1 Laboratório de Doenças Infecciosas, CIISA, Faculdade de Medicina Veterinária, R. Professor Cid dos Santos, Polo Universitário do Alto da Ajuda, 1300-477 Lisboa, Portugal
2 Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 ONF, UK
Correspondence
F. S. Boinas
fboinas{at}fmv.utl.pt
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
When the disease was endemic in the Iberian Peninsula, the most common transmission mechanism was considered to be direct contact with infected pigs including asymptomatic persistently infected pigs (Ordas et al., 1983; Perestrelo Vieira, 1993
). Other mechanisms of transmission include ingestion of infected pork products. In southern Portugal and Spain, the long-term persistence of ASFV in the field has also been associated with contact with the argasid tick Ornithodoros erraticus (Boinas et al., 2001a
; Perez-Sanchez et al., 1994
; Sanchez Botija, 1963
; Vigario & Caiado, 1989
). In one previous study, Sanchez Botija (1963)
reported that the viruses they isolated from O. erraticus were virulent in pigs and caused haemadsorption (HAD) in pig monocyte cultures. In other studies in the 1980s, ASFV was isolated from ticks in Alentejo in southern Portugal (Louza et al., 1989
) and Spain (J. M. Sanchez-Vizcaino, personal communication) but the properties of these viruses were not characterized.
Most ASF virus isolates cause HAD of erythrocytes to infected cells but there are several isolates that do not and these are referred to as non-haemadsorbing (non-HAD) isolates. This property is used as a diagnostic assay for virus isolation. The ASFV protein, CD2v (Kay-Jackson et al., 2004) responsible for haemadsorption to infected cells is encoded by a gene, EP402R, which is similar to the T-lymphocyte adhesion receptor CD2 (Borca et al., 1994
; Rodriguez et al., 1993
). The CD2v protein is incorporated into the membranes of virus particles as they bud through the membrane of infected cells, and is therefore probably also involved in the association of extracellular virus particles with red blood cells (Ruiz-Gonzalvo et al., 1996
). In blood from pigs infected with HAD isolates, but not non-HAD isolates, the majority of virus is associated with the red blood cell fraction (Borca et al., 1998
; Sierra et al., 1991
). Deletion of the gene encoding CD2v from the genome of a virulent HAD isolate delayed the onset of viraemia and dissemination of virus within infected pigs (Borca et al., 1998
). The CD2v protein is also suggested to have a role in impairment of lymphocyte proliferation in response to mitogens (Borca et al., 1998
).
Large deletions and insertions of DNA were found as a result of gain or loss of members of several different multigene families near the left and right genome termini (Almendral et al., 1990; Blasco et al., 1989a
, b
; Delavega et al., 1990
, 1994
; Gonzalez et al., 1990
; Rodriguez et al., 1994
; Yozawa et al., 1994
). The European, Caribbean and Cameroon isolates of ASFV are closely related to the Lisbon 60 isolate, which was the second introduction of the virus in Portugal. Other African isolates are more diverse. Viruses resembling Lisbon 57 and 60 are distributed throughout the west and central parts of West Africa (Bastos et al., 2003
; Ekue & Wilkinson, 2000
; Odemuyiwa et al., 2000
; Wesley et al., 1984
; Wilkinson et al., 1989
, 1993
). Isolates from Malawi and Zambia in East Africa are very different from these (Sumption, 1992
; Sumption et al., 1990
).
In this study, we identified two types of viruses collected from O. erraticus ticks inhabiting pig farms in southern Portugal during the period 1988 until 1993. One type is pathogenic for domestic pigs and causes HAD, and the second type is non-HAD and non-pathogenic. The ability of the isolates to cross-protect and be transmitted by direct contact was characterized, and preliminary characterization of the genomes was carried out. The implications of these findings in understanding the epidemiology of ASF are discussed.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The reference viruses used were a Malawi isolate (Lil 20/1) (Haresnape et al., 1988), Lisbon 57 and 60, Tomar 86 and 87 (supplied by J. Vigário, Laboratorio Nacional de Investigação Veterinária, Lisbon).
Preparation of virus samples from pigs.
Blood was collected from the anterior vena cava for whole blood in EDTA. The tissues collected for analysis were weighed and ground up in PBS with sterile sand in a pestle and mortar. Supernatants were collected and stored at 70 °C.
Detection of antibodies in pigs.
ELISA assay was used to detect antibodies against ASFV (Office International des Epizooties, 1996).
Isolation and titration of virus in pig cell cultures.
Viruses isolated from ticks were characterized by infecting cultures of pig bone marrow (PBM) cells (Malmquist & Hay, 1960; Plowright et al., 1968
) and pig blood leukocyte cultures (Martins et al., 1993
), and observed to detect haemadsorption of erythrocytes to infected cells and cytopathic effects. Titres of virus were determined as the amount of virus causing haemadsorbtion (for HAD isolates) or cytopathic effects (for non-HAD isolates) in 50 % of infected cultures (TCID50 ml1 or HAD50 ml1). These assays gave very similar titres.
HAD viruses isolated from ticks were passaged once, either in pigs or PBM cells. The non-HAD viruses from ticks were from the fourth passage in PBM cells.
Isolation of ASF viral DNA.
HAD isolates obtained from ticks were used to infect pigs and viral DNA was extracted from infected pig blood (Wesley et al., 1984). PBM cell cultures were infected with the non-HAD viruses from ticks and the HAD virus isolate Tomar 87. Viral DNA was purified from supernatants from infected cultures (Wesley & Pan, 1982
).
Restriction enzyme digestion of ASF virus DNA.
Viral DNA was digested with the restriction enzyme BamHI (Roche), according to the manufacturer's recommendations. After digestion, DNA fragments were end-labelled with [32P]dATP using the Klenow fragment of DNA polymerase I. Fragments were run on 0·6 % agarose gels. Gels were dried and exposed to X-ray film. To confirm the genomic location of the variable fragments, Southern blotting was carried out using probes prepared from clones of the BA71V isolate (Ley et al., 1984).
Infection of pigs.
Serum was collected from cross-bred Large White/Landrace pigs of 2030 kg live weight, before infection. Each pig was inoculated intramuscularly with 2 ml of suspension with virus titres of log HAD50 ml1 or TCID50 ml1 2·8 to 4·5. Clinical examination and rectal temperatures were recorded each day. Viraemia and antibodies were monitored weekly or when the temperature of pigs rose above 40 °C. This was carried out until the animals died or were euthanized or for a minimum period of 3 weeks. Experiments were carried out under Home Office Licence 90/00752.
Laboratory infection of ticks by feeding on pigs.
Feeding of O. erraticus ticks on viraemic pigs was carried out after they were anaesthetized with pentobarbital-Na (Sagatal) (Boinas, 1995).
Contact transmission.
Donor pigs were infected as described, and placed in direct contact with healthy recipient pigs. Both groups were monitored for rectal temperature and other clinical signs, viraemia and seroconversion for periods up to 49 days. In addition to this, in two experiments with the virus OUR T88/2 pharyngeal swabs and post-mortem were performed on all the donors. Virus titres were determined in selected lymphatic organs, lungs and, in one case, an ulcer on the rear hook.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Viruses isolated from the ticks were of two types: OUR T88/1, OUR T91/1, OUR T91/2, MAR T92/1, MAR T93/1 and MAR T93/2 caused HAD and the isolates OUR T88/2, OUR T88/3, OUR T88/4 and OUR T88/5 did not cause HAD (i.e. non-HAD). All of these non-HAD virus isolates were from one farm in Ourique district (farm 108) and these were isolated from ticks collected during three visits to the farm in 1988. Two HAD isolates were also obtained from farm 108 (OUR T91/1 and OUR T91/2) from ticks collected during a single visit in 1991. A single HAD isolate (OUR T88/1) was obtained in 1988 from farm 106 in Ourique and three HAD isolates (MAR T92/1, MAR T93/1 and MAR T93/2) were collected from one farm in Castro Verde district (farm 103) during two visits in 1992 and 1993. The isolates were from ticks collected between 268 and 1070 days after outbreaks of ASF had occurred.
Characterization of virus isolates by restriction enzyme site mapping of the virus genomes
Pigs were infected with HAD isolates and viral DNA was isolated from the red blood cell fraction of infected blood. BamHI digests of viral DNA were end-labelled with [32P] and separated by agarose gel electrophoresis. This identified several fragments that varied in length when genomes of different isolates were compared (Fig. 1a). A BamHI restriction enzyme site map of the Lisbon 60 isolate genome has previously been prepared (Ekue & Wilkinson, 2000
) and the restriction enzyme digest patterns were compared with those of the Lisbon 60 and 57 isolates. The genomes of the HAD isolates OUR T88/1 and OUR T91/1, which were isolated from ticks from different farms (farms 106 and 108), had identical fragment sizes (Fig. 1a
) and the total genome length was about 179 kbp. Genome locations of fragments, which differed in length from those in the Lisbon 60 isolate, were confirmed by probing Southern blots of restriction enzyme digests with cloned DNA fragments (Fig. 1b
). Isolates OUR T88/1, OUR T91/1 and OUR T91/2 could be distinguished from the Lisbon 60 isolate by difference in length of one fragment, J, in the right terminal region, which was 7·1 kbp in Lisbon 60 and 7·15 kbp in OUR T88/1, OUR T91/1 and OUR T91/2 isolates (Fig. 1b
and Fig. 2
). OUR T88/1, OUR T91/1 and OUR T91/2 could be distinguished by the mobility of four restriction enzyme fragments (B and C at the left end of the genome, L in the centre and O at the right end) from viruses isolated north of the River Tagus in 1986 from pigs (supplied by J. Vigario, Laboratorio Nacional de Investigacão Veterinaria, Lisbon) (Figs 1 and 2
) (P. J. Wilkinson, unpublished results). The digestion pattern of one of these isolates, Tomar 86 is shown in Fig. 1a
. Isolates MAR T92/1 and MAR T93/1 had identical restriction enzyme fragments and there was no difference in the sizes of the 22 restriction fragments when compared to the Lisbon 60 isolate (Fig. 1a
). Thus, the genomes analysed from the HAD isolates obtained from ticks were very similar to the Lisbon 60 isolate but differed significantly in several genome regions from isolates from north of the River Tagus including the Tomar 86 isolate. The genome of the HAD isolate MAR T93/2 was not analysed. The BamHI restriction enzyme fragment patterns of two non-HAD ASF virus isolate (OUR T88/2 and OUR T88/3) genomes differed in the size of the left terminal fragment, which was 19·4 kbp in the OUR T88/2 and 18·6 kbp in the OUR T88/3 isolate (Figs 1 and 2
). Thus, it is possible that these two isolates were derived from a common ancestor by gain or loss of sequences within the left terminal fragment. Both of these isolates differed from the genomes of the HAD isolates OUR T88/1, OUR T91/1 and OUR T91/2. In addition to having a larger left terminal fragment, both non-HAD virus isolates had a deletion of 9·6 kbp in the fragment adjacent to the left terminal fragment (fragment C), an insertion of 0·2 kbp in the central region (fragment L) and a deletion of 1·6 kbp from the right terminal fragment (fragment O) (Figs 1 and 2
).
|
|
Stability of virus genomes following passage in ticks
Virus was isolated from ticks that were kept alive in the laboratory for 2 years after feeding on a viraemic pig (log HAD50 ml1 7·0), which had been infected with a virulent HAD isolate from a pig (Tomar 87). Isolates from different ticks were characterized and all retained the HAD phenotype. Following growth of these viruses in PBM cells, viral DNA was purified. The fragments produced by BamHI digestion of genomes from three of these tick isolates (ticks 9, 16 and 18) had identical sizes, when compared to the virus isolate used to infect the pig (Tomar 87) (Fig. 3). This study indicated that major genome rearrangements did not occur over a 2 year period during virus replication in ticks and that the HAD phenotype was retained.
|
|
|
Pigs RY14, RY15, RZ7 and RZ10, which were infected with the isolate OUR T88/4 or that were challenged with the HAD isolate OUR T88/1 and recovered from infection, were re-challenged with the virulent Lisbon 57 isolate. Pigs were either resistant (pig RY15) or there was a delay of up to 14 days in the onset of typical acute disease (pigs RY14, RZ7 and RZ10) (Table 1). The only pig (RY15) that resisted Lisbon 57 challenge had previously been inoculated twice with OUR T88/1 isolate at 35 and 49 days p.i. This pig was subsequently challenged with a pathogenic HAD isolate from Malawi (Lil 20/1). In this pig, the onset of acute ASF was delayed and the pig was moribund and killed at 25 days post-challenge. All the pigs that succumbed to challenge had high viraemia at the time of death (log HAD50 ml1 6·06·2) and high titres of virus were isolated from the tissues (log HAD50 g1 6·08·8).
Contact transmission of HAD and non-HAD virus isolates between domestic pigs
HAD isolate OUR T88/1.
Two pigs were infected with the virulent HAD isolate OUR T88/1 (Fig. 4). Two non-infected pigs were exposed by contact with these infected donor pigs from the first day post-pyrexia, which was 4 days p.i. The infected donor pigs had viraemia of log HAD50 ml1 67 at this time. Contact was maintained between the donor and contact pigs for 2 days. Rectal temperatures of the contact pigs were monitored and, at 5 days after the first day of exposure to the infected pigs, rose above 40 °C. From day 6 after the first day of exposure, both contact pigs showed typical ASF clinical symptoms, developed viraemia of log HAD50 ml1 79 and, on the post-mortem, acute ASF lesions and high virus titres were detected in the organs.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The non-HAD virus isolates did not cause clinical disease but sporadic low viraemia was detected and virus was recovered from several lymph nodes (Table 2). Antibodies against ASFV were first detected from 8 days p.i. and persisted over the course of experiments (up to 49 days p.i.). Pigs infected with two of these non-pathogenic non-HAD virus isolates were protected against challenge with the pathogenic HAD virus (OUR T88/1), which was isolated from the same farm (Table 1
). Less effective protection was achieved when recovered pigs were challenged with more distantly related isolates. Only one pig resisted challenge with the Lisbon 57 isolate, although the onset of clinical disease was delayed in the other pigs challenged with this virus. The onset of clinical symptoms was also delayed when recovered pigs were challenged with the distantly related Malawi LIL 20/1 virus isolate. The Malawi virus genome differs considerably from that of European isolates (Dixon et al., 1994
; Sumption et al., 1990
; Yanez et al., 1995
). Thus, epitopes conferring protective immunity may not be conserved.
Previously non-HAD virus isolates were collected from pigs in the field from the south of Portugal where most of the herds with seropositive pigs were detected (Vigario & Caiado, 1989; Vigario et al., 1974
). Non-HAD strains were also frequently reported in Spain, with a total of 206 non-HAD isolates obtained in the period between 1965 and the first semester of 1974 (Sanchez Botija et al., 1977
). These non-HAD viruses are more difficult to isolate than HAD viruses since the viraemia they cause is sporadic and virus has mostly been isolated in small amounts from the pigs' organs. Infection of pig herds with non-pathogenic non-HAD isolates may account for some of the seropositive herds detected in the field in the absence of clinical symptoms. Our experiments showed that pigs infected with non-pathogenic non-HAD isolates can be protected from challenge with closely related virulent isolates. Thus, herds infected with non-pathogenic virus may at least be partially protected from infection with pathogenic virus. Our transmission experiments showed that the non-pathogenic non-HAD isolates were less efficiently transmitted to contact pigs (
4050 % transmission) than the virulent HAD isolate (100 % transmission). Nevertheless, contact of inapparent infected pigs with previously uninfected herds could have been an effective mechanism for maintaining ASF in the field in Portugal. The non-HAD isolates we obtained were from O. erraticus ticks. However, it is not clear how efficiently these viruses can infect and persist in the tick population. Pigs infected with non-HAD isolates developed only low and sporadic viraemia. Ticks, which feed on these pigs, may therefore not be efficiently infected with virus. Further studies are required to determine how efficiently the non-HAD isolates can replicate and persist in tick populations. The non-HAD isolates (OUR T88/2 and OUR T88/3) from ticks had large genomic deletions when compared with the HAD tick isolate OUR T88/1 and with the other Portuguese isolates. These deletions consisted of approximately 9 kbp from a region close to the left end of the genome and 1·6 kbp from the right terminal fragment. Although we have not precisely mapped these deletions, those on the left of the genome are in a region encoding members of multigene families 360 and 530. Variation in the number of copies of these multigene families in different ASFV isolates have previously been described (Almendral et al., 1990
; Delavega et al., 1990
; Gonzalez et al., 1990
; Yanez et al., 1995
; Yozawa et al., 1994
). Members of multigene families 360 and 530 have been demonstrated to act as virulence factors for domestic pigs (Tulman & Rock, 2001
; Zsak et al., 2001
). Deletion of the UK and DP71L genes from the right end of the genome has also been shown to reduce virulence of the virus for domestic pigs, although these genes are found in the genomes of all isolates analysed (Zsak et al., 1996
, 1998
). The low pathogenicity of the non-HAD isolates may be related to loss of virulence factors because of these larger genome deletions close to the left end of the genome or to smaller deletions or substitutions within genes encoding virulence factors elsewhere on the genome.
The loss of the HAD phenotype presumably results from mutations within the EP402R gene, which encodes the CD2v protein. The property of non-HAD does not determine the pathogenicity of an isolate. Non-HAD isolates causing up to 8090 % mortality have been isolated in outbreaks from pigs in South Africa and Madagascar (Gonzague et al., 2001; Pini & Wagenaar, 1974
). Non-HAD viruses of reduced virulence have also previously been isolated in Africa (Thomson et al., 1979
). Deletion of the gene encoding the CD2v protein from a virulent ASFV isolate delayed the onset of viraemia and the dissemination of virus within pigs, but did not reduce the mortality rate caused by the virus (Borca et al., 1998
). This suggests that the reduction in virulence of the non-HAD isolates is because of other factors. Expression of the CD2v protein on extracellular virus particles correlates with the association of the majority of virus with red cells in infected pig blood. Loss of the HAD phenotype may therefore be a factor in the low and sporadic viraemia observed in infections with the non-HAD isolates. However, other factors, which might result in reduced virus replication in blood or tissue macrophages, could also be important.
ASFV does not readily undergo major genome modifications when passaged in pigs. This was shown by the similarity of the genome following up to 20 experimental transmission in pigs or after 17100 passages in pig macrophage cultures (Blasco et al., 1989a, b
; Ekue et al., 1989
). After 10 passages in PBM cells, the Cameroon virus (CAM 82) became non-HAD and remained non-HAD in the following 17 passages in PBM cells (Ekue, 1989
). However, no differences were found in the BamHI and EcoRI restriction enzyme site maps of these genomes when compared to the virus before passage, and no differences in the pathogenicity of the isolate for pigs were reported.
The stability of the virus genome following passage in ticks has not previously been investigated. Here, we compared viruses obtained from three separate ticks, which had been fed on a viraemic pig 2 years previously. We did not detect differences in either restriction enzyme fragments or alteration in HAD phenotype, suggesting that the virus genome is stable in ticks. This type of analysis is relatively crude and there may be an accumulation of nucleotide substitutions in some critical genes, which we have not detected by restriction enzyme fragment analysis.
Viruses reported as attenuated have been isolated from nature in Portugal, but not further characterized other than for pathogenicity. It is known that the vaccine strain used extensively in the south of Portugal in the 1960s caused death of 3·4 % of the vaccinated animals, complications in a further 7 % and sometimes caused the occurrence of chronic carriers of the virus (Nunes Petisca, 1965a, b
). This virus was derived from the Lisbon 60 isolate after being passaged up to 150 times in PBM cells and always showed haemadsorption (Manso Ribeiro et al., 1963
).
The virus isolates characterized in this study were collected from areas where extensive vaccination took place in the 1960s (Manso Ribeiro et al., 1963). Since the introduction of the attenuated vaccine strain in the field in Portugal and Spain, chronic forms of ASF have been described and non-HAD virus isolates were also reported at this time (Vigario et al., 1974
). One possibility is that the non-pathogenic isolates we obtained from ticks are derived from this attenuated vaccine strain. Alternatively, they may have been derived from the original virulent isolate present in Portugal in 1960 and selected for as a consequence of their ability to persist in the field.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bastos, A. D., Penrith, M. L., Cruciere, C., Edrich, J. L., Hutchings, G., Roger, F., Couacy-Hymann, E. & Thomson, G. R. (2003). Genotyping field strains of African swine fever virus by partial p72 gene characterisation. Arch Virol 148, 693706.[Medline]
Blasco, R., Aguero, M., Almendral, J. M. & Vinuela, E. (1989a). Variable and constant regions in african swine fever virus DNA. Virology 168, 330338.[CrossRef][Medline]
Blasco, R., de la Vega, I., Almazan, F., Aguero, M. & Vinuela, E. (1989b). Genetic variation of african swine fever virus: variable regions near the ends of the viral DNA. Virology 173, 251257.[Medline]
Boinas, F. (1995). The role of Ornithodoros erraticus in the epidemiology of African swine fever in Portugal. In Department of Agriculture, pp. 240. Reading: University of Reading.
Boinas, F., Cruz, B., Portugal, F. C., Portugal, R., Mendes, S., Leitão, A., Martins, C. & Rosinha, A. (2001a). Evaluation of the role of Ornithodoros erraticus as a reservoir of African swine fever in Alentejo - Portugal. In Report on the Annual Meeting of the National Swine Fever Laboratories, pp. 9798. Lindholm, Denmark: European Commission.
Boinas, F., Pinheiro, C., Sobral, L. & Rosinha, A. (2001b). Erradicação de um surto esporádico de peste suína Africana em Portugal. In IX Congresso Internacional de Medicina Veterinária em Língua Portuguesa, pp. 267268. Edited by R. Dubois. Salvador da Baia, Brazil: Sociedade Brasileira de Medicina Veterinária e Sociedade de Medicina Veterinária da Baia.
Borca, M. V., Kutish, G. F., Afonso, C. L., Irusta, P., Carrillo, C., Brun, A., Sussman, M. & Rock, D. L. (1994). An African swine fever virus gene with similarity to the T-lymphocyte surface antigen CD2 mediates hemadsorption. Virology 199, 463468.[CrossRef][Medline]
Borca, M. V., Carrillo, C., Zsak, L., Laegreid, W. W., Kutish, G. F., Neilan, J. G., Burrage, T. G. & Rock, D. L. (1998). Deletion of a CD2-like gene, 8-DR, from African swine fever virus affects viral infection in domestic swine. J Virol 72, 28812889.
Comissão das Comunidades Europeias (1999). Decisão da Comissão de 3 de Dezembro de 1999 que diz respeito a certas medidas de protecção relativas à peste suína Africana em Portugal. Jornal Oficial das Comunidades Europeias L310, 7173.
de la Vega, I., Vinuela, E. & Blasco, R. (1990). Genetic variation and multigene families in African swine fever virus. Virology 179, 234246.[Medline]
de la Vega, I., Gonzalez, A., Blasco, R., Calvo, V. & Vinuela, E. (1994). Nucleotide sequence and variability of the inverted terminal repetitions of African swine fever virus DNA. Virology 201, 152156.[CrossRef][Medline]
Dixon, L. K., Twigg, S. R., Baylis, S. A., Vydelingum, S., Bristow, C., Hammond, J. M. & Smith, G. L. (1994). Nucleotide sequence of a 55 kbp region from the right end of the genome of a pathogenic African swine fever virus isolate (Malawi LIL20/1). J Gen Virol 75, 16551684.[Abstract]
Dixon, L. K., Costa, J. V., Escribano, J. M., Rock, D. L., Vinuela, E. & Wilkinson, P. J. (2000). Asfarviridae. In Virus Taxonomy. Seventh Report of the Internaitonal Committee on Taxonomy of Viruses, pp. 159165. Edited by M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle, R. B. Wickner. London: Academic Press.
Ekue, N. F. (1989). Epidemiology of African swine fever in Cameroon. In Department of Microbiology, pp. 231. Guildford: University of Surrey.
Ekue, N. F. & Wilkinson, P. J. (2000). Comparison of genomes of African swine fever virus isolates from Cameroon, other African countries and Europe. Rev Elev Med Vet Pays Trop 53, 229236.
Ekue, N. F., Wilkinson, P. J. & Wardley, R. C. (1989). Infection of pigs with the Cameroon isolate (Cam/82) of African swine fever virus. J Comp Pathol 100, 145154.[Medline]
Gonzague, M., Roger, F., Bastos, A., Burger, C., Randriamparany, T., Smondack, S. & Cruciere, C. (2001). Isolation of a non-haemadsorbing, non-cytopathic strain of African swine fever virus in Madagascar. Epidemiol Infect 126, 453459.[CrossRef][Medline]
Gonzalez, A., Calvo, V., Almazan, F., Almendral, J. M., Ramirez, J. C., de la Vega, I., Blasco, R. & Vinuela, E. (1990). Multigene families in African swine fever virus: family 360. J Virol 64, 20732081.[Medline]
Haresnape, J. M., Wilkinson, P. J. & Mellor, P. S. (1988). Isolation of African swine fever virus from ticks of the Ornithodoros moubata complex (Ixodoidea: Argasidae) collected within the African swine fever enzootic area of Malawi. Epidemiol Infect 101, 173185.[Medline]
Kay-Jackson, P. C., Goatley, L. C., Cox, L., Miskin, J. E., Parkhouse, R. M. E., Wienands, J. & Dixon, L. K. (2004). The CD2v protein of African swine fever virus interacts with the actin-binding adaptor protein SH3P7. J Gen Virol 85, 119130.
Ley, V., Almendral, J. M., Carbonero, P., Beloso, A., Vinuela, E. & Talavera, A. (1984). Molecular cloning of African swine fever virus DNA. Virology 133, 249257.[CrossRef][Medline]
Louza, A. C., Boinas, F. S., Calado, J. M., Vigario, J. D. & Hess, W. R. (1989). Role of invertebrate vectors and animal reservoirs in the maintenance of ASF in Portugal. Epidemiol Sante Anim 15, 89102.
Malmquist, W. A. & Hay, D. (1960). Hemadsorption and cytopathic effect produced by ASFV in swine bone marrow and buffy coat cultures. Am J Vet Res 21, 104108.[Medline]
Manso Ribeiro, J., Nunes Petisca, J. L., Lopes Frazao, F. & Sobral, M. (1963). Vaccination against ASF. Bull Off Int Epizoot 60, 921937.
Martins, C. L., Lawman, M. J., Scholl, T., Mebus, C. A. & Lunney, J. K. (1993). African swine fever virus specific porcine cytotoxic T cell activity. Arch Virol 129, 211225.[Medline]
Nunes Petisca, J. L. (1965a). Some morphological aspects of vaccination of swine against ASF (virus L) in Portugal. Bull Off Int Epizoot 63, 199237.
Nunes Petisca, J. L. (1965b). Studies of anatomical-pathology and histopathology on ASF (virus L) in Portugal. Bull Off Int Epizoot 63, 103142.
Odemuyiwa, S. O., Adebayo, I. A., Ammerlaan, W. & 7 other authors (2000). An outbreak of African swine fever in Nigeria: virus isolation and molecular characterization of the VP72 gene of a first isolate from West Africa. Virus Genes 20, 139142.[CrossRef][Medline]
Office International des Epizooties (1996). African Swine Fever. In OIE Manual, pp. 13714. Edited by OIE. Paris: OIE.
Ordas, A., Sanchez-Botija, C. & Diaz, S. (1983). Epidemiological studies on African swine fever in Spain. In Coordination of Agricultural research. African swine fever, pp. 6773. Commission of the European Communities edition. Edited by P. J. Wilkinson. Sardinia: European Commission.
Perestrelo Vieira, R. (1993). Evolution of African swine fever in Portugal. In Coordination of agricultural research. African swine fever, pp. 4351. Commission of the European Communities edition. Edited by A. Galo. Lisbon: European Commission.
Perez-Sanchez, R., Astigarraga, A., Oleaga-Perez, A. & Encinas-Grandes, A. (1994). Relationship between the persistence of African swine fever and the distribution of Ornithodoros erraticus in the province of Salamanca, Spain. Vet Rec 135, 207209.[Medline]
Pini, A. & Wagenaar, G. (1974). Isolation of a non-haemadsorbing strain of African swine fever (ASF) virus from a natural outbreak of the disease. Vet Rec 94, 2.[Medline]
Plowright, W., Parker, J. & Staple, R. F. (1968). The growth of a virulent strain of African swine fever virus in domestic pigs. J Hyg (Lond) 66, 117134.[Medline]
Rodriguez, J. M., Yanez, R. J., Almazan, F., Vinuela, E. & Rodriguez, J. F. (1993). African swine fever virus encodes a CD2 homolog responsible for the adhesion of erythrocytes to infected cells. J Virol 67, 53125320.[Abstract]
Rodriguez, J. M., Yanez, R. J., Pan, R., Rodriguez, J. F., Salas, M. L. & Vinuela, E. (1994). Multigene families in African swine fever virus: family 505. J Virol 68, 27462751.[Abstract]
Ruiz-Gonzalvo, F., Rodriguez, F. & Escribano, J. M. (1996). Functional and immunological properties of the baculovirus-expressed hemagglutinin of African swine fever virus. Virology 218, 285289.[CrossRef][Medline]
Sanchez Botija, C. (1963). Reservorios del virus da la Peste Porcina Africana. Bull Off Int Epizoot 60, 895899 (in Spanish).
Sanchez Botija, C., Ordaz, A., Solana, A., Gonzalvo, F., Olias, J. & Carnero, M. E. (1977). Peste Porcina Africana: observaciones sobre modificacion espontanea del virus de campo. An Inst Invest Vet 24, 717 (in Spanish).
Sierra, M. A., Gomez-Villamandos, J. C., Carrasco, L., Fernandez, A., Mozos, E. & Jover, A. (1991). In vivo study of hemadsorption in African swine fever virus infected cells. Vet Pathol 28, 178181.[Medline]
Sumption, K. J. (1992). Genotypic comparison of African swine fever virus isolates from Zambia and Malawi. In Department of Agriculture, pp. 324. Reading: University of Reading.
Sumption, K. J., Hutchings, G. H., Wilkinson, P. J. & Dixon, L. K. (1990). Variable regions on the genome of Malawi isolates of African swine fever virus. J Gen Virol 71, 23312340.[Abstract]
Thomson, G. R., Gainaru, M. D. & van Dellen, A. F. (1979). African swine fever: pathogenicity and immunogenicity of two non-haemadsorbing viruses. Onderstepoort J Vet Res 46, 149154.[Medline]
Tulman, E. R. & Rock, D. L. (2001). Novel virulence and host range genes of African swine fever virus. Curr Opin Microbiol 4, 456461.[CrossRef][Medline]
Vigario, J. D. & Caiado, J. (1989). Situazione epidemiologica in Portogallo. In Peste Suina Africana, pp. 123132. Edited by I. Z. S. p. l. Sardegna. Nuoro: Istituto Zooprofilattico Sperimentale Sardegna.
Vigario, J. D., Terrinha, A. M. & Moura Nunes, J. F. (1974). Antigenic relationships among strains of African swine fever virus. Arch Gesamte Virusforsch 45, 272277.[Medline]
Wesley, R. D. & Pan, I. C. (1982). African swine fever virus DNA: restriction endonuclease cleavage patterns of wild-type, Vero cell-adapted and plaque-purified virus. J Gen Virol 63, 383391.[Abstract]
Wesley, R. D., Quintero, J. C. & Mebus, C. A. (1984). Extraction of viral DNA from erythrocytes of swine with acute African swine fever. Am J Vet Res 45, 11271131.[Medline]
Wilkinson, P., Dixon, L., Sumption, K. & Ekue, F. (1989). Molecular epidemiology of African swine fever, In Abstracts of the Annual Meeting of the Association of Veterinary Teachers and Research Workers 1989, abstract B15, p. 18. Scarborough: Association of Veterinary Teachers and Research Workers.
Wilkinson, P. J., Dixon, L. K., Sumption, K., Ekue, F., Hutchings, G. H., Payne, A. & Boinas, F. (1993). Genetic variation and epidemiology of African swine fever in Europe and Africa. In IXth International Congress of Virology 1993, abstract, p. 223. Glasgow, Scotland: International Congress of Virology.
Yanez, R. J., Rodriguez, J. M., Nogal, M. L., Yuste, L., Enriquez, C., Rodriguez, J. F. & Vinuela, E. (1995). Analysis of the complete nucleotide sequence of African swine fever virus. Virology 208, 249278.[CrossRef][Medline]
Yozawa, T., Kutish, G. F., Afonso, C. L., Lu, Z. & Rock, D. L. (1994). Two novel multigene families, 530 and 300, in the terminal variable regions of African-swine-fever virus genome. Virology 202, 9971002.[CrossRef][Medline]
Zsak, L., Lu, Z., Kutish, G. F., Neilan, J. G. & Rock, D. L. (1996). An African swine fever virus virulence-associated gene NL-S with similarity to the herpes simplex virus ICP34.5 gene. J Virol 70, 88658871.[Abstract]
Zsak, L., Caler, E., Lu, Z., Kutish, G. F., Neiland, J. G. & Rock, D. L. (1998). A nonessential African swine fever virus gene UK is a significant virulence determinant in domestic swine. J Virol 72, 10281035.
Zsak, L., Lu, Z., Burrage, T. G., Neilan, J. G., Kutish, G. F., Moore, D. M. & Rock, D. L. (2001). African swine fever virus multigene family 360 and 530 genes are novel macrophage host range determinants. J Virol 75, 30663076.
Received 25 February 2004;
accepted 26 April 2004.