-labeled probes. The blots were washed twice in 2 X SSC/O.l% SDS at room temperature for 15 min and twice in 0.1 X SSC/O.l% SDS at 62°C. DNA hybrids were detected with an anti-Digoxigeninalkaline phosphatase conjugate and visualized by an enzyme-linked colour reaction according to the manufacturer’s protocol (DNA detection kit. non-radioactive, Boehringer).

I 1, 1

Vacciniavirus Elstree Hind111 I

1

I

II 1

6

4

1

8

9 10

I 11

1

Cowpoxvirus KR-2 Brighton Hind111 ,

I

I,,, 2

3

1

1

5 n 1okb

Fig. 1. Location of cloned sequences in the genomes of vacciniavirus The numbers refer to recombinant clones specified in Table I.

I

I

I 1 12 I4 13

Elstree and cowpoxvirus

KR-2 Brighton.

I. Renner-Miiller

et al. / Veterinatyv Microbiology

Table 2 Type of cytopathic effect induced by five different camelpoxvirus Elstree, and cowpoxvirus isolate OPV 85 in different cell lines

BHK 21 E-Derm HEP 2 L 929 MA 104 MDBK MDCK Vera RK 13

37s

45 (1995) 371-381

(CP)

isolates, vacciniavirus

(VV)

strain

CP-1

CP-MAU

CP-NIG

CP-SA

CP-5

vv

OPV 85

RC RC/RS GC RC GC RC RCiRS GC 0

RC RCIRS GC RC GC RC RCiRS GC 0

RC RCIRS RC RC RC RC RC/RS RC 0

RC RCIRS GC RC GC RC RCfRS GC 0

ND ND ND RC GC ND ND GC 0

RC SC RC RC RC SC SC RC RC

RC RC RC RC RC RC RC RC RC

RC = round cells: GC = giant cells: SC = strand cells; RS = replication done: 0 = no multiplication.

after repeated subcultivation;

ND = not

3. Results 3. I. Behmior

in cultured cells

All carnelpoxvirus isolates as well as vacciniavirus strain Elstree, and cowpoxvirus isolate OPV 85 replicated in all the cell cultures investigated (except CPV-isolates in RK 13 cells). Cytopathic effects (cpe) induced by CPV and cowpoxvirus tended to develop more slowly (up to 30 hours) and were less prominent compared to the cpe of vacciniavirus which could be seen already 12 hours post infection. Additionally, differences in the type of cpe were noted (Table 2). Usually, plaque formation could be seen with virus-infected cells contracted and rounded up. This was followed by vacuolization, lysis and finally detachment of the cells. Formation of large syncytia was observed only with some cell lines infected with CPV-isolates CP- I, CP-SA, CP-MAU, and CP-5. Coverslip preparations of infected Vero cells revealed typical multinucleate giant cells with more than 15 nuclei (Fig. 2). However, in CP-NIG-infected cells only 2-5 nuclei were involved in the formation of syncytia. Replication of CPV-isolates on E-derm and MDCK cells was poor, but adaptation by repeated subcultivation led to a cpe within 2-3 days post infection. In CPV-infected BHK cells stained with HE small intracytoplasmatic inclusions of irregular shape could be demonstrated in some of the cells. Similar results were obtained with vacciniavirus-infected cells. In contrast cowpoxvirus-infected BHK cells displayed large inclusions in the cytoplasm of almost every cell. These results are consistent with findings of Mahnel and Bartenbach ( 1973). In some cell lines, the type of the cpe caused by vacciniavirus was characterized by the production of “strand cells”. In this case with the cytoplasm of infected cells contracting the cell culture finally looks like a net. 3.2. Behavior on the chorioallantoic

membrane

On day 3 after infection. the diameter of the opaque white proliferative pock lesions on the CAM produced by CPV-isolates was at most 0.5 mm, significantly smaller than pocks

Fig. 2. Multinucleate giant cells induced in Vcro cells after mfcction with

post infection;

camelpoxvirusisolate

CP-MAU

(18 h

magnification ‘725 % 1.

induced by either vaccinia or cowpox virus (up to 3 mm). Two days later the diameter of the plaques was about I .S mm. No necrotic or hemorrhagic center developed. Sometimes a weak generalization without death of the embryo was observed. The ceiling temperature was determined to be in the range of 39 to 39S”C for all CPV-isolates. In contrast, the ceiling temperature determined for vaccinia and cowpox virus was above 39S”C. Infection with either virus led to a generalization. often accompanied with the death of the embryo. 3.3. Restriction

endonuclease

pattrrtl

Restriction fragments resulting from cleavage of CPV isolates CP- 1, CP-MAU, CP-NIG, CP-SA. and CP-5 DNA with endonuclease Hind111 or X!zoI are shown after separation by electrophoresis in a 0.6% agarose gel (Fig. 3). The electropherograms of three isolates (CP- 1, CP-MAU, and CP-5) were almost identical. Differences were noted in the size of two HindIII-fragments, marked with an arrow. These fragments were identified as the terminal fragments by their reactivity with probe pA7 (no. 1) which is specific for the inverted terminal repeat region. Terminal fragments of the other strains are marked accordingly. Although the HirzdIII and X/z01 electropherograms of all CPV-isolates resembled each-other, differences were observed readily by non-comigrating fragments. In the CP-NIG HindIll-pattern three fragments ( 18.0, 10. I, and 10.0 kb) are absent when compared with CP- 1, CP-MAU. and CP-5. Instead, a new fragment ( 13.0 kb) appeared. In CP-SA, the 18.0 kb fragment and the 2 molar 15.0 kb fragment are missing, and additional fragments accounting for 9.0, 12.5, and 16.0 kbp could be seen. In the XhoI electropherograms, minor variations were seen again in the CP-NIG and CP-SA isolates when compared with the

I. Renner-Midler

et al. /Veterinary

Microbiology

45 (1995) 371-381

377

Fig. 3. Eiectropherograms of HindIII (lane 1 to 7) and Xhol (lane 9 to 15) digested DNA from vacciniavirus strain Elstree ( lane 1 and 9), cowpoxvirus strain KR-2 Brighton (lane 2 and 10) 1and camelpoxvirus isolates CP1 (lane 3 and 11).CP-MAU (lane 4 and 12). CP-NIG (lane 5 and 13), CP-SA (lane 6 and 14). and CP-5 (lane 7 and 15). The 1 kbp ladder (Gibco/BRL. lane 8) was used as size marker. Terminal fragments are marked by an arrow.

other isolates. Summation of the molecular weight of Hind111 or X&I fragments of CP-NIG resulted in a smaller size of the genome ( 176 kbp) compared to CP-1, CP-MAU or CP-5 (200 kbp ) . The size of the genome of CP-SA accounted for 186 kbp. 3.4. Mapping of restriction fragments The Hind111 and XhoI sites in the genome of the five CPV-isolates could be mapped by cross-hybridization with Digoxigenin-labeled probes derived from vacciniavirus Elstree and cowpoxvirus KR-2 Brighton (table 1) . Published maps and the general assumption that cross-hybridizing fragments represent equivalent regions in the genome enabled mapping of restriction sites for all CPV-isolates. Fig. 4 displays the physical map location of

378

Hind111 CP-1

CP-MAU

CP-NIG

CP-SA

CP-5

Elstree

XhOI CP-1

IIll

m-MALI

CP-NIG

URK

F

I,,, URK

F

E /

B

I

/ I

IV//

GNIP

C

C

E

B

I,,! GNLP

E

B

G

IllI

III/

QOIF

CP-SA

III nQ?

CP-5

II/l

URK

Elstree

I

F

, , , GLJ!

Brighton,

/ D

I

I

i!

E

B

I

/

P

1

,111 GLJO

III1

GNLP

C

c

C

I I Ill

A II

JI

I

1 E

IIJN

FMKO

1 ,,,I

B

GNXPC

/I

II NJ

f

II

I

MJ

// C

N

I

D

I 1 1 HH

QT H

A

A

A

I I

KPR

I,, /I,

,

0

Ill 0 ISV

in

I

D

I,

,I, )I I, fl H 0 ISV'

I

0

/! I XI

II,

A

D

t H

/I

I

D

HJ

PS H RN

I,,

A

,I

QTH

OISV

I

,

B

E

A

III

I H

1, / LO

E

I

n IO!&

Fig. 4. Physical maps ( HindIII. Xhol ) oC camelpoxwrus ( CPV ) isolates fromIran (CP-I ).Mauritania ( CPMAU ). Niger (CP-NIG ). Saudi-Arabia ( CP-SA ). and Dubai ( CP-5 ) The additional HirldIII bite present in all CPV Isolates is marked by an arrow. Published maps (Espositoand Knight. 1985) of vacciniaviru strain Elstree and cowpoxvms strain KR-2 Brighton are shown below

Hind111 and XhnI restriction fragments compared to the published maps of Elstree and KR3 Brighton. Earlier studies on OPV-DNA indicated a high degree of midregion DNA sequence conservation, whereas the terminal regions and the lengths of the genome showed speciesspecific variations (Esposito and Knight. 198.5). Our results were consistent with these data, but an additional Hind111 site which has not been described for any OPV-species tested so far, could be mapped in the midregion (Fig. 4, see arrowhead). Furthermore, the occurrence of small XhoI-fragments at either terminus seems to be a feature specific for CPV.

I. Renner-Miilferet

al. / Veterinct~ Micmbiolog~

45 (1995) 371-381

379

Differences within CP-isolates were also obvious. Comparison of the maps (Fig. 4) suggested the existence of a large deletion (about 25 kbp) in the right terminus of CP-NIG and two smaller sized deletion in both terminal ends of CP-SA (accounting for 15 kbp) .

4. Discussion This paper is concerned with the characterization of OPV-isolates from camels. The different geographic origin of the five isolates investigated (Iran, Saudi-Arabia, Mauritania, Niger, and Dubai) represents almost the entire region of camel raising countries. After infection of the chorioallantoic membrane, all isolates were found to produce small opaque white pocks with a ceiling temperature of about 39°C. This clearly separate CPV from vaccinia
380

1. Renner-Miiiler

et al. / Vereritzary Microbiolqp

4.5 (I 995J371-381

Acknowledgements We thank Mrs. Laura Chaudhuri and Mrs. Gudrun Z6ller for excellent technical help.

References Al-Falluji. M.M.. Tantawi. H.H., and Shony, M.O.. 1979. Isolation, identificationand characterization of camelpox virus in Iraq. J. Hyg. Camb., 83: 267-272. Baxby. D.. 1972. Smallpox-like viruses from camels in Iran. Lancet, Nov. 18, 2, 1063-1065. Baxby. D.. 1974. Differentiation of smallpox and camelpox viruses in cultures of human and monkey cells. I. Hyg. Camb.. 72: 25 l-254. Baxby. D.. 1975. Identification and interrelationships of variola/vaccinia subgroup of poxviruses. Prog. Med. Virol. 19: 2 15-246. Baxby. D.. Ramyar. H.. Hessami. M., and Ghaboosi, B., 1975. Response of camels to intradermal inoculation with smallpox and camelpox viruses. Infect. Immun. 11:617-621. Buchnev, K.N. and Sadykov. R.G.. 1969. On camelpox in Kazakhstan (in Russian 1. Tr. Nauchno-issledov. Vet. Instituta Alma-Ata 15: 12. Chauhan, R.S. and Kaushik. R.K., 1987. Isolation of camelpoxvitus in India. Brit. Vet. J., 143: 581-582. Czemy, C.-P. and Mahnel, H.. 1990. Structural and functional analysis of orthopoxvirus epitopes with neutralizing monoclonal antibodies. J. Gen. Viral.. 71: 2341-2352. Davies, F.G., Mungai, J.N., and Shaw, T., 1975. Characteristics of a Kenyan camelpox virus. J. Hyg. Camb., 75: 381-385. Downie. A.W., 1939. A study of the lesions produced experimentally by cowpoxvirus. J. Path. Bact., 48: 361379. El-Kenawy, A., Abdel-Galil, Y., El-Mekkawi. M., and Enany, M., 1989. Studies on camel pox virus isolated from camels in Sharkia govemorate. J. Egyptian Vet. Med. Assoc.. 49: 389-395. Esposito, J.J. and Knight, J.C., 1985. Orthopoxvirus DNA: a comparison of restriction profiles and maps. Virology, 142: 23&25 I. Fenner. F.. Wittek, R., and Dumbell, K.R. (Editors), 1989. The orthopoxviruses. Academic Press, San Diego. Francki. RIB.. Fouquez. CM., Knudson. D.L.. and Brown, F., 1991. Classification and nomenclature of viruses. Arch. Viral., Suppl. 2. Springer Verlag. Wien & New York. Goebel. S.J., Johnson, G.P., Perkus, M.E.. Davis. S.W.. Winslow, J.P., and Paoletti, E.. 1990. The complete DNA sequence of vaccinia virus. Virology, 179,247-266. Hafez, S.M., Al-Sukayran, A., dela Cruz. D., Mazloum. KS., Al-Bokmy. A.M., Al.Mukayel, A., and Amjad, A.M.. 1992. Development of a live cell culture camelpox vaccine. Vaccine. 10: 533-539. Hanahan, D., 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol., 166: 557-580. Hattori, M. and Sakaki. Y., 1986. Dideoxy sequencing method using denatured plasmid templates. Anal. Biochem.. 152: 232-238. Jezek, Z.. Kriz. B.. and Rothbauer. V., 1983. Camelpox and its risk to the human population I. Hyg. Epidemiol. Microbial. Immun.. 27: 2942. Mackett. M. and Archard, L.C., 1979. Conservation and variation in orthopoxvirus genome structure. J. Gen. Virol., 45: 683-701. Mahnel H. and Bartenbach, G., 1973. Classification of camelpoxvirus. J. Vet. Med. B, 20: 572-576. Marennikova, S.S., Shenkman. L.S.. Shelukhina, E.M., and Maltseva. N.N.. 1974. Isolation of camelpox virus and investigation of its properties. Acta Viral., 18: 423428. Mayr, A., Bachmann, P.A., Bibrack, B., and Wittmann, G. (Editors), 1974. Virologische Arbeitsmethoden. Band I. Gustav Fischer Verlag, Stuttgart. p 220. Meyer, H., Sutter. G.. and Mayr, A., 1991. Mapping of deletion of the genome of the highly attenuated vaccinia virus MVA and their influence on virulence. J. Gen. Virol.. 72: 1031-1038. Meyer, H. and Rziha. H.-J., 1993. Characterization of the gene encoding the A-type inclusion protein of camelpox virus and sequence comparison with other orthopoxviruses. J. Gen. Viral., 74: 1679-1684.

I. Rerwwr-Miiller

et nl. / Veterinup

Microhinlog~

45 (1995) 371-381

381

Munz. E.. Schillinger. D.. Reimann. M., and Mahnel. H., 1986. Electron microcopical diagnosis of Ecthyma Contagiosum in camels (Camelus dromedarius): First report of the disease in Kenya. J. Vet. Med. B, 33: 7317. Munz, E.. Moallin. A.S.M.. Mahnel, H.. and Reimann, M.. 1990. Camel papillomatosis in Somalia. J. Vet. Med. B, 37: 191-196. Nasemann, Th.. Mayr. A.. Schaeg. G.. Kimmig. W. und Mahnel, H.. 1987. Infektion eines Madchens mit Kuhpockenvirus. Hautarzt. 38: 414418. Nguyen-ba.vy. Richard. D., and Gillet, J.P.. 1989. Characterization of an orthopoxvirus of camels from Niger. Rev. l&v. Med. Vet. Pays Trop.. 42: 19-25. Ramyar. H. and Hessami. M.. 1972. Isolation. cultivation and characterization of camelpox virus. J. Vet, Med. B. 19: 1X2--189. Sambrook. J.. Fritsch. E.F.. and Maniatis, T., 1989. Molecular clonmg. A laboratory manual. Cold Spring Harbor Laboratory Press, New York. Southern, E.M.. 1975. Detection of specific sequences among DNA fragments separeted by gel electrophoresis. J. Mol. Biol.. 98: 503-518. Tantawi. H H., 1974. Comparative studies on camelpox. sheeppox and vaccinia viruses. Acta Virol.. IS: 347351. Tantawi. H H.. El-Dahaby. H.. and Fahmy. LX, 1978. Comparative studies on poxvirus strains isolated from camels. Acta. Viral.. 32: 45 1457. Wilson, R.T., Araya. A.. and Melaku, A., 1990. The one-humped camel. An analytical and annotated Bibliography 1980-1989. Animal Production and Health Section, Joint FAO/IAEA, Division for Nuclear Techniques in Food and Agriculture. Wien. Austria. Wittek. R.. 1982. Organization and expression of the poxvirus genome. Experientia. 38: 285-297.