93

Virus Research, 17 (1990) 93-104 Elsevier

VIRUS

00601

Interaction

Antonio

of African swine fever virus with macrophages

Alcami,

Angel

Centro de Biologia Molecular (CSIC-UAM),

L. Carrascosa

and

Eladio

Viiiuela

Facultad de Ciencias, Universidad Autbnoma, Madrid, Spain

(Accepted

21 May 1990)

Summary Morphological data obtained by electron microscopy have shown that African swine fever virus adapted to VERO cells enters swine macrophages, its natural host cell, by a mechanism of receptor-mediated endocytosis. Binding studies with 3Hlabeled virus and competition experiments with UV-inactivated virus have shown that the virus entry that leads to a productive infection in swine macrophages is mediated by saturable binding sites on the plasma membrane. The virus also penetrated into rabbit macrophages that do not produce infectious virus and initiated the synthesis of some early viral proteins; however, the viral replication cycle was aborted since viral DNA synthesis did not occur. The interaction of ASF virus particles with rabbit macrophages was mediated by nonsaturable binding sites, suggesting that the lack of specific receptors in these cells may be related to the absence of a productive infection. A similar abortive infection was detected in macrophages from other virus-resistant animal species. African binding;

swine fever virus; Abortive infection

Virus entry;

Receptor-mediated

endocytosis;

Saturable

Introduction African al., 1983;

swine fever (ASF) is a disease of domestic pigs (Hess, 1981; Wardley et Vifiuela, 1985; Viiiuela, 1987) caused by an icosahedral cytoplasmic

Correspondence to: E. Vifiuela, Centro de Biologia Universidad Autbnoma, 28049 Madrid, Spain.

0168-1702/90/$03.50

Molecular

0 1990 Elsevier Science Publishers

(CSIC-UAM),

B.V. (Biomedical

Facultad

Division)

de Ciencias,

94

deoxyvirus with lipid envelopes (Breese and De Boer, 1966; Almeida et al., 1967; Moura Nunes et al., 1975; Enjuanes et al., 1976b; Carrascosa et al., 1984). ASF virus multiplies in pigs and related animal species (Vitiuela, 1985) where it replicates in peripheral monocytes and macrophages (Maurer et al., 1958; Malmquist and Hay, 1960) and in a small fraction of polymorphonuclear leukocytes (Casal et al., 1984). VERO-adapted ASF virus enters susceptible cells by receptor-mediated endocytosis (Geraldes and Valdeira, 1985; Valdeira and Geraldes, 1985; Alcami et al., 1989b) and virions attach to VERO cells, but not to virus-resistant L cells, through saturable binding sites on the plasma membrane (Alcami et al., 1989a). The possibility that virus adsorption to the host cell in natural infections is not mediated by specific receptors could account for the absence of neutralizing antibodies in infected pigs (De Boer, 1967). For this reason, we have investigated the interaction of ASF virus particles with swine macrophages, the natural host cell of the virus, and with macrophages from virus-resistant animal species, in order to know whether specific cellular receptors are involved in the infection of primary cultures. In this paper we report that ASF virus adapted to VERO cells enters swine macrophages by a receptor-mediated endocytosis mechanism and that saturable binding sites are directly involved in the infection process. We also show that an abortive infection takes place in macrophages from virus-resistant animal species.

Materials and Methods Cells and virus

Alveolar macrophages were obtained by broncho-alveolar lavage from miniature pigs (Sachs et al., 1976), according to the method described by Carrascosa et al. (1982), or from New Zealand White rabbits, according to Myrvik et al. (1961). Peripheral blood monocytes were obtained from miniature pigs or White Leghorn chickens. Human monocytes were prepared from blood extracted from healthy volunteers. Monocytes were purified by adherence to tissue culture plates of peripheral blood mononuclear cells obtained by sedimentation of heparinized blood on Ficoll-Paque (Pharmacia) as described (Boyum, 1968). Macrophages and monocytes were cultured in Dulbecco’s modified Eagle (DME) medium supplemented with 30% homologous serum, except for rabbit macrophages, where DME medium was supplemented with 10% homologous serum. To study the early steps of infection of swine macrophages, we have chosen the VERO-adapted ASF virus strain (BA71V) (Garcia-Barren0 et al., 1986), which maintains the capacity to infect swine macrophages (Enjuanes et al., 1976a), due to the availability of highly purified preparations of virus particles, necessary to perform binding and competition experiments. To test for possible differences between the adapted and the non-adapted virus strain (BA71) (Garcia-Barren0 et al., 1986) the latter has also been used occasionally. Purified BA71V particles (Carrascosa et al., 1985), either unlabeled or labeled with [ 3H]thymidine ( 3H-BA71V)

95

to a specific activity of about 1 x lo4 cpm/hg of protein, were used for binding and competition experiments, and for electron microscopy. The PFU to particle ratio in these preparations is 1: 50 (Carrascosa et al., 1985) and the amount of virus particles used in the experiments was calculated from the protein concentration of the purified virus preparations. UV-inactivated purified BA71V (UV-BA7lV) was obtained as described (Alcaml et al., 1989a). To study viral protein and DNA synthesis in the course of the infection, extracdlular ASF virus concentrated by high-speed centrifugation was used. The infections were carried out with an adsorption period of 1 h at 37 o C. Virus binding Cell cultures grown in Microtest II plates containing 2-3 x lo4 cells per cell were incubated with 1.5-2.0 x lo4 cpm of 3H-BA’71V for 2 h at 4°C in 50 ~1 of DME medium in which the sodium bicarbonate was replaced by 25 mM HEPES (N-2-hydroxyethylpiperazine-N ‘-Zethanesulfonic acid) and supplemented with serum as indicated before. After the adsorption period, cell monolayers were washed twice with cold phosphate-buffers saline and solubilized with 1% Nonidet-Pi. Finally the trichloroacetic acid (TCA)-insoluble radioactive material in the washings and in the extracts of cell monolayer was determined in a liquid scintillation counter. Assay of viral protein synthesis Cell cultures were pulse-labeled with [ ?$]methionine (1200 Ci/mmol; Amersham, U.K.) at different times of infection and the extracts immunoprecipitated by either non-immune rabbit antiserum or anti-ASF virus infected VERO cell rabbit antiserum, preadsorbed to VERO cell proteins, as described (Alcami et al., 1989b). The analysis of proteins by polyacrylamide gel electrophoresis and the quantitation of the radioactive protein bands have been described (Alcami et al., 1989b). Synthesis of viral DNA To study the kinetics of viral DNA synthesis, cells grown in 24-well plates were pulse-labeled for 2 h with 10 PCi of [3H]thymidine (40-60 Ci/mmol; Amersham, U.K.) per ml. Macrophages and monocytes were then washed twice with phosphate-buffered saline and solubilized with 1% Nonidet-P40. The TCA-insoluble radioactive material in the cell extract was determined.

Results

Electron microscopy of ASF virus penetration into swine ~acrophages Qu~titative electron microscopy analysis of BA7lV-infected swine macrophages at early times of infection indicates that BA71V enters the cell by a receptor-media-

96

80

TIME AFTER ADSORPTION, min Fig. 1. Entry of BA71V into swine macrophages. Swine macrophages were infected with BA71V at higb multip~c~ty of infection (1.3 X 103 PFU per cell) for 2 h at 4°C. After two washes with medium, the cells were incubated at 37 o C for various times, thin-sectioned and examined by electron microscopy. Panel A shows virns particles ad.sorbed to the plasma membrane (a), in membrane invaginations (b) or in cytoplasmic vesicles (c), as weli as cores in the cytoplasm {arrows) (d). The bar represents 200 nm. Panel B shows the quantitation of viral and subviral particles at different steps of internalization. Striped bars represent virus in coated pits (b) and virus in vesicles which contained only one virion (c). Black bars represent viral cores. Data were obtained from the identification of 500 virus particles at each indicated time of infection.

97

A no

I

100 ADDED VIRUS

200 PARTKLES

ix lo-%/CELL

300

100

I

200

UNLABELED tx

300

VIRUS

I

I

400

500

PARTItlES

lo-~I/cELt

Fig. 2. Binding of 3H-BA71V to swine macrophages. (A) Dose-response curve of binding of kBA71V to swine macrophages. Increasing amounts of 3H-BA71V were incubated in duplicate at 4O C with swine macrophages and the binding of labeled virus was determined after 2 h. Adsorption was carried out in the absence (0, total binding) or presence (A,, nonsaturable binding) of an excess of unlabeled virus (5 x 10’ virus particles per cell). Saturable binding (0) was calculated after subtraction of nonsaturable binding from total binding. The mean value of the standard errors was 7.9%. (B) Competition by unlabeled virus of 3H-BA71V binding to swine macrophages. Swine macropbages were incubated for 2 h at 4O C with a constant amount of 3H-BA71V (4 x lo3 virus particles per cell) in the presence of increasing amounts of unlabeled virus (01, and total virus binding was determined. The percentages refer to the amount of bound 3H-BA71V in the absence of competitor virus.

ted endocytosis mechanism (Fig. 1). Quantitation of the viral and subviral particles during the first 90 mm of infection showed the occurrence of a temporal flow of virus particles from the surface (Fig. lB, a) through membrane invaginations (Fig. lB, b) into vesicles (Fig. lB, c). Viral cores did not appear until 45 min postadsorption (Fig. lB, d). A similar study of BA71-infected swine macrophages showed that the non-adapted virus strain also enters the cell by endocytosis {not shown).

Saturable binding sites for ASF vim in swine mucrophages Binding experiments of ‘H-BA7lV to swine macrophages, carried out at 4OC to prevent penetration, showed the existence of saturable binding sites for BA71V on the plasma membrane (Fig. 2A). The competition of the binding of tritiated virus by increasing amounts of unlabeled virus (Fig. 2B), and not by bovine serum albumin (not shown), confirmed the existence of a specific saturable interaction of virions with swine macrophages. Total virus binding was only completed up to 608, probably due to a high nonsaturable binding at the multiplicity of infection used.

100 ‘3 90 2

ao-

? * ;

60-

d m 5 40-

L”

K7 2 z

z

zo-

-0 I

100

,--0--y too

300

400

INACTIVATESVIRUS PARTICLES ix tO-3!/CELL

s 6 E 20E YI ml u

“\ QLO-, I 100

I 200

-0 I 300

l

LOO

INACTIVATES VIRUS PARTICLES tx 10-')/CELL

‘Fig. 3. Inhibition of viral protein and DNA synthesis by competitor inactivated virus. Swine macrophages were either mock-infected or infected with a constant amount of infectious BA71V (6 PFU per cell) in the presence of increasing amounts of UV-BA71V. To analyze the early viral protein synthesis, cells were pulse-labeled with [ 3SS]methionine from 2 to 4 h postinfection in the presence of the indicated amounts of UV-BA71V. Extracts were then prepared, immunoprecipitated with rabbit antiserum against ASF virus-infected VERO cells, and analyzed by polyacrylamide gel electrophoresis. In parallel experiments, cells were pulse-labeled with [3H]thymidine, in the absence or in the presence of UV-BA71V, for 2 h at different times after infection and the TCA-insoluble radioactive materiat was determined as described in Materials and Methods. (A) Electrophoretic analysis of immunopr~ipitated proteins and quantitatio~ of the early viral protein ~34. (9) ~3H]th~idine incorporated into TCA-insoluble material from 8 to 10 h postinfection, when maximum incorporation was obtained in the absence of UV-BA71V, The results obtained with mock-infected swine macrophages (M, 8) and with cells incubated with the highest dose of UV-BA71V (UV, 0) are indicated.

Inhibition of viral expression in swine macrophages

by inactivated

viru.s

The synthesis of the early viral protein ~34 and viral DNA (Fig. 3), as well as that of late viral proteins (not shown), were inhibited by concentrations of inactivated virus that competitively prevent the interaction of virions with saturable binding sites. Since the incorporation of [“%]methionine into TCA-insoluble material was inhibited about 50% in uninfected swine macrophages incubated with high doses of inactivated virus (not shown), we investigated the possible toxic effect of high amounts of UV-BA71V on viral replication. The. addition of UV-inactivated virus at late times of infection did not cause the total in~bition of the infectious cycle, obtaining 40% inhibition of the early viral protein synthesis when inactivated virus was added 1 h after the infectious virus (not shown). Similar percentage of inhibition of DNA replication was obtained when inactivated virus was added 5 h after the infectious virus (not shown).

99

Adsorption and ~enetrativn of ASF virus into rabbit macro~hages The binding of increasing amounts of 3H-BA71V to rabbit macrophages at 4“C was similar in the absence or in the presence of an excess of unlabeled virus (not shown) indicating non-saturability. The lack of competition of high doses of unlabeled virus (up to 6 x lo5 virus particles per cell) of the binding at 4’ C of a constant amount of 3H-BA71V to rabbit macrophages (not shown) confirmed the absence of saturable binding sites for ASF virus particles in these cells. The 3H-BA71V binding at 4’ C was about twofold higher in swine macrophages than in rabbit macrophages. This value increased about sixfold when the binding was performed at 37 o C (not shown).

SWINE MOCK

RABBIT

INFECTED -- 2-4

MOCK

5-7

2-4

abc

abc

INFECTED 2-4

5-7

Fig. 4. Viral protein synthesis in BA71V-infected rabbit macrophages. Swine and rabbit macrophages were mock-infected or infected with 20 PFU of BA71V per cell and pulse-labeled for 2 h with [35S]methionine at the indicated times after infection. The polyacryiamide gel electrophoresis of radioactive proteins before (a) or after the immunoprecipitation by non-immune (b) or anti-virus infected VERO cells (c) rabbit serum is shown. Molecular weights are indicated. K, 1000 daltons.

100

Electron microscopy was used at early times of infection to determine whether BA71V bound to nonsaturable sites of rabbit macrophages can enter the cell. Similar percentages of virus particles inside cytoplasmic vesicles and cores in the cytoplasm were observed both in swine and rabbit macrophages. However, virions in coated pits were not seen in the latter (not shown). ASF virus expression

in rabbit macrophages

To determine whether viral cores in the cytoplasm of rabbit macrophages are able to initiate the genome expression, viral protein and DNA synthesis in BA’IlVinfected rabbit macrophages were analyzed. _M

0 -----

50

100

200

300

uv

INACTIVATED VIRUS PARTICLES (x lo-')/CELL Fig. 5. Early viral protein synthesis in BA71V-infected macrophages in the presence of UV-inactivated virus. Swine (0) and rabbit (0) macrophages were infected with a constant amount of infectious BA71V (4 PFU per cell) in the presence of increasing amounts of UV-BA7lV. Cells were pulse-labeled from 2 to 4 h postinfection with [35S]methionine, in the presence of UV-BA71V, and the extracts were immunoprecipitated with rabbit antiserum against ASF virus-infected VERO cells. The electrophoretic analysis of the immunoprecipitated proteins and the quantitation of the viral protein p34 are shown. The synthesis of protein p34 in rabbit macrophages was 2% from that obtained in swine macrophages. The autoradiography was overexposed in the case of rabbit macrophages for a better quantitation of the protein band. Percentages refer to the expression level obtained in the absence of competitor virus. The results obtained in the mock-infected macrophages (M) and in the macrophages incubated with the highest dose of UV-BA71V used (UV) are also shown.

101

BA71V caused strong inhibition of protein synthesis in rabbit macrophages (not shown). This inhibition was higher and occurred earlier (94% at 5-7 h postinfection) than that observed in susceptible swine macrophages (60% at S-10 h postinfection). Electrophoretic analysis of the proteins immunoprecipitated with serum against virus-infected VERO cells showed the synthesis of two early viral proteins, p34 and ~38 (Fig. 4). The densitometry of the bands corresponding to these proteins showed that the expression levels of p34 and ~38 were 2 and 38, respectively, from those obtained in swine macrophages. The early viral proteins p24 and p26 were also detected in rabbit macrophage extracts, although they did not immunoprecipitate (Fig. 4). Infection of rabbit macrophages with BA71V was abortive since neither viral DNA synthesis nor virus production were detected, although a strong cytopathic effect was observed (not shown). Similar results were obtained with the non-adapted virus strain (BA71) (not shown). Early viral protein synthesis in infected rabbit macrophages in the presence of UV-inactivated virus Fig. 5 shows that the synthesis of the early viral protein p34 in BA71V-infected rabbit macrophages was not affected by the presence of amounts of UV-BA71V capable of saturating the specific cellular receptor and of inhibiting the infection in swine macrophages. As with swine macrophages, the highest dose of inactivated virus used inhibited the incorporation of [35S]-methionine into TCA-insoluble material by about 50% (not shown). ASF virus infection of chicken and human monocytes The infection of chicken and human monocytes with BA71V, as in rabbit macrophages, was abortive. The virus induced a strong inhibition of cellular protein synthesis and a marked cytopathic effect. There was a weak expression of the early viral proteins ~24, ~26, p34 and ~38, but no viral DNA synthesis was detected (not shown).

Discussion

The analysis by electron microscopy of the different steps of the entry pathway showed that BA71V and BA71 enter swine macrophages by a mechanism of receptor-mediated endocytosis, as described for other animal viruses (Buk~nskaya, 1982; Dimmock, 1982; Marsh, 1984; Patterson and Oxford, 1986) and for infection of monkey kidney cells by ASF virus (Geraldes and Valdeira, 1985; Valdeira and Ceraldes, 1985; Alcami et al., 1989b). The sequential appearance of virus particles in the different internalization steps suggests that virus uncoating takes place from intracellular vacuoles, where whole virus particles were accumulated after internalization.

102

The presence of specific, saturable binding sites for BA71V on the plasma membrane of swine macrophages is supported by binding experiments of tritiated virus in the presence of unlabeled virus. The number of cellular receptor sites per swine macrophage was about 1 X 104, estimated from the number of virions needed to saturate binding sites. This value is similar to that described in VERO cells (Alcami et al., 1989a) and to those reported for other animal viruses (lo4 to lo5 sites per cell) (Lonberg-Holm, 1981; Tardieu et al., 1982). Saturable binding, however, is one of two different virus-cell interactions detected, since virus particles can bind also to nonsaturable sites on the plasma membrane, as it has been described in other systems (Perrin et al., 1982; Schlegel et al., 1982; Tardieu et al., 1982). The inhibition of early viral protein synthesis and viral DNA replication by doses of inactivated virus that were able to compete with the interaction of virus particles with specific receptors on the plasma membrane suggests that only virus bound to saturable sites can productively infect swine macrophages. Therefore, virus bound to nonsaturable sites does not infect swine macrophages, although we do not know whether these virions are able to enter the cell. The decrease of cellular protein synthesis in swine macrophages by high doses of UV-inactivated virus could account for the inhibition of virus replication under these conditions. However, the addition of high amounts of UV-BA71V at late times of infection produced only a slight inhibition of the infectious cycle, suggesting that UV-BA71V inhibits an early step of the infection, probably the specific interaction with cellular receptors. The interaction of BA71 with swine macrophages could be different from that of BA71V, since the latter has been adapted to cells that were initially resistant to the infection. It is possible that a mutation in the virus attachment protein allowed the virus to interact with a new receptor during adaptation to VERO cells, as described for picornavirus (Reagan et al., 1984), and probably maintains the affinity for the receptor in swine macrophages. Because of the unavailability of highly purified preparations of the non-adapted virus strain, no binding and competition experiments could be performed to test this hypothesis. In nature, ASF virus is very specific for swine and related species. The resistance of several animal species seems to be due to the failure of ASF virus to replicate in their macrophages (Enjuanes et al., 1977). The non-specific interaction of BA71V with rabbit macrophages gave rise to an abortive infection, since viral genetic expression was restricted to a low synthesis of some early viral proteins (~24, ~26, ~34, ~38). The absence of virus particles in coated-pits and the failure of high doses of UV-inactivated virus to inhibit early viral protein synthesis would support the notion that BA71V attachment and entry into rabbit macrophages is not mediated by a saturable component. The abortive infection in rabbit macrophages, also observed in BA71-infected rabbit macrophages and in BA71V-infected chicken and human monocytes, could be due to a post-attachment restriction. It could be argued that interaction with specific receptors is necessary not only for virus adsorption and penetration, but also for activation of a component in the virus particle required for complete expression of the viral genome or for an appropriate virus uncoating, as it has been reported in picornavirus (Crowell et al., 1981).

103

The requirement of a receptor-mediated interaction with the host cell to get a productive infection implies that, in natural infections, ASF virus might be neutralized with antibodies directed against the virus attachment protein, which would block the specific interaction of the virus particle with the target cell and, therefore, the infection.

Acknowledgements

We are grateful to C. San Martin and P. Gonzalez for assistance with electron microscopy. This work was supported by grants from the Comision Interministerial de Ciencia y Tecnologia, Junta de Extremadura, European Economic Community, and by an institutional grant of Fundacion Ramon Areces.

References AlcamI, A., Carrascosa, A.L. and Vifmela, E. (1989a) Saturable binding sites mediate the entry of African swine fever virus into Vero cells. Virology 168, 393-398. Alcami, A., Carrascosa, A.L. and V&uela, E. (1989b) The entry of African swine fever virus into Vero cells. Virology 171, 68-75. Almeida, J.C., Waterson, A.P. and Plowright, W. (1967) The morphological characteristics of African swine fever virus and its resemblance to Tipula iridescent virus. Arch. Ges. Virusforsch. 20, 392-396. Boyurn, A. (1968) Isolation of mononuclear cells and granulocytes from human blood. J. Clin. Lab. Invest. 21, 77-89. Breese, S.S. and De Boer, C.J. (1966) Electron microscopy observations of African swine fever virus in tissue culture cells. Virology 28, 420-428. Bukrinskaya, A.G. (1982) Penetration of viral genetic material into host cell. Adv. Virus Res. 27, 141-204. Carrascosa, J.L., Carazo, J.M., Carrascosa, A.L., Garcia, N., Santisteban, A. and ViImela, E. (1984) General morphology and capsid fine structure of African swine fever virus particles. Virology 132, 160-172. Carrascosa, A.L., Del Val, M., Santaren, J.F. and Viiiuela, E. (1985) Purification and properties of African swine fever virus. J. Virol. 54, 337-344. Carrascosa, A.L., Santaren, J.F. and Viiiuela, E. (1982) Production and titration of African swine fever virus in porcine alveolar macrophages. J. Virol. Methods 3, 303-310. Casal, I., Enjuanes, L. and Vihuela, E. (1984) Porcine leukocyte cellular subsets sensitive to African swine fever virus in vitro. J. Virol. 52, 37-46. Crowell, R.L., Landau, B.J. and Siak, J.S. (1981) Picomavirus receptors in pathogenesis, p. 169-184. In: K. Lonberg-Holm and L. Philipson (Eds), Virus Receptors: Part 2, Animal Viruses. Receptors and recognition series B, vol. 8. Chapman and Hall, London. De Boer, C.J. (1967) Studies to determine neutralizing antibody in sera from animals recovered from African swine fever and laboratory animals inoculated with African virus and adjuvants. Arch. Ges. Virusforsch. 20, 164-179. Dimmock, N.J. (1982). Initial stages in infection with animal viruses. J. Gen. Virol. 59, l-22. Enjuanes, L., Carrascosa, A.L., Moreneo, M.A., and Vihuela, E. (1976a). Titration of African swine fever (ASF) virus. J. Gen. Virol. 32, 471-477. Enjuanes, L., Carrascosa, A.L. and Viiiuela, E. (1976b) Isolation and properties of the DNA of African swine fever (ASF) virus. J. Gen. Virol. 32, 479-492. Enjuanes, L., Cubero, I. and Viiiuela, E. (1977) Sensitivity of macrophages from different species to African swine fever (ASF) virus. J. Gen. Virol. 34, 455-463.

104 Garcia-Barreno, B., Sanz, A., Nogal, M.L., Vihuela, E. and Enjuanes, L. (1986) Monoclonal antibodies of African swine fever virus: antigenic differences among fields virus isolates and viruses passaged in cell culture. J. Virol. 58, 385-392. Geraldes, A. and Valdeira, M.L. (1985) Effect of chloroquine on African swine fever virus infection. J. Gen. Virol. 66, 1145-1148. Hess, W.R. (1981). African swine fever: a reassessment. Adv. Vet. Sci. Comp. Med. 25, 39-69. Lonberg-Holm, K. (1981) Attachment of animal viruses to cells: an introduction, p. I-20. In: K. Lonberg-Holm and L. Philipson (Eds.), Virus Receptors: Part 2, Animal Viruses. Receptors and recognition series B, vol. 8. Chapman and Hall, London. Malmquist, W.A. and Hay, D. (1960) Hemadsorption and cytopathic effect produced by African swine fever virus in swine bone marrow and buffy coat cultures. Am. J. Vet. Res. 21, 104-109. Marsh, M. (1984) The entry of enveloped viruses into cells by endocytosis. Biochem. J. 218, l-10. Maurer, F.D., Griesemer, R.A. and Jones, T.C. (1958) The pathology of African swine fever, a comparison with hog cholera. Am. J. Vet. Res. 19, 517-539. Moura Nunes, J.F., Vigario, J.D. and Terrinha, A.M. (1975) Ultrastructural study of African swine fever virus replication in cultures of swine bone marrow cells. Arch. Virol. 49, 59-66. Myrvik, Q.N., Leake, E.S. and Fariss, B. (1961) Studies on pulmonary alveolar macrophages from the normal rabbit: a technique to procure them in a high state of purity. J. Immunol. 86, 128-132. Patterson, S. and Oxford, J.S. (1986) Early interactions between animal viruses and the host cell: relevance to viral vaccines. Vaccine 4, 79-90. Perrin, P., Portnoi, D. and Sureau, P. (1982) Etude de l’adsorption et de la penetration du virus rabique: interactions avec les cellules BHK21 et des membranes artificielles. Ann. Virol. (Inst. Pasteur) 133E, 403-422. Reagan, K.J., Goldberg, B. and Crow&, R.L. (1984) Altered receptor specificity of coxsackievirus B3 after growth in rhabdomyosarcoma cells. J. Virol. 49, 635-640. Sachs, D.H., Leight, G., Cone, J., Schwarz, S., Stuart, L. and Rosenberg, S. (1976) Transplantation in miniature swine. I. Fixation of the major histocompatibility complex. Transplantation 22, 559-567. Schlegel, R., Willingham, M.C. and Pastan, I. (1982) Saturable binding sites for vesicular stomatitis virus on the surface of Vero cells. J. Virol. 43, 871-875. Tardieu, M.R., Epstein, R.L. and Weiner, H.L. (1982) Interaction of viruses with cell surface receptors. Int. Rev. Cytol. 80, 27-61. Valdeira, M.L. and Geraldes, A. (1985) Morphological study on the entry of African swine fever virus into cells. Biol. Cell 55, 35-40. Viiiuela, E. (1985) African swine fever virus. Curr. Top. Microbial. Immunol. 116, 151-170. Vihuela, E. (1987) Molecular biology of African swine fever virus. In: Y. Becker, (Ed.), African Swine Fever. Martinus Nijhoff Publishing, Boston. Wardley, R.C., De M. Andrade, C., Black, D.N., Castro Portugal, F.L., Enjuanes, L., Hess, W.R., Mebus, C., Ordas, A., Rutili, D., Sanchez Vizcaino, J., Vigario, J.C., Wilkinson, P.J., Moura Nunes, J.F. and Thomson, G. (1983) African swine fever virus. Arch. Virol. 76, 73-90. (Received

22 January

1990; revision

received

18 May 1990)

Interaction of African swine fever virus with macrophages.

Morphological data obtained by electron microscopy have shown that African swine fever virus adapted to VERO cells enters swine macrophages, its natur...
1MB Sizes 0 Downloads 0 Views