key: cord-0007766-m9nlx6bn
authors: Juarrero, Mercedes Gonzalez; Mebus, Charles A.; Pan, Reyes; Revilla, Yolanda; Alonso, Jose M.; Lunney, Joan K.
title: Swine leukocyte antigen and macrophage marker expression on both African swine fever virus-infected and non-infected primary porcine macrophage cultures()
date: 2002-11-12
journal: Vet Immunol Immunopathol
DOI: 10.1016/0165-2427(92)90049-v
sha: 5e84b922f73fd289cf25b78ba06cdafd999bb1e0
doc_id: 7766
cord_uid: m9nlx6bn

Swine leukocyte antigens (SLA) and a macrophage specific marker were monitored on porcine macrophages cultured with or without macrophage colony stimulatory factor (M-CSF) and on cells infected with African swine fever virus (ASFV). SLA expression was maximal either in the total cell extract or on the cell surface at 3–4 days of culture; after 4 days these values began to decrease. Fluorescence analyses of immunostained macrophages cultured with or without M-CSF indicated a major upward shift in the number of SLA Class I molecules on individual macrophages whereas for SLA Class II both a novel expression of Class II and an upward shift in the number of molecules per cell were evident. Infection of 3-day-old macrophage cultures with three different isolates of ASFV resulted in minor changes in surface expression of SLA Class I, SLA Class II, and macrophage markers. No differences in infection with ASFV was observed whether macrophages were SLA Class II positive or negative, nor was there blocking by anti-SLA Class I or Class II monoclonal antibodies of ASFV infection of cultured macrophages.

Host resistance to viral disease depends in part on the appropriate recognition by the T-cell antigen receptor of both antigens, e.g. a viral peptide and either Class I or Class II antigens of the major histocompatibility complex (MHC) on the surface of the antigen presenting cell (APC) (Unanue, 1984; Bjorkman et al., 1988; Geppert and Lipsky, 1989) . Reduced stimulation of T-cells may result from inadequate expression of either or both of these antigens, due to down-regulation of MHC antigen by viral infection of cells (Bernards et al., 1983; Jennings et al., 1985; Brown et al., 1988) or altered association of viral antigens with MHC antigens (Kane et al., 1989 ) .

African swine fever (ASF) is caused by a large enveloped DNA virus of the family Iridoviridae (Breese and DeBoer, 1966 ) sharing many properties with poxvirus (Vifiuela, 1987) . African swine fever virus (ASFV) infects cells of the monocytic/phagocytic system (Hess, 1981 ) causing some functional alterations (Martins et al., 1988) . The disease in domestic swine results in significant economic loss to the pig industry in endemic areas. Mortality in ASFVinfected pigs correlates with the virulence of the virus isolates (Pan and Hess, 1984) and occurs within 5-15 days of infection (Wardley et al., 1987) . Although ASFV induces a strong antibody response, the antibody has no neutralizing activity and virus clearance mediated by antibody-specific mechanisms does not occur until after the peak of viremia is over. Thus, antibody does not seem to play a major role in determining survival of pigs infected with ASFV (Wardley et al., 1987) . Specific immune mechanisms of ASFV clearance from infected pigs are poorly understood, hence, the prospects for development of vaccines are poor.

Studies of kinetics of the synthesis of the ASFV-induced polypeptides in Vero cells indicate two early and one late class of proteins (Santaren and Vifiuela, 1986) . Although chemical studies on ASFV antigen expression on the surface of infected Vero cells (Santaren and Vinuela, 1986; Revilla et al., 1988) , infected IB-RS cells (Wardley et al., 1985) or infected porcine macrophages (Whyard et al., 1985) have been reported recently, the exact time course of ASFV antigen expression on the surface of infected cells is poorly characterized. We have reported changes in the expression of swine MHC or swine leukocyte antigens (SLA) in spleen tissue sections from animals infected with different isolates ofASFV (Gonzalez Juarrero et al., 1992 ) . Since macrophages are important APC (Unanue, 1984) , a decrease in SLA expres-sion during ASFV infection of the porcine macrophage could limit the recognition of ASFV antigens by the immune system.

Long-term blood mononuclear cell cultures require the use of a supernatant from murine L929 cells as a growth factor, or macrophage colony stimulatory factor (M-CSF) (Genovesi et al., 1989) . The present study analyzed SLA expression in both ASFV-infected and non-infected primary porcine macrophage cultures. Our objectives were to analyze the effects of M-CSF on the expression of SLA in factor-stimulated cultures and to characterize the effect of ASF viral replication on SLA expression.

Yorkshire pigs, 6-12 months old, were conventionally maintained in the PIADC colony, and were bled from the ear into heparin (10 IU ml -I ) for these experiments. The highly virulent ASFV isolate, Lisbon 60 (L60) (Manso Ribiero and Rosa Azevedo, 1961 ) , the moderately virulent isolate, Dominican Republic-II (DR-II) (Mebus, 1988) and non-hemadsorbing ASFV isolate (NHV) (Vigario et al., 1974) were grown on porcine macrophages. These three isolates replicate with similar kinetics when grown in vitro; their titer, expressed as 50% hemadsorption units (HADso) ml-1 or 50% cytopathic effect (c.p.e.) units ml-I, was determined on porcine macrophage cultures as described by Martins et al. ( 1988 ) .

Cultures of porcine macrophages were prepared from heparinized blood essentially as described by Martins et al. ( 1988 ) . Briefly, the leukocyte-rich fraction, prepared by Dextran flotation of blood, was centrifuged on a Ficoll-Hypaque gradient and the interface layer, containing peripheral blood mononuclear cells (PBMC), collected, washed and cultured in complete medium (RPMI-1640, 2 mM glutamine, 25 mM Hepes, 0.025% sodium bicarbonate, 10% autologous plasma and 10% fetal bovine serum (Hyclone Lab, UT)) that had been prescreened for low/no endotoxin. Macrophages grown with M-CSF were prepared essentially as described by Genovesi et al. (1989) in complete medium with 20% L929 cultured fibroblast supernatant. PBMC at 2 × 106 cells ml-i were cultured in 150 cm 2 flasks and incubated at 37°C in 5% CO2 atmosphere. For long term cultures, cells were washed in phosphatebuffered saline (PBS) and cultured in fresh complete medium without autologous plasma. Macrophages were purified by rinsing off adherent cells with cold calcium magnesium free PBS and then eluting adherent cells with PBS containing 0.03% EDTA for 20 min on ice. Eluted macrophages were pooled, washed with Hanks balanced salt solution, and resuspended in complete medium.

Three day old porcine macrophage cultures were harvested, and aliquots were cultured either without viruses or were adsorbed, in cell suspension, with 10 HADso units or 10 c.p.e, units of ASFV per cell for 1 h at 37°C. After three washes with RPMI-1640, the cells were incubated in complete medium with 2% fetal calf serum for the times indicated in the appropriate figures. Aliquots from each infected culture and from control cells (without virus) were taken at different time points, washed and used for antibody staining experiments. Control macrophage were also cultured with 0-2.5 ng ml-1 lipopolysaccharide ( LPS ) to assess endotoxin effects. Viability before and after infection was higher than 70% for all cultures as determined by Trypan blue exclusion.

The monoclonal antibodies (mAbs) used in these studies are listed in Table 1, along with their known reactivity against swine PBMC or ASFV antigens. Aliquots of 2-4 × 106 macrophages in 500/A of RPMI-1640 were frozen and thawed three times using liquid nitrogen and tap water and microfuged for 20 min. The resultant supernatants were analyzed for protein content by Lowry assay (Lowry et al., 1951 ) and aliquots of these supernatants were frozen at-70 °C until use. Duplicate samples ( 10/.tg per sample) of each extract were applied onto presoaked nitrocellulose membranes (Schleicher and Whyard et al., 1985 Whyard et al., 1985 Schuell A83, 2 mM). Nonspecific binding was blocked by 3 h incubation at room temperature with washing buffer (PBS containing 5% non-fat milk, 0.05% Tween 20, 0.01% sodium azide). Individual nitrocellulose strips were incubated overnight at 4°C with each of the mAb ascites, at 1:200-1:500 dilution in washing buffer. The strips of nitrocellulose paper were incubated with ~2sI rabbit anti-mouse lg, kindly provided by T. Whyard, PIADC, NY. The strips were washed, air dried and either autoradiographed directly, or the spots from nitrocellulose strips cut and radioactivity counted in a Beckman gamma counter.

lmmunofluorescence Cells were stained by indirect immunofluorescence and analyzed with a flow cytometer (FACSCAN, Becton-Dickinson, Sunnyvale, CA ). Briefly, cells were washed twice with PBS and non-specific binding was blocked by incubating for 30 rain at 4°C with PBS containing 5% normal rabbit serum. The cells were incubated with aliquots of the mAb supernatants listed in Table l, for 40 min at 4 ° C, washed with PBS and incubated for another 40 rain with fluoresceinated rabbit F (ab')2-anti-mouse Ig. After washing, fluorescence data was collected on 10 000 light scatter gated macrophages using a FACSCAN Consort 30 program. Percentage of positive cells was calculated after setting the binding in the control samples, incubated with medium or mAb MSA4, to 5%.

The percentage of ASFV-infected cells was determined on aliquots of cells infected with each ASFV isolate. Cells were spun onto glass slides (cytospun) and fixed with methanol/acetone (50:50 v/v) at -20°C for l0 min and then air dried. The cells were preincubated with l : 5 dilution of normal swine serum for 20 rain at room temperature, washed with PBS and incubated with fluoresceinated swine IgG against ASFV. The percentage of ASFV-infected cells was determined using a fluorescent microscope.

The procedure for dual antibody labeling has been described in detail by Gonzalez Juarrero et al. ( 1991 ) and was adapted for lightly fixed cytospun cells. Briefly, 2-4 × 104 macrophages in 200 #l of RPMI-1640 were cytospun and fixed with 1% paraformaldehyde, 0.1 M lysine, 0.01 M sodium m-periodate (PLP buffer) (pH 7.4) for l0 min at room temperature. Non-specific binding was blocked by 1% normal pig serum in PBS for 30 rain prior to 1 h incubation with mAb culture supernatant or medium as a control. Between all steps TBS (0.85% NaCI, 0.1 M Tris, pH=7.8) washes were performed. Alkaline phosphatase coupled rabbit anti-mouse Ig conjugate (Dako Co., Copenhagen, Denmark) was reacted with cells. Finally substrate, naphthol AS-MX, was added and positive staining was visualized in blue. The second staining was performed by incubating cells with biotinylated porcine IgG anti-ASFV prior to blocking endogenous peroxidase with 0.1% phenylhydrazine. Peroxidase coupled avidin biotin complex (Vector Labs, Burlingame, CA) was added followed by the substrate, 3-amino-9-ethylcarbamizole, in the presence of H202. Positive reaction for this staining was red. Slides were then mounted in glycerol gelatin GG (Sigma) and examined under a light microscope.

Macrophages at different intervals of culture were tested for total antigen expression by immunoblotting assays and for cell surface antigen expression of macrophage-specific markers and SLA by immunostaining and flow cytometric analyses. The time course assessment of total antigen expression in macrophage cultures, examined by immunoblotting assays, showed that macrophages cultured without M-CSF exhibited maximal expression of SLA Class I ( mAb PT85a, 74-11 -10 ) and macrophage markers (mAb 74-22-15 ) by Day 3 of culture ( Fig. 1 (m) ). Similar results were found for SLA Class II; in addition, at Day 4 there was a greater decrease in SLA-DR (mAb MSA3 and 40D) than SLA-DQ (mAb THI6) antigen expression. In a series of experiments, the day of maximum expression for these antigens varied but the peak of reactivity was consistently found at Days 3 and 4 of culture (data not shown). A major decrease of SLA Class I, Class II and macrophage markers was observed by Day 6 of culture without M-CSF ( Fig. 1 (A) ). The same macrophages cultured in the presence of M-CSF ( Fig. 1 (B) ) also exhibited maximal expression of SLA Class I and Class II at Day 3 although the macrophage marker (mAb 74-22-15 ) exhibited no differential expression during the time of culture. At Days 6 and 15 of culture, and in contrast with macrophages cultured without M-CSF, SLA Class I expression was still pronounced whereas expression of SLA Class II was minimal when compared to control (mAb MSA4) values ( Fig. I(B) ).

Quantitative analyses of cell surface expression of SLA and macrophage specific markers in both types of cultures was obtained using immunostaining and flow cytometric analyses, reporting two parameters: the percentage of positive cells and the intensity of fluorescence or mean channel fluorescence. The total number of SLA Class I positive cells was not changed but rather the number of antigens per cell, as indicated by the mean channel intensity of fluorescence of the cell population, was increased with M-CSF, a change of 300 channels or more ( Fig. 2 (B) ) and without M-CSF, a change of 150 channels or more ( Fig. 2 (D) ). These increases in marker expression peaked at 3- 4 days of culture and thereafter decreased, in agreement with the results shown by immunoblotting (Fig. 1 ) . Changes in the expression of SLA Class II on macrophages during culture were reflected both in the percentage of cells that express Class II antigens as well as in the intensity of marker expression. Macrophages cultured without M-CSF increased from 40% (THI6, SLA-DQ) or 65% (40D, SLA-DR) Class II positive at 1 day of culture to 60% and 80% positive, respectively, at 3 or 4 days of culture with a mean fluorescence increase of approximately 100 channels for SLA-DR and 30 channels for SLA-DQ ( Fig. 2A,2B ) . These increases were reversed by 6 days of culture. When macrophages were cultured with M-CSF they exhibited a less pronounced increase in the percentage of SLA II positive cells, although these still peaked at 3-4 days of culture ( Fig. 2 (C) ). The relative intensity of SLA Class II expression was changed little in macrophages cultured with M-CSF, although the remaining Class II positive cells may be slightly brighter for SLA Class II expression at 6 days of culture (Fig.  2 (D) ). Incubation with LPS endotoxin caused no such increase in SLA I or II expression (data not shown). Overall, the changes in SLA Class II expression varied significantly between experiments. Thus, macrophages harvested after 1 day of culture ranged from 30 to 75% positive for SLA Class II expression, however, the maximum percentage of Class II positive cells invariably occurred at 3-4 days of culture with or without M-CSF. 

Macrophage antigen expression was assessed using mAb 74-22-15 which reacts weakly with the immunoblotted antigen. When extracts from non-infected macrophages were compared with extracts from macrophages infected for 4, 8, 12, or 24 h with L60, DR-II or NHV isolates there was no apparent difference in 74-22-15 antigen expression (data not shown). When surface antigen expression was assessed by immunostaining of intact macrophages at 8 and 12 h after ASFV infection, there was no decrease in the percentage of 74-22-15 positive cells (Fig. 3 ) , however, there was a decrease in the intensity of antigen expression at 8 h after infection with all of the isolates, which was most pronounced with the NHV isolate (Table 2) . At 12 h post-infection, however, the expression of the 74-22-15 antigen was increased in the ASFVinfected cells when compared to the non-infected cells harvested at the same time point, although the surface 74-22-15 expression of all aliquots appeared to be decreased.

Immunoblot analyses of total SLA Class I expression in extracts of macrophages infected with ASFV for 4, 8, 12, or 24 h indicated that there were no major differences (data not shown). When surface expression of intact macrophages was analyzed a different result was obtained; all macrophages expressed the SLA Class I marker but at a much lower intensity for L60 and NHV-infected macrophages. At 8 and 12 h post-infection, ASFV infected macrophages had less SLA Class I on the surface than non-infected macrophages (Figs. 3, 4(A,B) ). Since the infected cells expressed less macrophage antigen at 8 h post-infection there could have been a general decrease in expression of surface antigens: to assess this, the relative intensity of the two markers, i.e. macrophage antigen and SLA, were compared as a ratio of the mean channel of fluorescence intensity of SLA relative to 74-22-15. The ratio calculations indicated that SLA Class I expression was reduced in macrophages infected for 8 h with the L60 or NHV ASFV isolates relative to the same macrophages cultured in medium alone (Table 2 ) . Similar ratios were found at 12 h post-infection. For the moderately virulent DR-II ASFV isolate, however, SLA Class I expression was increased at both time points using either measure of SLA Class I intensity.

The wide degree of variation of SLA Class II expression on cultured macrophages makes studies of the effect of viral infection on SLA Class II expression more difficult. The immunoblotting profiles showed that total Class II expression is decreased after 8 h of culture in both infected and non-infected macrophages (data not shown). Comparison with ASFV-infected cells revealed apparently higher levels of SLA Class II expression at 8 h post-infection but no apparent differences at 12 or 24 h. Assessment of surface antigen expression indicated that the intensity of SLA-DQ expression decreased at 8 and 12 h after infection ( Fig. 4(C,D) ). Similar results were found for SLA- DR expression based on both mean channel and ratio determinations ( Table  2) .

Viral antigen expression was demonstrated during the course of infection with ASFV. Porcine macrophages expressing viral proteins intracellularly were detected as early as 4 h post-infection using either the immunoblot assay or staining of permeabilized cells with porcine IgG against-ASFV ( Fig. 5 ) . Macrophage surface expression of ASF viral antigens could not be determined at early times post-infection using different anti-ASFV mAb (Table I ) or polyo clonal porcine IgG against ASF viral proteins. Using fluorescence immuno- Fig. 5 . Dual expression of SLA Class II and ASFV antigens. Dual antibody staining of porcine macrophages infected with ASFV L60 isolate for 12 h. Cytospun macrophages were stained with anti-SLA Class II mAb, MSA3, and anti-mouse IgG coupled to alkaline phosphatase (blue) and then with biotinylated porcine IgG against ASFV antigen coupled to biotin and avidin peroxidase (red). Original magnification × 200.

staining, there was positive surface staining for ASFV antigens between 12 and 24 h post-infection but on only 25-30% of infected cells as compared to 90% of the same macrophages expressing intracellular viral proteins observed by staining cells after permeabilization (Fig. 5) . This discrepancy could be explained by the lack of mAb, and swine antisera, reactive with ASFV epitopes expressed on the infected macrophage surface.

The ability of mAb against SLA to block ASFV infection was studied by incubating porcine macrophages, prior to infection with ASFV L60, with mAb against SLA Class I (PT85a), Class II (MSA3), or T-cells (MSA4). At 12 h post-infection cells were stained with fluoresceinated swine IgG against ASFV and the number of infected cells was counted for each sample. No differences in the percentage of infected cells was found when macrophages were treated prior to infection with mAb against SLA Class I, Class II, or without mAb (data not shown).

The possibility that ASFV preferentially infected SLA Class II positive macrophages was studied by the dual antibody staining technique using mAb against SLA Class II and swine IgG against ASFV. At 12 h post-infection with ASFV L60 two color immunostaining showed that both macrophages expressing SLA Class II antigens (blue) and macrophages not expressing SLA Class II antigens were infected by ASFV (red) (Fig. 5) . Moreover, the percentage of ASFV infected cells was always higher (90%) than the percentage (30-70%) of cells expressing SLA Class II before infection. When cells were analyzed at 4, 8, 12, or 24 h after infection with the L60, DR-II or NHV isolates, no preferential infection of SLA Class II positive or negative cells was found at any time point (data not shown ).

Our results indicate that expression of SLA Class I and Class II is greatly enhanced on porcine macrophages in cultures independent of M-CSF. The up-regulation, which is maximal at 3 to 4 days of culture for SLA Class I and Class II, is found for the total, immunoblotted SLA as well as for the antigens expressed on the cell surface. Fluorescence analyses verified that there is a major upward shift in the number of SLA Class I molecules on each macrophage whereas for SLA Class II there is both a novel expression of SLA Class II and an upward shift in the number of molecules per cell, which is greatest on macrophages cultured without M-CSF. By 6 days of culture these increases are reversed. These results indicate that 3-day-old macrophage cultures should be suitable stimulators of immune responses and the best targets for SLAmediated cytolytic activities.

These changes in expression of SLA Class I and Class II markers correlate with the growth, survival and functional data of such cultured swine macrophages ( Martins et al., 1988; Genovesi et al., 1989 ) . While maximal cell proliferation occurs between 4 and 6 days of culture with M-CSF, the peak of SLA expression was restricted to 3 to 4 days of culture. In cultures with M-CSF, cell proliferation declined by 6-8 days of culture and the expression of SLA Class I and Class II declined at 6 and 15 days of culture, respectively. The expression of MHC Class II antigens on monocytes and tissue macrophages from other animal species such as murine and human, also decreased after prolonged culture (Gonwa and Stobo, 1984; Toews et al., 1984) .

After infection with NHV and L60 ASFV isolates macrophages exhibited decreased Class I antigen expression at 8 or 12 h post-infection; in contrast, an increase of SLA Class I was seen on cells infected with ASFV DR-II. Interestingly, these changes in SLA Class I expression do not seem to correlate with the virulence of ASFV isolates (Pan and Hess, 1984) . The general losses in 74-22-15, SLA Class I and Class II could be attributed to virus mediated membrane turnover as virus infects the cells and moves to the replication centers in the cytoplasm. However, this would not explain the increase in SLA Class I expression on DR-II-infected macrophages. This isolate may express a unique function, possibly associated with other moderately virulent ASFV isolates, in that it causes up-regulation of SLA Class I expression similar to that observed after infection with radiation-leukemia virus, adenovirus 12, or coronavirus (Brown et al., 1988 ) . The fact that the non-virulent NHV isolate does not also stimulate SLA I expression may be due to the NHV isolate being related to the virulent L60 isolate or to it requiring other cells, e.g. Tcells, to secrete factors to influence macrophage antigen expression. The apparent recovery of some SLA antigen expression at 12 h post-infection may be due to a concomitant decrease in viral protein synthesis which in Vero cells is maximal at 8 h post-infection (Tabares et al., 1980) . The decrease in SLA Class I surface expression found in macrophages infected with ASFV NHV or L60 was not detectable when total extracts of these macrophages were analyzed by immunoblot analyses. This may indicate that the relative loss of surface SLA is small compared to the total cell associated SLA and therefore not detectable by the assay system. It is more likely that the deficit in surface SLA expression is due to a reduction in the cycling of the antigens back to the cell surface. This is supported in part by an electron microscopic examination of macrophages which reveals that SLA Class I and Class II molecules are associated with internal ASF viral particles detected by mAb and immunogold staining (M. Gonzalez Juarrero et al., unpublished data, 1988 ) . As these viral particles bud from the macrophage surface at later times in the infection, they may carry these SLA proteins in the viral envelope, similar to the earlier report of ASFV budding from Vero cells (Carrascosa et al., 1985) . A decrease in expression of SLA Class II may lead to a decrease in antigen presentation, a mechanism favorable for survival of the virus.

Some viruses, such as lactic dehydrogenase virus (Inada and Mims, 1985 ) , Semliki Forest virus (Helenius et al., 1978 ) and cytomegalovirus (Grundy et al., 1987) are reported to use MHC antigens as viral receptors. In vitro studies of pig monocytes infected with ASFV showed that both Class II positive and negative pig monocytes were equally likely to be infected with ASFV. Moreover, treatment of these monocytes before infection with mAb against SLA Class I or Class II caused no decrease in the number of ASFV-infected cells. These results indicated that there is no preferential infection of SLA Class II positive cells by ASFV and that at least the epitopes recognized by the SLA Class II mAb are not involved as receptors for ASFV. Washington State University, and Dr. Craig Hammerberg, University of Michigan, for their generous gifts of hybridoma cell lines and T. Whyard for his generous gift of monoclonal antibodies against ASFV proteins, and Dr. Eladio Vifiuela for his support of this research.

Tumorigenicity of cells transformed by adenovirus type 12 by evasion of T cell immunity

The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens

Chemical structure of ASFV investigated by electron microscope

Regulation of H-2 class I gene expression in virally transformed and infected cells

Purification and properties of African swine fever virus

The development and analysis of species specific and cross reactive monoclonal antibodies to leukocyte differentiation antigens and antigens of the major histocompatibility complex for use in the study of the immune system in cattle and other species

In vitro induction of swine peripheral blood monocyte proliferation by the fibroblast-derived murine hematopoietic growth-factor CSF-I

Antigen presentation at the inflammatory, site

Differential expression of la molecules by human monocytes

Modulation of splenic macrophagcs, and swine leukocyte antigen (SLA) and viral antigen expression following African swine fever virus (ASFV) inoculation

Betazmicroglobulin enhances the infectivity of cytomegalovirus and when bound to the virus enables Class I HLA molecules to be used as a virus receptor

Characterization of monoclonal antibodies against swine leukocytes

Human (HIA-A and HLA-B) and murine (H-2K and H-2D) histocompatibility antigens are cell surface receptors for Semliki Forest virus

African swine fever: a reassessment

la antigens and Fc receptors of mouse peritoneal macrophages as determinants of susceptibility to lactic dehydrogenasc virus

Effect of herpes simplex virus types I and 2 on surface expression of class I major histocompatibility complex antigens on infected cells

Cytolytic T-lymphocyte response to isolated class I H-2 proteins and influenza peptides

Protein measurement with the folin phenol reagent

Sharing of Ia antigens between species. IV. Interspecies cross-reactivity of monoclonal antibodies against polymorphic mouse Ia determinants

Reapparition de la peste porcine Africaine an Portugal

Modulation of porcine peripheral blood-derived macrophage functions by in vitro infection with African swine fever virus (ASFV) isolates of different virulence

African Swine Fever

Virulence in African swine fever: Its measurement and implications

Preparation and characterization of monoclonal antibodies reactive with porcine PBL

A protein of molar mass 12 KDa incorporates into the membrane of ASF virus-infected cells

African swine fever virus induced polypeptides in Vero cells

Proteins specified by African swine fever virus. Analysis of the viral structural proteins and antigenic properties

The accessory cell function of human alveolar macrophages in specific T cell proliferation

Antigen presenting function of macrophages

Antigenic relationship among strains of Africa swine fever virus

Molecular biology of African swine fever virus

The host response to African Swine fever virus

The role of antibody in protection against African swine fever virus

Monoclonal antibodies against African Swine Fever Viral antigens

The authors thank B. Skuba for her technical assistance and S.E. Perlman for his library assistance. The authors are also grateful to Dr. William Davis,