Immune responses of infants to infection with respiratory viruses and live attenuated respiratory virus candidate vaccines

Respiratory viruses such as respiratory syncytial virus (RSV), the parainfluenza viruses (PIV), and the influenza viruses cause severe lower respiratory tract diseases in infants and children throughout the world. Experimental live attenuated vaccines for each of these viruses are being developed for intranasal administration in the first weeks or months of life. A variety of promising RSV, PIV-3, and influenza virus vaccine strains have been developed by classical biological methods, evaluated extensively in preclinical and clinical studies, and shown to be attenuated and genetically stable. The ongoing clinical evaluation of these vaccine candidates, coupled with recent major advances in the ability to develop genetically engineered viruses with specified mutations, may allow the rapid development of respiratory virus strains that possess ideal levels of replicative capacity and genetic stability in vivo. A major remaining obstacle to successful immunization of infants against respiratory virus associated disease may be the relatively poor immune response of very young infants to primary virus infection. This paper reviews the immune correlates of protection against disease caused by these viruses, immune responses of infants to naturally-acquired infection, and immune responses of infants to experimental infection with candidate vaccine viruses.     

Intra-host versus inter-host selection: viral strategies of immune function impairment

We investigate the evolution of viral strategies to counteract immunological attack. These strategies can be divided into two classes: those that impair the immune response inside or at the surface of a virus-infected cell and those that impair the immune response outside an infected cell. The former strategies confer a "selfish" individual selective advantage for intra-host competition among viruses. The latter strategies confer an "unselfish" selective advantage to the virus population as a group. A mutant, defective in the gene coding for the extracellular immune function-impairment strategy, may be protected from immune attack because the wild-type virus in the same host successfully impairs the host's immune function. Such "unselfish" defense strategies are neutral with respect to intra-host competition. We present simple models of viral intra-host and combined inter- and intra-host evolution. We show that selfish strategies can evolve by intra-host evolution. Unselfish strategies may evolve if inter-host selection pressures outweigh intra-host selection, suggesting that such strategies can only evolve in viruses with low mutation rates.     

Modelling the dynamics of LCMV infection in mice: conventional and exhaustive CTL responses

Lymphocytic choriomeningitis virus (LCMV) infection in mice provides an example of an extraordinarily dynamic process with an extreme sensitivity of phenotype of infection to parameters of virus/host interaction. A mathematical model is developed to examine the dynamics of virus-specific cytotoxic T lymphocyte (CTL) response for LCMV infection in mice. The model, formulated by a system of nonlinear delay-differential equations, considers the interacting populations of viruses, precursor CTLs, terminally differentiated effector CTLs and total virus antigen load. Clonal elimination of virus-specific cytotoxic T cells in high-dose LCMV-Docile infection represents an example of the classical phenomenon--high zone tolerance. To describe both conventional and exhaustive CTL responses in the acute phase of LCMV-D infection two mechanisms are invoked: the high virus antigen load inhibition of T-cells proliferation via energy induction and the activation-induced cell death by apoptosis. Parameters of the model, characterizing the rates of virus and CTL production and elimination in spleen, are estimated by assimilating with the model data on the LCMV-D infection in C57BL/6 mice for low-, moderate- and high-dose infections. It is suggested that not only the clonal expansions have to be described in mathematical models as being virus regulated but also the later phases of primary immune response. Down-regulation of the primary CTL response is controlled by a network of mechanisms inducing anergy and apoptosis in activated T cells. The model is used to investigate the effect of variations in virus and CTL response parameters on LCMV infection outcome and suggest predictions for experimental studies, in particular the phenotype of LCMV-WE infection in C57BL/6 as a function of initial virus doses.     

Mechanisms of antigenic variation in influenza virus

Human influenza A viruses evolve rapidly by antigenic shift and antigenic drift. Antigenic shift occurs by gentic reassortment between currently circulating human viruses and influenza viruses of other origin and by re-emergence of a previously circulating virus. The segmental structure of the virus genome enables reassortment in multiply infected cells and also promotes multiple infection because it results in yielding noninfectious particles which randomly lack some genome segment and only become infectious by complementation with others. Antigenic drift is due to an accumulation of nonsynonymous substitutions in the genes encoding the HA and NA proteins. The limited nature of infection to the respiratory epithelium which is the border of the immune system capacitates the virus to reinfect and grow under the partial immune pressure which results in selecting and expanding antigenic mutants.     

Construction and characterization of a triple-recombinant vaccinia virus encoding B7-1, interleukin 12, and a model tumor antigen

BACKGROUND: Construction of recombinant viruses that can serve as vaccines for the treatment of experimental murine tumors has recently been achieved. The cooperative effects of immune system modulators, including cytokines such as interleukin 12 (IL-12) and costimulatory molecules such as B7-1, may be necessary for activation of cytotoxic T lymphocytes. Thus, we have explored the feasibility and the efficacy of inclusion of these immunomodulatory molecules in recombinant virus vaccines in an experimental antitumor model in mice that uses Escherichia coli beta-galactosidase as a target antigen. METHODS: We developed a "cassette" system in which three loci of the vaccinia virus genome were used for homologous recombination. A variety of recombinant vaccinia viruses were constructed, including one virus, vB7/beta/IL-12, that contains the following five transgenes: murine B7-1, murine IL-12 subunit p35, murine IL-12 subunit p40, E. coli lacZ (encodes beta-galactosidase, the model antigen), and E. coli gpt (xanthine-guanine phosphoribosyltransferase, a selection gene). The effects of the recombinant viruses on lung metastases and survival were tested in animals that had been given an intravenous injection of beta-galactosidase-expressing murine colon carcinoma cells 3 days before they received the recombinant virus by intravenous inoculation. RESULTS: Expression of functional B7-1 and IL-12 by virally infected cells was demonstrated in vitro. Lung tumor nodules (i.e., metastases) were reduced in mice by more than 95% after treatment with the virus vB7/beta/IL-12; a further reduction in lung tumor nodules was observed when exogenous IL-12 was also given. Greatest survival of tumor-bearing mice was observed in those treated with viruses encoding beta-galactosidase and B7-1 plus exogenous IL-12. CONCLUSION: This study shows the feasibility of constructing vaccinia viruses that express tumor antigens and multiple immune cofactors to create unique immunologic microenvironments that can modulate immune responses to cancer.     

Heterologous expression of human transketolase

The outcome of a virus infection is strongly influenced by interactions between host immune defences and virus 'antidefence' mechanisms. For many viruses, their continued survival depends on the speed of their attack:their capacity to replicate and transmit to uninfected hosts prior to their elimination by an effective immune response. In contrast, the success of persistent viruses lies in their capacity for immunological subterfuge: the evasion of host defence mechanism by either mutation (covered elsewhere in this issue, by Gould and Bangham, pp. 331-338) or interference with the action of host cellular proteins that are important components of the immune response. This review will focus on the strategies employed by persistent viruses against two formidable host defences against virus infection: the CD8+ cytotoxic T lymphocyte (CTL) and natural killer (NK) cell responses.     

The Leeuwenhoek Lecture, 1997. Marek's disease herpesvirus: oncogenesis and prevention

There are a number of neoplasias for which a herpesvirus is an essential part of the aetiology. Of these, Marek's disease is the most common and provides excellent opportunities for the study of a herpesvirus-induced tumour both experimentally and under natural conditions in the field. Marek's disease is caused by an alpha herpesvirus; it differs from the other oncogenic herpesviruses which are gamma herpesviruses. It is a ubiquitous virus in poultry populations of the world and is highly cell-associated and contagious, yet only a proportion of infected fowl develop tumours. Evidence is presented to suggest that at least one of the reasons for a wide variation in the incidence of the disease is a temporal interplay between virulent viruses and viruses of low or no virulence. The viral genes associated with the oncogenicity of Marek's disease virus (MDV) are discussed and it is concluded that it is likely that several genes are involved. Finally, a brief history of vaccination to control Marek's disease is given and mode of action discussed. It is concluded that the mechanism of protection is mainly through an antiviral cell mediated immune response, resulting in a lowered challenge virus burden. Marek's disease viruses over the past 40 years have been evolving greater oncogenicity, some of which are not adequately controlled by the vaccines that are currently available. It is suggested that for MDV to produce tumours, there is a need for the cytolytic infection phase and that infection must be with an MDV which possesses a functional gC, ICP4 for maintaining latency which allows the expression of at least the 1.8 kb family, pp38, meq, and possibly pp14 genes, for maintaining the tumour state and possibly initiating this state. Intervention in this process reduces the chance of tumour formation and incidence in a population which can occur through natural or man-mediated infection with non-pathogenic MDVs.     

Immunization with nonstructural proteins promotes functional recovery of alphavirus-infected neurons

The encephalitic alphaviruses are useful models for understanding virus-neuron interactions. A neurovirulent strain of Sindbis virus (NSV) causes fatal paralysis in mice by infecting motor neurons and inducing apoptosis of these nonrenewable cells. Antibodies to the surface glycoproteins suppress virus replication, but other recovery-promoting components of the immune response have not been recognized. We assessed the effect on the outcome of NSV-induced encephalomyelitis of immunization of mice with nonstructural proteins (nsPs) by using recombinant vaccinia viruses. Mice immunized with vaccinia virus expressing nsPs and challenged with NSV initially developed paralysis similar to unimmunized mice but then recovered neurologic function. Mice preimmunized with vaccinia virus expressing structural proteins were completely protected from paralysis. Mice immunized with vaccinia virus alone showed paralysis with little evidence of recovery. Vaccinia virus expressing only nsP2 was as effective as vaccinia virus expressing all the nsPs. Protection provided by immunity to nsPs was not associated with a reduction in virus replication or with improved antibody responses to structural proteins. Protection could not be passively transferred with nsP immune serum. The depletion of T cells at the time of NSV infection decreased protection. The data show that antiviral immune responses can improve the ability of neurons to survive infection and to recover function without altering virus replication.     

Resistance to and recovery from lethal influenza virus infection in B lymphocyte-deficient mice

In the adaptive immune response to most viruses, both the cellular and humoral arms of the immune system play complementary roles in eliminating virus and virus-infected cells and in promoting recovery. To evaluate the relative contribution of CD4+ and CD8+ effector T lymphocytes in virus clearance and recovery, we have examined the host response to lethal type A influenza virus infection in B lymphocyte-deficient mice with a targeted disruption in the immunoglobulin mu heavy chain. Our results indicate that naive B cell-deficient mice have a 50- 100-fold greater susceptibility to lethal type A influenza virus infection than do wild type mice. However, after priming with sublethal doses of influenza, immune B cell-deficient animals show an enhanced resistance to lethal virus infection. This finding indicates that an antibody-independent immune-mediated antiviral mechanism accounts for the increased resistance to lethal virus challenge. To assess the contribution of influenza-specific CD4+ and CD8+ effector T cells in this process, defined clonal populations of influenza-specific CD4+ and CD8+ effector T cells were adoptively transferred into lethally infected B cell-deficient mice. Cloned CD8+ effectors efficiently promoted recovery from lethal infection, whereas cloned CD4+ T cells conferred only partial protection. These results suggest that memory T lymphocytes can act independently of a humoral immune response in order to confer resistance to influenza infection in immune individuals. The potential implications of these results for vaccination against human influenza infection are discussed.     

Specific inhibition of granzyme B by parainfluenza virus type 3

T-cell-mediated cytotoxicity is an important means of defense against viral pathogens; however, several viruses possess mechanisms to disrupt cytotoxicity, thereby allowing them to avoid immune clearance. These viruses have been shown to inhibit cytotoxicity by interfering with the capacity of T lymphocytes to specifically recognize infected cells. An alternative mechanism for virally induced cytotoxic dysfunction is identified in this report. We show that parainfluenza virus type 3, a negative-stranded RNA virus, can inhibit cytotoxicity by causing a defect in the cytotoxic effector apparatus. This defect is identified as a selective inhibition of granzyme B mRNA.     

Cancer-related neuropathic pain

Infection by the human immunodeficiency virus (HIV) causes depletion of CD4-positive lymphocytes with consequent immunodeficiency. HIV infection also causes, by direct or indirect mechanisms, both reactive and neoplastic changes in lymphoid tissues. In primary infection reactive changes are a direct response to HIV. Later in the course of the disease there are reactive changes in lymph nodes and extranodal lymphoid tissues which are likely to be largely an indirect effect of HIV infection, being a response to opportunistic infection by other organisms. There is also an increased incidence of autoimmune phenomena in HIV-infected subjects which is likely to be consequent, at least in part, on impaired control of the proliferation of self-reactive B-cell clones. A second mechanism of immune damage of blood cells, probably operating in the case of HIV-related immune thrombocytopenic purpura, is that of cellular damage by immune complexes containing antiviral antibodies. Lymphoid neoplasms associated with HIV infection include non-Hodgkin's lymphoma, Hodgkin's disease and, uncommonly, plasma cell dyscrasias. HIV-associated lymphomas have distinct clinicopathological features and generally a poor prognosis. As for reactive lymphoid lesions, induction of neoplasia is likely, in the majority of cases, to be an indirect rather than a direct effect of the virus. The combination of chronic B-cell stimulation and impaired T-cell function is important, and interaction of lymphoid cells with virus-infected stromal cells may also play a role. Infection by oncogenic viruses such as the Epstein-Barr virus and human herpes virus 8 is also aetiologically important. In rare cases of T-cell lymphoma, HIV may be directly oncogenic.     

Protective heterologous antiviral immunity and enhanced immunopathogenesis mediated by memory T cell populations

A basic principle of immunology is that prior immunity results in complete protection against a homologous agent. In this study, we show that memory T cells specific to unrelated viruses may alter the host's primary immune response to a second virus. Studies with a panel of heterologous viruses, including lymphocytic choriomeningitis (LCMV), Pichinde (PV), vaccinia (VV), and murine cytomegalo (MCMV) viruses showed that prior immunity with one of these viruses in many cases enhanced clearance of a second unrelated virus early in infection. Such protective immunity was common, but it depended on the virus sequence and was not necessarily reciprocal. Cell transfer studies showed that both CD4 and CD8 T cell populations from LCMV-immune mice were required to transfer protective immunity to naive hosts challenged with PV or VV. In the case of LCMV-immune versus naive mice challenged with VV, there was an enhanced early recruitment of memory phenotype interferon (IFN) gamma-secreting CD4(+) and CD8(+) cells into the peritoneal cavity and increased IFN-gamma levels in this initial site of virus replication. Studies with IFN-gamma receptor knockout mice confirmed a role for IFN-gamma in mediating the protective effect by LCMV-immune T cell populations when mice were challenged with VV but not PV. In some virus sequences memory cell populations, although clearing the challenge virus more rapidly, elicited enhanced IFN-gamma-dependent immunopathogenesis in the form of acute fatty necrosis. These results indicate that how a host responds to an infectious agent is a function of its history of previous infections and their influence on the memory T cell pool.     

Clinical aspects of perinatal hypoxic-ischemic brain injury

Feline panleukopenia virus (FPV) and canine parvovirus (CPV) are autonomous parvoviruses which infect cats or dogs, respectively. Both viruses cause an acute disease, with virus replicating for less than seven days before being cleared by the developing immune responses. The viruses have a broad tropism for mitotically active cells. In neonatal animals the viruses replicate in a large number of tissues, and FPV infection of the germinal epithelium of the cerebellum leads to cerebellar hypoplasia, while CPV may infect the hearts of neonatal pups, causing myocarditis. In older animals the virus replicates systemically, primarily in the primary and secondary lymphoid tissues, and also in the rapidly replicating cells of the small intestinal epithelial crypts. A transient panleukopenia or relative lymphopenia is often observed after FPV or CPV infection, respectively. Whether the reduction in cell numbers in vivo is due to virus replicating in and killing cells, or due to other indirect effects, is not known. However, FPV kills both erythroid and myeloid colony progenitors in in vitro bone marrow cultures, and it has been suggested that virus replication in the myeloid cells in vivo could lead to the reduced neutrophil levels seen after FPV infection of cats.     

Evolutionary dynamics of genetic variation in Epstein-Barr virus isolates of diverse geographical origins: evidence for immune pressure-independent genetic drift

The question whether immune pressure exerted by cytotoxic T lymphocytes (CTLs) can influence the long-term evolution of genetically stable viruses such as Epstein-Barr virus (EBV) has generated considerable scientific interest, primarily due to its important implications for the overall biology of the virus. While arguing for a role of CTLs in the evolution of viruses, it is important to differentiate between genetic variation in virus and immune recognition of these variant virus by CTLs. To assess the role of genetic selection in the long-term evolution of EBV, we have analyzed a large panel of type 1 EBV isolates from African, Southeast Asian, Papua-New Guinean (PNG), and Australian Caucasian individuals. Seven different regions of the EBV genome, which include nine CTL epitopes restricted through a range of HLA class I alleles, were sequenced and compared. Although numerous nucleotide changes were identified within these isolates, comparison of synonymous and nonsynonymous substitutions in the CTL epitope indicated that the genetic variation was generated mostly independently of immune selection pressure. Surprisingly, an inverse correlation between genetic variation within certain CTL epitopes and the frequency distribution of HLA alleles that present the CTL epitopes was seen, suggesting that the evolutionary pressures on the CTL epitopes of the virus may be toward their conservation rather than their inactivation. Furthermore, molecular evolutionary genetic analysis of nucleotide sequences revealed that viral isolates from PNG are evolving as a lineage distinct from isolates from African, Southeast Asian, and Australian Caucasian individuals.     

Evolutionary pattern of human respiratory syncytial virus (subgroup A): cocirculating lineages and correlation of genetic and antigenic changes in the G glycoprotein

The genetic and antigenic variability of the G glycoproteins from 76 human respiratory syncytial (RS) viruses (subgroup A) isolated during six consecutive epidemics in either Montevideo, Uruguay, or Madrid, Spain, have been analyzed. Genetic diversity was evaluated for all viruses by the RNase A mismatch cleavage method and for selected strains by dideoxy sequencing. The sequences reported here were added to those published for six isolates from Birmingham, United Kingdom, and for two reference strains (A2 and Long), to derive a phylogenetic tree of subgroup A viruses that contained two main branches and several subbranches. During the same epidemic, viruses from different branches were isolated. In addition, closely related viruses were isolated in distant places and in different years. These results illustrate the capacity of the virus to spread worldwide, influencing its mode of evolution. The antigenic analysis of all isolates was carried out with a panel of anti-G monoclonal antibodies that recognized strain-specific (or variable) epitopes. A close correlation between genetic relatedness and antigenic relatedness in the G protein was observed. These results, together with an accumulation of amino acid changes in a major antigenic area of the G glycoprotein, suggest that immune selection may be a factor influencing the generation of RS virus diversity. The pattern of RS virus evolution is thus similar to that described for influenza type B viruses, expect that the level of genetic divergence among the G glycoproteins of RS virus isolates is the highest reported for an RNA virus gene product.     

Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences

Despite the rapid mutational change that is typical of positive-strand RNA viruses, enzymes mediating the replication and expression of virus genomes contain arrays of conserved sequence motifs. Proteins with such motifs include RNA-dependent RNA polymerase, putative RNA helicase, chymotrypsin-like and papain-like proteases, and methyltransferases. The genes for these proteins form partially conserved modules in large subsets of viruses. A concept of the virus genome as a relatively evolutionarily stable "core" of housekeeping genes accompanied by a much more flexible "shell" consisting mostly of genes coding for virion components and various accessory proteins is discussed. Shuffling of the "shell" genes including genome reorganization and recombination between remote groups of viruses is considered to be one of the major factors of virus evolution. Multiple alignments for the conserved viral proteins were constructed and used to generate the respective phylogenetic trees. Based primarily on the tentative phylogeny for the RNA-dependent RNA polymerase, which is the only universally conserved protein of positive-strand RNA viruses, three large classes of viruses, each consisting of distinct smaller divisions, were delineated. A strong correlation was observed between this grouping and the tentative phylogenies for the other conserved proteins as well as the arrangement of genes encoding these proteins in the virus genome. A comparable correlation with the polymerase phylogeny was not found for genes encoding virion components or for genome expression strategies. It is surmised that several types of arrangement of the "shell" genes as well as basic mechanisms of expression could have evolved independently in different evolutionary lineages. The grouping revealed by phylogenetic analysis may provide the basis for revision of virus classification, and phylogenetic taxonomy of positive-strand RNA viruses is outlined. Some of the phylogenetically derived divisions of positive-strand RNA viruses also include double-stranded RNA viruses, indicating that in certain cases the type of genome nucleic acid may not be a reliable taxonomic criterion for viruses. Hypothetical evolutionary scenarios for positive-strand RNA viruses are proposed. It is hypothesized that all positive-strand RNA viruses and some related double-stranded RNA viruses could have evolved from a common ancestor virus that contained genes for RNA-dependent RNA polymerase, a chymotrypsin-related protease that also functioned as the capsid protein, and possibly an RNA helicase.     

Microbial parasites versus developing T cells: an evolutionary 'arms race' with implications for the timing of thymic involution and HIV pathogenesis

The thymus attempts to ensure that T cells which emerge from it are able to discriminate self from nonself. As such, it is a potential 'backdoor' through which microbial parasites can enter, manipulate the host into perceiving them as 'self', and thereby avoid immune surveillance. It is proposed that the host has evolved to overcome this parasitic strategy by rapidly producing large numbers of long-lived T cells very early in life (closing the backdoor), before the developing individual has significant contact with infectious organisms, and while still under the protection of its mother's intact immune system. Hence the capacity of the thymus to function efficiently early in the lifespan would have been strongly favored by natural selection. It is well established in evolutionary biology that strong selection favoring enhanced early function easily accommodates, through pleiotropy, the accumulation of later occurring negative effects, and it is through this process that thymic involution and subsequent immune system senescence may have evolved. Once a large pool of competent T cells has been produced, even those microbes capable of contaminating the thymus usually can be eliminated, or at least contained. However, microbes that both destroy peripheral T cells (particularly peripheral T cells that are activated against them), and contaminate the thymus (leading to deletion of potential replacements of the destroyed peripheral cells), may be able to eventually overcome the immune system, thus producing disease after a long period of apparent latency. Human immunodeficiency virus, which is initially well controlled by the immune system, may become unleashed via this process.     

Sequential addition of temperature-sensitive missense mutations into the PB2 gene of influenza A transfectant viruses can effect an increase in temperature sensitivity and attenuation and permits the rational design of a genetically engineered live influenza A virus vaccine

We have previously described a strategy for the recovery of a synthetic influenza A virus wild-type (wt) PB2 gene (derived from influenza A/Ann Arbor/6/60 [AA] virus) into an infectious virus. It was possible to introduce an attenuating temperature-sensitive (ts) mutation at amino acid residue 265 of the AA wt PB2 gene and to rescue this mutant gene into infectious virus. Application of this new technology to influenza A virus vaccine development requires that multiple attenuating mutations be introduced to achieve a satisfactorily attenuated virus that retains the attenuation (att) phenotype following replication in vivo. In this report, we demonstrate that putative ts mutations at amino acids 112, 556, and 658 each indeed specify the ts and att phenotypes. Each of these mutations was introduced into a cDNA copy of the AA mutant mt265 PB2 gene to produce three double-mutant PB2 genes, each of which was rescued into an infectious virus. In general, the double-mutant PB2 transfectant viruses were more ts and attenuated in the lower respiratory tracts of hamsters than the single-mutant transfectant viruses, and the ts phenotype of two of three double-mutant PB2 transfectant viruses was stable even after prolonged replication in the upper respiratory tracts of immunocompromised mice. Two triple-mutant PB2 transfectant viruses with three predicted amino acid substitutions resulting from five nucleotide substitutions in the cDNA were then generated. The triple-mutant PB2 transfectant viruses were more ts and more attenuated than the double-mutant PB2 transfectant viruses. These results indicate that sequential introduction of additional ts mutations into the PB2 gene can yield mutants that exhibit a stepwise increase in temperature sensitivity and attenuation compared with the preceding mutant(s) in the series. Furthermore, the level of temperature sensitivity of the transfectant viruses correlated significantly with the level of attenuation of these viruses in hamsters. Although the triple-mutant PB2 transfectant viruses were attenuated in hamsters, intranasal administration of these viruses elicited a vigorous serum hemagglutination-inhibiting antibody response, and this was associated with resistance of the lower respiratory tract to subsequent wt virus challenge. These observations suggest the feasibility of using PB2 reverse genetics to generate a live influenza A virus vaccine donor strain that contains three attenuating mutations in one gene. It is predicted that reassortant viruses derived from such a donor virus would have the properties of attenuation, genetic stability, immunogenicity, and protective efficacy against challenge with wt virus.     

Changes in swine macrophage phenotype after infection with African swine fever virus: cytokine production and responsiveness to interferon-gamma and lipopolysaccharide

Cytokines produced by cells of the immune system, including macrophages, can influence inflammatory responses to viral infection. This has been exploited by viruses, which have developed strategies to direct the immune response towards ineffective responses. African swine fever virus (ASFV) is a double-stranded DNA virus that infects macrophages of domestic swine. In this study, primary cells of monocyte macrophage lineage were obtained from the lungs, peritoneum or blood of domestic swine and, after infection with ASFV, supernatants were tested for cytokines using biological assays. The cytokine transforming growth factor-beta (TGF-beta) was detected after infection of macrophage preparations, but tumour necrosis factor (TNF) and interleukin-1 (IL-1) were not detected. ASFV-infected and uninfected macrophage populations were also tested to assess their ability to respond to cytokines by enhancing production of superoxide in the respiratory burst mechanism. Responses to interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) were suppressed in macrophage populations infected with virus, even at low multiplicities of infection. Addition of TGF-beta to uninfected macrophages resulted in a similar suppression of response, but antibody to TGF-beta did not prevent suppression induced by virus. These results are discussed in relation to the pathology of African swine fever.