ABSTRACT
While infections with the group B coxsackieviruses are generally asymptomatic, these viruses occasionally cause acute and chronic inflammatory diseases of the pancreas and heart. Chronic inflammatory diseases are of major clinical concern, since they predispose affected individuals to cancer. For example, chronic pancreatitis, which can develop from acute pancreatitis, is a major risk factor for pancreatic cancer, a disease with an exceedingly poor prognosis. It is crucial that we understand the mechanisms underlying the development of acute pancreatitis and the progression to chronic disease, if we are to identify new therapeutic targets to prevent the deterioration of the exocrine pancreas. We have developed a mouse model that allows us to explore the mechanisms by which CVB4 causes acute and chronic pancreatitis. The present review develops the idea that disease progression is a continuum, from acute inflammatory disease to chronic inflammatory disease to cancer, and that accompanying changes in gene expression are both quantitative and qualitative.
Subject(s)
Coxsackievirus Infections/genetics , Enterovirus B, Human , Pancreatic Neoplasms/genetics , Pancreatitis, Chronic/virology , Pancreatitis/genetics , Pancreatitis/virology , Acute Disease , Animals , Coxsackievirus Infections/immunology , Coxsackievirus Infections/virology , Disease Models, Animal , Disease Progression , Enterovirus B, Human/genetics , Enterovirus B, Human/pathogenicity , Gene Expression Profiling , Gene Expression Regulation , Humans , Macrophages/immunology , Mice , NF-kappa B/metabolism , Pancreatic Neoplasms/virology , Pancreatitis/immunology , Pancreatitis, Chronic/genetics , Pancreatitis, Chronic/immunologyABSTRACT
The immunogenicity of soluble peptides can be improved by expression within recombinant microorganisms. The immunogenicity of a peptide expressed within a capsid protein of an attenuated coxsackievirus B4 was evaluated. The insertion site was chosen based on its antigenic structure. A foreign peptide was inserted into a region of the VP1 capsid protein that was identified as a T helper cell epitope. A recombinant virus containing ten amino acids of ovalbumin sequence was genetically stable and retained the biological and physical characteristics of the parental virus. The recombinant was able to elicit a T helper cell response against ovalbumin sequences. This study shows, for the first time, that coxsackievirus can be used as an expression vector and that insertion of heterologous peptides into an immunogenic region is a viable strategy for inducing T helper cell responses against foreign sequences. The implications of this work are that the attenuated coxsackievirus variant may be useful as a vaccine vector for expressing T helper cell epitopes that are important in inducing protective immunity.
Subject(s)
Capsid/immunology , Enterovirus B, Human/immunology , Recombinant Proteins/immunology , Amino Acid Sequence , Animals , Base Sequence , Capsid/genetics , Cell Line , Chimera/genetics , Chimera/immunology , DNA, Recombinant/genetics , Enterovirus B, Human/genetics , Enterovirus B, Human/physiology , Genetic Vectors , In Vitro Techniques , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Ovalbumin/genetics , Ovalbumin/immunology , Recombinant Proteins/genetics , T-Lymphocytes/immunology , Transfection , Virus ReplicationABSTRACT
The ultimate goal in the treatment of HIV-infected persons is to prevent disease progression. A strategy to accomplish this goal is to use chemotherapy to reduce viral load followed by immunotherapy to stimulate HIV-specific immune responses that are observed in long-term asymptomatic individuals. An effective, live, recombinant virus, expressing HIV sequences, would be capable of inducing both CTL and CD4(+) helper T cell responses. To accomplish these goals, the viral vector must be immunogenic yet retain its avirulent phenotype in a T cell-deficient host. We have identified a coxsackievirus variant, CB4-P, that can induce protective immunity against a virulent variant. In addition, the CB4-P variant remains avirulent in mice lacking CD4(+) helper T cells, suggesting that CB4-P may be uniquely suited as a viral vector for a therapeutic HIV vaccine. Two strategies designed to elicit CTL and CD4(+) helper T cell responses were used to construct CB4-P/HIV recombinants. Recombinant viruses were viable, genetically stable, and retained the avirulent phenotype of the parental virus. In designing a viral vector for vaccine development, an issue that must be addressed is whether preexisting immunity to the vector would affect subsequent administration of the recombinant virus. Using a test recombinant, we showed that prior exposure to the parental CB4-P virus did not affect the ability of the recombinant to induce a CD4(+) T cell response against the foreign sequence. The results suggest that a "cocktail" of coxsackie/HIV recombinants may be useful as a therapeutic HIV vaccine.
Subject(s)
AIDS Vaccines/therapeutic use , Enterovirus/immunology , Genetic Vectors/immunology , HIV Core Protein p24/immunology , HIV Infections/therapy , Animals , Base Sequence , DNA, Viral , Enterovirus/pathogenicity , Gene Expression , Genetic Variation , HIV Core Protein p24/genetics , HeLa Cells , Humans , Mice , Mice, Inbred BALB C , Mice, Knockout , Mice, Transgenic , Molecular Sequence Data , Polyproteins/immunology , Vaccines, Attenuated , Vaccines, Synthetic/therapeutic use , VirulenceABSTRACT
While coxsackievirus infections have been linked to several autoimmune diseases, very little is known about the immunogenicity of the coxsackieviruses. Using two genetically related variants of coxsackievirus B4, CB4-P and CB4-V, the relationship between virulence and antigenicity was examined. The virulent variant, CB4-V, was shown to be more antigenic than the avirulent CB4-P variant. The increased antigenicity of CB4-V was due to a single amino acid substitution in the VP1 capsid protein (a threonine residue at amino acid position 129), a site that had been previously identified as a major determinant of viral virulence. Thr-129 of VP1 is predicted to lie within a conformational B cell epitope. In addition, a nearby linear B cell epitope spanning residues 68 to 82 of VP1 was identified as a potential serotype-specific, neutralization antigenic site. The linear and conformational B cell epitopes of coxsackievirus B4 may be analogous to antigenic sites 1 and 1B of poliovirus. To address whether the increased antigenicity of CB4-V influenced the severity of disease, mouse strains that differ in their outcome to viral infection were analyzed. Mice that developed the most severe disease and succumbed to infection were more immunoresponsive than mice that survived infection with CB4-V. The data suggest that immune-mediated mechanisms play a role in the severity of CB4-V induced disease.
Subject(s)
Antigens, Viral/immunology , Capsid/genetics , Capsid/immunology , Enterovirus B, Human/immunology , Enterovirus B, Human/pathogenicity , Point Mutation/genetics , Amino Acid Sequence , Amino Acid Substitution/genetics , Animals , Antigens, Viral/chemistry , Antigens, Viral/genetics , Capsid/chemistry , Coxsackievirus Infections/immunology , Coxsackievirus Infections/mortality , Coxsackievirus Infections/virology , Enterovirus B, Human/chemistry , Enterovirus B, Human/genetics , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/genetics , Epitopes, B-Lymphocyte/immunology , Genetic Variation/genetics , Immunoglobulin G/immunology , Mice , Mice, Congenic , Mice, Inbred Strains , Models, Molecular , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/immunology , Protein Conformation , Sequence Alignment , Threonine/genetics , Threonine/immunology , Virulence/immunologyABSTRACT
By using a model of coxsackievirus B4-induced disease, the question of whether tissue damage is due to the virus or to immune-mediated mechanisms was addressed. Both viral replication and T-cell function were implicated in contributing to the severity of disease. Three stages (I to III) of disease, which correspond to periods of high viral titers, low viral titers, and no infectious virus, have been identified. Stage I disease is considered to be primarily the result of viral replication. Immunopathological mechanisms appear to contribute to the severity of stage II and III disease. To investigate the role of T cells in contributing to the severity of disease, viral infection in CD8 knockout (ko) mice and CD4 ko mice was analyzed. CD8 T-cell responses appear to be beneficial during early, viral disease but detrimental in later disease when viral titers are diminishing. CD4 ko mice, unlike the parental strain, survived infection. Viral replication was lower in the CD4 ko mice. Was survival due to decreased viral replication or to the lack of T-helper-cell function? To investigate further the role of T helper cells in contributing to tissue damage, viral infection in two additional ko strains (interleukin-4 [IL-4] ko and gamma interferon ko strains) was examined. A clear correlation between viral replication and the outcome of infection was not observed. The absence of IL-4, which may influence T-helper-cell subset development, was advantageous during early viral disease but deleterious in later disease. The results suggest that T-cell-mediated immunity is both beneficial and detrimental during coxsackievirus B4 infection.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Coxsackievirus Infections/immunology , Enterovirus/immunology , Animals , Coxsackievirus Infections/physiopathology , Cytotoxicity, Immunologic , Female , Male , Mice , Mice, KnockoutABSTRACT
Using a semi-quantitative multiplex reverse transcription-polymerase chain reaction assay, we examined cytokine mRNA expression for interleukin-1alpha (IL-1alpha), IL-2, IL-10, IL-12p40, tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) in skin samples obtained from C4-deficient (C4D) guinea-pigs inoculated intradermally with virulent Treponema pallidum (VTP). Controls included unmanipulated animals, guinea-pigs injected with T. pallidum-free rabbit inflammatory testicular fluid (ITF) alone, or mixed with heat-killed organisms (HKTP). The expression of IL-1alpha, IL-12p40, and TNF-alpha mRNA [T helper type 1 (Th1)] remained within the normal range in both infected and control animals throughout the experimental period. However, a significant increase (P<0.05) in IL-10 mRNA (Th2) was found exclusively in the VTP-inoculated animals from 3 to 30 days post-infection. Another unique characteristic of the inflammatory response in infected guinea-pigs was the appearance, between 11 and 30 days post-inoculation, of a substantial number of eosinophils in addition to infiltrating mononuclear cells. The results showed a local Th2 response which is consistent with an inadequate immune response. This is reflected by the lengthy and incomplete clearance of the pathogen from the local site of entry and the chronic infection of distant organs.
Subject(s)
Cytokines/genetics , Skin/immunology , Syphilis/immunology , Treponema pallidum , Animals , Eosinophilia/immunology , Gene Expression , Guinea Pigs , Interleukin-1/genetics , Interleukin-10/genetics , Interleukin-12/genetics , Interleukin-2/genetics , Male , Rabbits , Reverse Transcriptase Polymerase Chain Reaction/methods , Transforming Growth Factor beta/genetics , Tumor Necrosis Factor-alpha/geneticsABSTRACT
The authors report, for the first time, the cloning, characterization and sequencing of guinea pig cDNAs for interleukin (IL)-2, IL-10, IL-12p40, and transforming growth factor beta (TGF-beta). Partial cDNAs for two additional cytokines, IL-1alpha and TNF-alpha, whose sequences are present in the GenEMBL database, were also cloned. The IL-10 clone is a full-length cDNA, while the remaining clones are partial cDNAs. The guinea pig cDNA sequences have high identity with their mouse and human counterparts. Northern blot analysis revealed that the guinea pig transcripts range in size from 1.0 kb to 2.2 kb. The constitutive expression of cytokines in two strains of guinea pig (C4D, Albany) that differ in susceptibility to infection with Treponema pallidum was examined. Since susceptibility to T. pallidum is also age dependent, both neonates and adults were examined. Spontaneous cytokine expression was examined in peripheral blood, skin, spleen, lymph node, brain, and peritoneal cells. In skin, lymph node, and peripheral blood, very low levels of IL-1alpha, IL-12p40, tumour necrosis factor alpha (TNF-alpha), and TGF-beta and moderate levels of IL-2 and IL-10 were observed. Cytokine gene expression was not observed in spleen and brain. Peritoneal cells expressed only TGF-beta. Age- and strain-associated differences were not observed, except for IL-12p40, which was elevated in guinea pigs resistant to T. pallidum infection (C4D neonates, Albany adults).
Subject(s)
Cytokines/genetics , Gene Expression Regulation , Adult , Animals , Base Sequence , Cloning, Molecular , Cytokines/biosynthesis , Cytokines/blood , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Guinea Pigs , Humans , Mice , Molecular Sequence Data , Organ Specificity , Polymerase Chain ReactionABSTRACT
Two genetically similar variants of coxsackievirus B4, CB4-P and CB4-V, cause distinct disease syndromes in mice. A multidisciplinary approach was used to examine the events occurring in situ. The CB4-P variant induced acute pancreatitis, followed by repair of the exocrine tissues, while the CB4-V variant induced chronic pancreatitis, characterized by extensive destruction of the exocrine tissues. Since CB4-V replicated more efficiently than CB4-P in vivo, the more extensive tissue injury associated with CB4-V infection could be explained as the result of a higher level of viral replication. However, the fact that CB4-V replicated more efficiently in a mouse strain that survives infection than in a strain that succumbs to infection suggests that immune-mediated mechanisms as well as viral cytolysis may contribute to pancreatic tissue injury. To address the role of the immune system in virus-induced pancreatitis, the cell types within the inflammatory infiltrate were analyzed by flow cytometry. B cells (34 to 75%) were the most abundant, followed by T cells (10 to 30%), natural killer cells (4 to 8%), and macrophages (0 to 6%). Recruitment (and perhaps proliferation) of B and T cells to the pancreatic tissues was influenced by viral strain. Differential recruitment of T and B cells may reflect altered antigenic sites between CB4-P and CB4-V. The viral sequence that affected T- and B-cell recruitment was identified as a threonine residue at position 129 of the VP1 capsid protein.
Subject(s)
B-Lymphocytes/immunology , Capsid/immunology , Coxsackievirus Infections/immunology , Enterovirus B, Human/immunology , Pancreatitis/virology , T-Lymphocytes/immunology , Animals , Antigens, Viral/immunology , Cells, Cultured , Flow Cytometry , HeLa Cells , Heart/virology , Humans , Immunophenotyping , Killer Cells, Natural/immunology , Macrophages/immunology , Mice , Pancreatitis/immunology , Spleen/immunology , Spleen/virology , Virus ReplicationABSTRACT
Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease whose etiology is complex. Both genetic susceptibility, which is polygenic, and environmental factors, including virus infections, appear to be involved in the development of IDDM. In this review, we have tried to balance the discussion of diabetes by examining both immunological and virological perspectives. Several mouse models, including viral and non-viral models, have been used to study diabetes. For this review, we include lessons gleaned from the non-obese diabetic (NOD) mouse and from mouse models of coxsackievirus- and encephalomyocarditis-virus-induced diabetes. Finally, we present a multi-stage model in which several viral infections, including the coxsackieviruses, are postulated to play a role in the autoimmune destruction of pancreatic beta cells.
Subject(s)
Coxsackievirus Infections , Diabetes Mellitus, Type 1 , Enterovirus , Animals , Autoimmunity , Disease Models, Animal , Humans , Mice , Mice, Inbred NODABSTRACT
While alcohol abuse and biliary disease can result in the development of pancreatitis, the factors that contribute to the idiopathic form of the disease are not well understood. I propose that coxsackievirus infections account for a subset of cases of pancreatitis of unknown etiology. Evidence to support this concept is derived from serological studies, case reports and animal models. In reviewing the available data, it is obvious that the relationship between coxsackievirus infection and the development of pancreatitis is not a simple one. Many elements contribute to the development of the disease including the strain of the infecting virus, the genetic predisposition of the host and additional environmental factors that maintain the disease process. Studies that show an association between coxsackievirus infection and acute pancreatitis in humans are given additional support by the extensive data from mouse studies demonstrating that some serotypes (B4,B3) are tropic for the exocrine pancreas. Some viral strains may cause limited pancreatic tissue injury which is compatible with tissue repair followed by full restoration of pancreatic function. Other viral strains may cause more extensive tissue damage giving rise to chronic pancreatitis which, on a genetic background that predisposes to autoimmunity, may result in an autoimmune chronic pancreatitis. A multi-disciplinary approach is required to increase our understanding of the complex relationship between coxsackievirus infection and pancreatic diseases. Such studies should address the biology of viral replication, the immune response to infection, the role of viruses in the development of autoimmunity, the biology of pancreatic tissue injury and the underlying repair process.
Subject(s)
Coxsackievirus Infections/virology , Pancreatitis/virology , Acute Disease , Animals , Autoimmune Diseases/virology , Chronic Disease , Enterovirus/genetics , Enterovirus/isolation & purification , Enterovirus/physiology , Enterovirus B, Human , Humans , Mice , Pancreatitis/etiology , Pancreatitis/pathologySubject(s)
Coxsackievirus Infections/virology , Enterovirus B, Human/genetics , Enterovirus B, Human/pathogenicity , Virulence/genetics , Animals , Base Sequence , Chromosome Mapping , Coxsackievirus Infections/genetics , Heart/virology , Humans , Mice , Mice, Inbred C3H , Molecular Sequence Data , Pancreas/virology , Sequence AlignmentABSTRACT
Coxsackieviruses B (CVBs) are etiological agents of human inflammatory myocardial disease. The genetics of the coxsackieviral virulence phenotype in mice are now beginning to be understood with the availability of infectious cDNA copies of CVB genomes. Investigations to date with CVB3 and CVB4 have shown that sites within a non-translated region and in the capsid proteins can affect the virulence phenotype. The relative importance of these sites to expression of the phenotype remains unclear.
Subject(s)
Cardiomyopathy, Dilated/virology , Coxsackievirus Infections/virology , Enterovirus B, Human/genetics , Enterovirus B, Human/pathogenicity , Myocarditis/virology , Aged , Animals , Base Sequence , Humans , Mice , Molecular Sequence Data , VirulenceABSTRACT
In analyzing the molecular determinants of virulence of coxsackievirus B4, chimeric viruses were constructed from avirulent and virulent viruses. The vCB424 recombinant contained a single nucleotide substitution on an avirulent genetic background, resulting in replacement of Ser-16 of VP4 with Arg-16. Mice infected with vCB424 displayed an intermediate phenotype.
Subject(s)
Capsid Proteins , Capsid/genetics , Coxsackievirus Infections/virology , Enterovirus B, Human/genetics , Enterovirus B, Human/pathogenicity , Point Mutation , Amino Acid Sequence , Amylases/blood , Animals , Arginine , Chimera , Coxsackievirus Infections/pathology , Coxsackievirus Infections/physiopathology , Enterovirus B, Human/ultrastructure , Genotype , Mice , Pancreas/pathology , Pancreas/virology , Phenotype , Restriction Mapping , Serine , Viral Plaque Assay , Virulence/geneticsABSTRACT
The genetic determinants of plaque size of two variants of coxsackie-virus B4, CB4-P and CB4-V, were identified using a panel of recombinant, chimeric viruses. When grown in monkey kidney cells, CB4-V yielded large plaques with an average size of 1.0 cm while CB4-P yielded small plaques with an average size of 0.4 cm. Two genetic domains, the 5' untranslated region and the VP4 gene sequence, independently influenced plaque size. Recombinant viruses containing the CB4-P genetic background with point mutations in either the VP1 or VP2 coding sequences had small plaque phenotypes. However, two additional chimerics containing the CB4-P genetic background with either a point mutation in the VP4 sequence or four substitutions in the 5' untranslated region, had large plaque phenotypes. Plaque size correlated with growth kinetics under single-step conditions. Large-plaque variants replicated to higher titers than small-plaque variants. Comparison of the growth kinetics of the recombinant viruses revealed some differences in viral replication. These data suggest that both the 5' untranslated region and arg-16 of VP4 influence viral replication but at different stages of the replication cycle.
Subject(s)
Capsid/genetics , Enterovirus B, Human/growth & development , Enterovirus B, Human/genetics , Animals , Capsid Proteins , Cell Line , Chimera , Genes, Viral , Genetic Variation , Haplorhini , Kidney , Phenotype , Point Mutation , Recombination, Genetic , Restriction Mapping , Viral Plaque AssayABSTRACT
RNA extracted from purified Pichinde virion preparations yields four major species, two of which appear to represent the viral genome while the other two appear to be cellular ribosomal 28S and 18S RNA. The molecular weights of these RNA species have been estimated by gel electrophoresis following denaturation by either methylmercury or glyoxal/dimethyl sulfoxide. The values determined by the two methods are in close agreement. The average molecular weights of L and S Pichinde viral RNA are 2.63 x 10(6) to 2.83 x 10(6) to 1.31 x 10(6), respectively.