Virus-specific cellular blastogenesis and interleukin-2 production in swine after recovery from African swine fever

Animals recovered from viral diseases represent an important model to study the host cellular and humoral immune responses to the etiologic agents. This is particularly important for African swine fever virus (ASFV) infections in which antibodies have little or no virus-neutralizing effect. Pigs surviving experimental infection with the naturally occurring low-virulent, nonhemadsorbing ASFV/NH/P68 (NHV) isolate did, however, exhibit virus-specific T-cell activities, as measured by a variety of assays. A strong virus-induced, antigen-specific blastogenic response was observed only with blood mononuclear cells (BMC) from ASF-recovered swine, whereas cells from recovered and naive swine responded similarly to the mitogens concanavalin A and phytohemagglutinin. The ASFV-induced blastogenesis was dependent on virus dose and on the presence of adherent cells. Blood mononuclear cells cultured with antigenically related hemadsorbing ASFV isolates of different virulence characteristics, the highly virulent L60 isolate and moderately virulent DRII isolate, exhibited a similar magnitude of blastogenesis to cells infected with the low-virulent NHV isolate. Virus-infected cells proved to be an efficient inducer of interleukin-2 (IL-2) activity to cells from recovered swine, but not from naive swine, whereas T-cell-specific lectins induced production of similar amounts of IL-2 activity from cells of naive and recovered swine. Correlated with the appearance of virus-induced IL-2 activity in the culture supernatant was the induction of promiscuous killing in cells exposed to prolonged (7 days) virus stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early in vitro detection of interleukin-2-like activities by pretreatment with allogeneic blood lymphocytes in miniature swine.

In order to determine the means of monitoring the immunological status of allograft recipients in miniature swine, an assay was developed to measure interleukin (IL)-2 production in vitro by pretreatment of donor peripheral blood lymphocytes (PBL). Miniature swine were given 0 to 4 weekly intravenous transfusions of 5-10 X 10(7) donor PBL incompatible at major histocompatibility complex (MHC) and assayed in vitro for donor specific immune IL-2-like activities. The results are summarized as follows: (1) IL-2-like activity in 24 hr and 48 hr supernatants from mixed lymphocyte cultures (MLC) with MHC-incompatible PBL was detected without pretreatment. The 48 hr MLC supernatant exhibited a high IL-2-like activity compared with the 24 hr; (2) IL-2-like activity after only one transfusion with MHC-incompatible PBL was higher than that without pretreatment; (3) IL-2-like activity in 4 weekly transfusions was detectable slightly earlier than that without pretreatment or three transfusions with MHC-incompatible PBL.     

In vitro and in vivo association of African swine fever virus with swine erythrocytes

The association of African swine fever virus (ASFV) with swine erythrocytes in vivo, in high titers, was verified by inoculating 30 pigs with 17 ASFV isolates and assaying their plasma and washed erythrocyte fractions for residual virus. Viral antigens were specifically localized on the surface of in vitro and in vivo swine erythrocytes, using the fluorescent antibody technique and 3 monoclonal antibodies specific for ASFV. The same monoclonal antibodies immunoprecipitated virus-specific polypeptides of molecular weights 13 kd and 73 kd from ASFV-infected Vero cells. Erythrocytes from viremic swine infected with Lisbon-60, Dominican Republic, Badajoz-M98, or Cameroon isolates of ASFV were studied by transmission electron microscopy. Virus was found in membrane depressions at the surface of erythrocytes. These surface depressions resembled stages of smooth surfaced pits. Erythrocytes from viremic pigs were fragile osmotically.     

Classical swine fever virus in plasma and peripheral blood mononuclear cells of acutely infected swine

The distribution of classical swine fever virus (CSFV) in plasma, monocytes, T and B lymphocytes in peripheral blood was monitored during experimentally induced acute classical swine fever infection in piglets. Six piglets were infected with 10(3.8) TCID50 of virus and blood samples taken up to 18 days post-inoculation (p.i.). Infectious virus was detected in monocytes, T and B lymphocytes to similar titres in five of the six infected piglets. Infectious virus was detected earlier in plasma than in any of the mononuclear cell subpopulations. No significant difference was observed in the period of time in which virus could be isolated from the three cell subpopulations. While a progressive lymphopenia developed, a marked B cell depletion was observed. However, B cells were apparently replaced by non-IgM-bearing mononuclear cells, as the proportion 'total lymphocyte/total leucocytes' remained unaltered throughout the experiment. Virus titres in plasma and peripheral blood mononuclear cells showed a tendency to increase as the disease progressed to its outcome.     

Frequency of association of noncytopathic bovine viral diarrhea virus with bovine neutrophils and mononuclear leukocytes before and after treatment with trypsin

Enriched populations of neutrophils and mononuclear leukocytes from 9 cattle persistently infected with noncytopathic bovine viral diarrhea virus were analyzed for frequency of association with virus, using flow cytometric procedures. Trypsinization of neutrophils decreased the frequency of viral association from 0.82% to 0.49%. Similar treatment of mononuclear leukocytes decreased the frequency of viral association from 5.53% to 4.81%. Results of immunocytochemical procedures to locate viral antigen were inconclusive for neutrophils, but viral antigen was found in the cytoplasm of mononuclear leukocytes. A distinct and highly pure population of eosinophils was identified during flow cytometric analysis of neutrophil populations from 2 of 9 cattle.     

Increased porcine circovirus type 2 replication in porcine leukocytes in vitro and in vivo by concanavalin A stimulation

Previously, it was shown that modulation of the immune system enhances porcine circovirus type 2 (PCV2) replication in pigs. In the present study, the effect of the mitogen concanavalin A (ConA) on PCV2 replication was investigated. Since ConA induces T-lymphocyte activation and initiates the production of interferon-gamma (IFN-gamma), a cytokine that enhances PCV2 replication in porcine epithelial and monocytic cell lines in vitro, it was examined if the effects observed with ConA were mediated by IFN-gamma. In an in vitro study, ConA but not IFN-gamma enhanced PCV2 replication in peripheral blood mononuclear cells (PBMC). Up to 2.08% and 0.96% of PBMC were antigen positive for PCV2 strains 1121 and Stoon-1010, respectively, and a low virus production was observed. PCV2-infected PBMC were identified as CD4(+) (40%), CD8(+) (54%) and IgM(+) (11%). In a subsequent in vivo study, caesarean-derived colostrum-deprived piglets were injected with ConA or IFN-gamma 12h before inoculation and every 3 days for 9 days after inoculation with strain 1121. PCV2 was isolated from inguinal lymph node biopsies from 10 days post-inoculation (dpi) in ConA-treated pigs and from 15dpi in non-treated and IFN-gamma-treated pigs. ConA increased PCV2 replication levels, but disease was not observed. Half of the ConA-treated and IFN-gamma-treated pigs showed a delayed humoral immune response, but this delay did not result in increased PCV2 replication in these pigs. These experiments demonstrated that ConA enhances PCV2 replication in PBMC in vitro and in lymphoid tissues in vivo.     

Inhibition of natural killer activity in porcine mononuclear cells by African swine fever virus

The coincubation at 37 degrees C for 24 hours of swine peripheral blood mononuclear cells with African swine fever virus inhibited in part the natural killer activity shown by cells incubated without the virus. This inhibition depended on the dose of the virus and on the time that cells were incubated with it. When the virus preparation was fractionated by ultracentrifugation, most of the inhibitory activity was found in the sedimented fraction, where viral particles were present; however, the loss of inhibitory activity in respect to the whole virus preparation indicated that some inhibitory activity was present in the supernatant fraction, probably as factors released by infected cells. Most of the inhibitory activity shown by the sedimented fraction was lost when the virus was inactivated by ultraviolet radiation, indicating an active role of virus infectivity in the inhibition.     

Detection of Mycobacterium bovis infection and production of interleukin-2 by in vitro stimulation of badger lymphocytes

The Eurasian badger (Meles meles) is considered to be an important wildlife reservoir for Mycobacterium bovis infection of cattle in Ireland and in GB. However, rapid diagnosis of tuberculosis in live badgers has been constrained through a lack of suitable immuno-diagnostic reagents for detection of M. bovis-infected animals. To date, there have been no reports of cytokine activity in badgers that might be associated with specific immune responses to M. bovis infection. In this study, nine badgers were removed from an area with a persistent tuberculosis problem in cattle herds and tuberculosis was confirmed in four of the animals by "post-mortem" examination and M. bovis culture. In preliminary investigations of interleukin-2 (IL-2) activity, we were able to demonstrate that lymphoblasts prepared from badger peripheral blood mononuclear cells (PBMCs) proliferated when cultured in the presence of human recombinant IL-2 (HrIL-2). Supernatants derived from purified protein derivative of tuberculin (PPD-bovine) stimulated PBMC cultures also induced blastogenesis of badger-derived lymphoblasts. The results demonstrate that badger lymphocytes are responsive to HrIL-2 and that PPD-bovine stimulation of badger PBMC results in production of bio-active IL-2.     

Phenotypic analysis of peripheral leukocytes in piglets infected with classical swine fever virus.

The phenotypic changes in circulating leukocytes in swine fever influenced by classical swine fever virus (CSFV) infection with different strain virulence was studied in piglets. The phenotypic differences were measured by monoclonal antibodies specific for porcine differentiation antigens. The pattern of phenotypic change varied with the virulence of CSFV. Infection with virulent, but not the attenuated strain of CSFV resulted in the dramatic early loss of CD8-bearing T lymphocytes from the circulation. A similar trend was also seen in the gammadelta T-cell compartment following infection with the highly virulent strain, Washington. The loss of circulating B-lymphocytes was consistent with the failure to generate neutralising antibody. These observations contrasted the finding that the number of leukocytes expressing the CD4 surface antigen increased in piglets infected with CSFV. These data provide preliminary information on the potential range of leukocyte changes produced in piglets following infection with CSFV.     

Inhibitory effects of ribavirin alone or combined with human alpha interferon on feline infectious peritonitis virus replication in vitro

The antiviral activities of ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide; virazole), either alone or in combination with recombinant human leukocyte (alpha) interferon (rHuIFN-alpha), were evaluated against feline infectious peritonitis virus (FIPV) in feline kidney-cell cultures. The 50% inhibitory dose (ID50) of ribavirin for uninfected, rapidly dividing cells was approximately 17 micrograms ml-1 whereas the ID50 for FIPV was 2.5 micrograms ml-1. The therapeutic index (TI) of ribavirin (i.e. the ratio of the minimum cell-toxic dose to minimum virus-inhibitory dose) was 6.8. Although a dose-dependent inhibition of viral infectivity occurred at non-toxic doses, maximum antiviral effects (greater than or equal to 4 log10 reduction in FIPV) occurred at cytotoxic doses. When low or moderate doses of ribavirin were combined with either 10 or 100 U of rHuIFN-alpha ml-1, the resulting antiviral effects were significantly greater than the sum of the observed effects from either ribavirin or rHuIFN-alpha alone. Significant synergistic interactions with rHuIFN-alpha occurred at ribavirin doses of 1, 5, 12.5 and 25 micrograms ml-1. Synergistic combinations of rHuIFN-alpha and ribavirin produced up to an 80-fold or a 200-fold relative increase in FIPV antiviral activities compared with that produced by equivalent doses, respectively, of ribavirin or rHuIFN-alpha alone. In cell growth studies, the addition of either 10 or 100 U of rHuIFN-alpha ml-1 to test doses of ribavirin did not increase the anticellular effect observed with ribavirin alone; seemingly, the potentiation of ribavirin antiviral activity by rHuIFN-alpha was independent of any additive cytotoxic effects. Potentially, synergistic combinations of the two antiviral agents in vivo may decrease the therapeutic dose of ribavirin required for inhibition of FIPV and thus reduce drug toxicity.     

In vitro and in vivo 2',5'-oligoadenylate synthetase activity induced by recombinant DNA-derived bovine interferon alpha I1 in bovine alveolar macrophages and blood mononuclear cells.

Biological responses to recombinant DNA-derived bovine interferon alpha (rBoIFN-alpha I1) by bovine alveolar macrophages were examined by measuring viral yield reduction and 2',5'-oligoadenylate synthetase (2',5'-OAS) production by IFN-treated cells. In vitro IFN pretreatment of alveolar macrophages reduced viral yield in cultures challenged exposed with parainfluenza-3 virus, compared with control cultures. In vitro treatment of alveolar macrophages with IFN also resulted in increased 2',5'-OAS activity. The 2',5'-OAS activity was measured in alveolar macrophages and blood mononuclear leukocytes of calves injected IM with 3.6 x 10(6) U of rBoIFN-alpha I1/kg of body weight. The IFN action was monitored by measuring 2',5'-OAS activity of blood mononuclear leukocytes beginning 6 days before and ending 24 hours after IFN treatment. The 2',5'-OAS activity in the blood mononuclear leukocytes sharply increased 24 hours after IFN treatment, indicating response to IFN. The alveolar macrophages collected from the same calves 24 hours after IFN administration also had increased 2',5'-OAS activity, compared with alveolar macrophages from the same calves collected 6 days before treatment. Increased 2',5'-OAS activity indicates: a possible mechanism of IFN action in cattle that may be responsible for viral yield reduction; potential use of high enzyme activity as a marker for IFN induction; and potential use of 2',5'-OAS activity as a marker for determining effects of IFN on bovine macrophages and other cells of the bovine immune system.     

Virus antigen expression and alterations in peripheral blood mononuclear cell subpopulations after classical swine fever virus infection.

Depletion in the number of lymphocytes and viral persistence are thought to be the most important outcomes of classical swine fever virus (CSFV) infection. To define the change in peripheral blood mononuclear cells (PBMC) and virus replication in leukocytes after CSFV infection, 8-week old pigs were infected with the LPC vaccine strain or virulent CSFV (HCV-YL strain). Changes in the relative number of PBMCs were analyzed by flow cytometry. The results showed a significant increase in the relative percentage of monocytes in PBMCs during acute CSFV infection of naive pigs (p < 0.05). Monocyte frequencies were not changed in LPC-vaccinated pigs and control pigs. There was also a significant decrease in the number of IgM+ cells (p < 0.05) and a slight decrease in the number of CD4+ lymphocytes after 5 days of infection. There was no change in the frequency of CD8+ lymphocytes in PBMCs after infection. To define which subpopulation of PBMCs was the target for CSFV infection, PBMC populations from CSFV infected pigs were separated and stained for virus antigen expression. Alveolar macrophages (AM) were also studied. The results showed that CSFV replicated in all PBMC subpopulations: CD4+, CD8+, and IgM+ lymphocytes, and monocytes as well as AMs. However, virus antigen expression was more intense in monocytes and AMs. The infection of lymphocytes may, therefore, contribute to the depletion in their numbers after infection and lead to defective antibody production during virulent CSFV infection.     

Transmissible gastroenteritis virus antibody production in vitro by porcine peripheral blood leukocytes.

The purpose of this study was to demonstrate the in vitro production of transmissible gastroenteritis virus (TGEV)-specific antibodies by peripheral blood leukocytes (PBL) harvested from piglets infected with TGEV. Piglets were infected with the virulent Purdue strain of TGEV and at intervals postinfection their PBL were cultivated in the presence of TGEV antigen, control antigen or pokeweed mitogen (PWM). The culture supernatants were tested for TGEV antibodies by a fixed cell enzyme immunoassay. Antibodies were never found in the supernatants of unprimed PBL cultures from control piglets, nor in cultures stimulated with control antigen, and antibodies were produced more frequently in response to stimulation of primed PBL with viral antigen than with PWM. In PBL cultures stimulated with viral antigen, TGEV antibodies of the IgG class were produced more frequently than IgA class antibodies. Optimal antibody responses were produced by PBL harvested two weeks after infection and cultivated at a concentration of 10(7) cells/mL for five days.     

Frequency of association of noncytopathic bovine viral diarrhea virus with mononuclear leukocytes from persistently infected cattle

All mononuclear leukocytes and T lymphocyte-enriched and B lymphocyte-enriched subpopulations of mononuclear leukocytes collected from 8 cows persistently infected with 1 of 3 isolates of noncytopathic bovine viral diarrhea virus were tested for association with virus. For all persistently infected cows, approximately 4.4% of all mononuclear leukocytes, 5.4% of T lymphocyte-enriched, and 2.1% of B lymphocyte-enriched subpopulations of mononuclear leukocytes were associated with virus. Differences between leukocyte populations in percentages of leukocytes associated with virus were real (P less than 0.05). Among virus isolates, significant differences in percentages of leukocytes associated with virus were not detected.     

In vitro stimulation of pig lymphocytes after infection and vaccination with Aujeszky's disease virus

Mitogenic and antigenic lymphocyte stimulation were examined in Aujeszky's disease virus (ADV) infected pigs and in pigs vaccinated with modified live ADV. Neither infection nor vaccination had any effect on lymphocyte responsiveness to phytohaemagglutinin (PHA), pokeweed mitogen (PWM) or concavalin A (Con A). ADV antigen-responsive lymphocytes began to appear in the peripheral blood between 7 and 14 days after inoculation and could still be demonstrated in blood and spleen of infected pigs at 174 days after infection. In vaccinated pigs, sensitized peripheral blood lymphocytes could be detected up to at least 35 days after revaccination. Pre-incubation of ADV antigen with specific antibody markedly reduced lymphocyte stimulation. Non-immunized pigs showed no lymphocyte response to ADV antigen. Infected pigs exhibited no lymphocyte reactivity against antigens of non-infected cells.     

In vitro interferon production by bovine tissues: induction with infectious bovine rhinotracheitis virus.

The interferon-inducing ability of infectious bovine rhinotracheitis (IBR) virus was determined in tissue cultures of bovine origin inoculated with untreated and ultraviolet (UV) irradiated IBR viruses. Interferon was assayed by the plaque-reduction method in bovine fetal kidney (BFK) cell cultures, using vesicular stomatitis virus as challenge virus. Highest interferon concentrations were produced by cultures of bovine fetal (BF) spleen cells and aveolar macrophage cultures derived from adult cattle. Moderate interferon concentrations were produced by peripheral blood leukocyte (PBL) suspension cultures from adult cattle with serum-neutralizing antibodies against IBR virus. Cultures of PBL from 1 cow without detectable serum-neutralizing antibodies against IBR virus did not produce detectable interferon in response to IBR virus. Cultures of PBL from cattle with or without detectable serum-neutralizing antibodies against IBR virus produced interferon when stimulated with phytohemagglutinin (PHA). Low levles of viral inhibitors were detected infrequently in monolayer cultures of BFK and BF nasal mucosa inoculated with UV-irradiated IBR virus and in BF tracheal organ cultures inoculated with untreated IBR virus. Interferon was not detected in fluids collected from IBR virus-exposed monolayer cultures of primary and secondary BF lung, secondary BF tracheal mucosa, secondary BF liver, secondary BF adrenal, and PBL in the 4th and 7th passages. The antiviral inhibitors from BF spleen, bovine alveolar macrophage, and PBL cultures induced with IBR virus, as well as inhibitors from PBL cultures induced with PHA, had the usual properties of interferon.