EMC Virus Infection Research Articles

Increase of Kynurenic Acid after Encephalomyocarditis Virus Infection and Its Significances

The moust symptoms of piglets infected with Encephalomyocarditis virus (EMCV) are related to breeding difficulty, circulation insufficiency, depression and occurrence of high lethality. An increase of tryptophan metabolism in the periphery and in the central nervous system (CNS) in human and non-human subjects with inflammatory diseases has been suggested. We investigated an alterations of tryptophan metabolite i.e. kynurenic acid (KYNA) level in the serum of piglets after EMC virus infection. In addition, we investigated the markers of immune stimulation i.e. neopterin and β2-microglobulin. KYNA was determined by high performance liquid chromatography method, while neopterin and β2-microglobulin by ELISA method. Piglets with an age of 8 weeks were infected intranasal and orally with the EMC virus. Blood samples were collected before virus inoculation at day 0 (control) and at 1, 2, 3 and 4 days post inoculation (DPI) and piglets as control subjects were used, too. In EMCV infected piglets we observed a time dependent alteration of investigated parameters. KYNA level increased significantly and at 3 DPI was 341% of CO, p<0.001 and at 4 DPI an enhancement was 242% of CO, p<0.001, respectively. Neopterin increased moderately after EMCV infection and at 4 DPI was 130% of CO, p<0.05. Serum β2-microglobulin was slightly lowered and at 4 DPI was 86% of CO, p<0.05. Present data indicate an marked increase of kynurenine metabolism in the periphery after EMCV infection and an moderate activation of immune system. A marked increase of KYNA and a moderate enhancement of neopterin indicate sensibility of kynurenine metabolism to EMCV infection. Lowering of ß2-microgobulin might relate to development of events leading to the lethality. We suggest that due to viral infection an increase of KYNA might contribute to the inpairment of organs in the periphery and CNS function and might participate by sudden death.

Enhancement of antiviral immunity by a peptide antagonist of SOCS (89.9)

Abstract Suppressors of cytokine signaling (SOCS) are negative regulators of both innate and adaptive immunity via inhibition of signaling by cytokines such as interferons (IFNs). We have developed a small peptide antagonist of SOCS-1 that corresponds to the activation loop of the Janus kinase JAK2. SOCS-1 inhibits both type I and type II IFN activities by binding to the kinase activation loop via the kinase inhibitory region (KIR) of the SOCS. The antagonist, pJAK2(1001-1013), inhibited the replication of vaccinia virus and EMC virus in cell culture, suggesting that it possesses broad antiviral activity. In addition, pJAK2(1001-1013) protected mice against lethal vaccinia and EMC virus infection. pJAK2(1001-1013) increased the intracellular level of the constitutive IFNβ, which may play a role in the antagonist antiviral effect at the cellular level. pJAK2(1001-1013) also synergizes with IFNs as per IFN gamma mimetic to exert a multiplicative antiviral effect at the level of transcription, the cell, and protection of mice against lethal viral infection. pJAK2(1001-1013) binds to the KIR of both SOCS-1 and SOCS-3 and blocks their inhibitory effects on the GAS promoter. In addition to a direct antiviral effect and synergism with IFN, the SOCS antagonist also exhibits adjuvant effects on the humoral and cellular arms as well as an enhancement of polyI:C activation of TLR3. The SOCS antagonist thus presents a novel and effective approach to enhancement of host defense against viruses.

An age-related change in susceptibility of rat brain to encephalomyocarditis virus infection.

Rats were inoculated intraperitoneally (i.p.) or intracerebrally (i.c.) with 1 x 10(4) plaque forming units (PFU)/animal of the D variant of encephalomyocarditis virus (EMC-D) at 2, 4, 7, 14, 28 or 56 days of age for virological and histopathological examination. In the i.p.-inoculation study, neither viral replication nor lesions were detected in the animals inoculated at 28 and 56 days of age. In the animals inoculated when younger than 14 days of age, lesions were restricted to the brain although viral replication was detected in the brain, heart and pancreas. The brain lesions were characterized by acute meningoencephalitis with neuronal necrosis in the cerebral cortex, hippocampus and thalamus, and viral RNA was detected in degenerated and/or intact neurons. In the i.c.-inoculation study, similar age-related changes in susceptibility of rat brain to EMC-D infection were observed, but a minor difference was that viral replication and lesions were still detected in the hippocampus of some animals inoculated at 28 days of age. These results suggest that an age-related decrease in the susceptibility of rat brain to EMC virus infection may reflect an age-related change in the susceptibility of neurons themselves as well as in maturation of the immune system.

Variation in serum creatine phosphokinase activity as indicated in two-phase EMC-D virus-induced myocarditis.

In this study, myocardial damage in the D-variant of encephalomyocarditis (EMC-D) virus-induced myocarditis has been investigated consecutively by measuring serum creatine phosphokinase (CPK) activity. CPK activity in 8 week-old male BALB/cAJcl mice inoculated with EMC-D virus increased to a peak at 4 or 5 days postinoculation (DPI) and then gradually decreased. The CPK activity rose again after 7 DPI until it reached a second peak. In view of the kinetics of CPK activity, two-phase (early and late phase) myocardial damage in EMC virus infection were considered. In the late phase, an increase in cellular infiltration in the myocardium and a decrease in viral titer in the heart were observed. It was therefore suspected that the increase in CPK in the late phase may be caused by cellular infiltration, but not by viral replication. In our results, we suggested that a serial measurement of serum CPK activity might be a useful method for throwing more light on the myocardial damage caused by the autoimmune response. We also used a pathological (TUNEL) method to detect apoptotic cells and some apoptotic myocytes in the myocardium in late phase EMC virus-induced myocarditis.

EMC virus infection in baboons as a model for studies on antiviral substances

EMC virus causes a lethal infection in baboon monkeys within 4–8 days following subcutaneous injection with 10 4–10 8 pfu of virus. The infection is accompanied by viremia, invasion of heart muscle and of brain. Monkeys infected with 10 6 pfu of EMC virus were treated with human leukocyte interferon. The interferon was injected intramuscularly first 0, 0.5, 6 and 24 h post-infection, then twice daily with a dose of 3 × 10 6 units for 5 consecutive days. All the monkeys treated with interferon remained alive and healthy. Animals infected with EMC virus, but not treated with interferon died within 6 days with evidence of myocarditis. The EMC virus-interferon interaction in baboon monkeys seems to provide a useful primate model system for testing the prophylactic and therapeutic antiviral activity of interferons or other antiviral substances.

Studies on the mode of action of single-stranded polynucleotides which are antiviral against encephalomyocarditis virus infection of mice.

Poly(dI) and poly(dC) administered separately or sequentially show no antiviral effects against EMC virus infection of mice, whereas poly(rI) and poly(rC) are antiviral in such treatment regimens without evidence of interferon induction. The antiviral effects of poly(rI) and poly(rC) appear to depend on single-strandedness because their antiviral effects are decreased by annealing to poly(dC) and poly(dI) respectively. This decrease in antiviral effect would not seem to be due to an adverse effect of polydeoxyribonucleotides on EMC virus infection because the polydeoxyribonucleotides have no effect on the antiviral activity of another single-stranded RNA, E. coli tRNA.

Anti-viral effects of single-stranded polynucleotides against avirulent Semliki Forest virus infection of mice and avirulent infection of rats with encephalomyocarditis virus.

Single-stranded polynucleotide preparations, which neither induce detectable interferon nor affect immune responses, suppress development of antiviral antibodies in mice infected with an avirulent strain of SFV. On a weight basis the antiviral activity of a mixture of poly(I) and poly(ho5C)-copolymer is greater than that of tRNA and similar antiviral effects are observed against a related virulent strain of SFV. EMC virus causes and avirulent infection of rats and development of EMC virus antibodies (routinely determined by assaying the protective effect of rat serum against EMC virus infection of mice) is suppressed when the rats are treated with tRNA or the mixture of poly(I) and poly(ho5C)-copolymer. This suppression of antibodies to EMC virus appears to reflect reduction of virus replication. Treatments of 6 mg/rat i.p. or i.v. 6 hours before infection confer essentially the same antiviral effect as 3 times these polynucleotide doses administered during 3 days immediately post infection. These results with avirulent infections indicate that the previously reported antiviral effects of the single-stranded polynucleotides are not simply due to modifications of the tissue pathology which leads to death in the case of virulent virus infections.

A cell-free model of the encephalomyocarditis virus-induced inhibition of host cell protein synthesis

When preincubated extracts from Krebs-II cells were supplemented with total poly(A)-containing RNA isolated from encephalomyocarditis virus-infected Krebs-II cells, two phenomena characteristic of EMC virus infection of these cells in vivo were observed: (i) preferential translation of viral rather than cellular, mRNA, and (ii) a general inhibition of protein synthesis, relative to the synthesis in samples where the poly(A)-containing RNA from uninfected cells served as the template. The first effect could not be explained by an irreversible functional inactivation of cellular templates and seemed to result from a direct interference of viral RNA with the translation of cellular mRNA. The second effect was due primarily to the presence of double-stranded RNA.

Investigation of the Anti-viral Mechanism of Poly I and Poly C Against Encephalomyocarditis Virus Infection in the Absence of Interferon Induction in Mice

Protection of mice against EMC virus infection by poly C and poly I has already been distinguished from interferon mediated protection in several ways. Transfer of serum from EMC virus infected and poly C or poly I treated mice to donor mice that were then infected shows that the anti-viral effect of the single-stranded polynucleotides is not due to boosting interferon produced by infection itself in the way that inferferon can be 'primed' in vitro. Mice surviving infections of more than I X LD100 as a result of poly C or poly I treatment show no protection against re-infection 15 days after the first infection, indicating no long-term stimulation of immune responses to the virus. Mice treated with an immunosuppressive regime of cytosine arabinoside can be protected against EMC virus infection with poly C and poly I treatment and athymic 'nude' mice can also be protected. The possibility of IgM stimulation by poly C and poly I seems unlikely from experiments in which serum was transferred from mice treated with the polynucleotides and an inactivated EMC 'vaccine' to recipient mice which were then challenged with infectious virus. Protection of mice against EMC virus by the single-stranded polynucleotides is abolished by administration of silica to the mice, implying an involvement of macrophages in the protective effects of poly C and poly I. The possibility that the polynucleotides stimulate clearance of virus particles, at least from immunologically responsive regions of the mouse, has been discounted by the inability of polynucleotide treatment to suppress 'vaccine' mediated protection of mice. These results indicate that macrophages are involved in the anti-viral effects of poly C and poly I either because they inhibit replication of the virus in macrophages or because direct anti-viral properties of macrophages are activated by the polynucleotides.

ENCEPHALOMYOCARDITTS VIRUS INFECTION OF PIGS 1. An Outbreak in New South Wales

Substantial pig mortalities due to heart disease caused by EMC virus infection are described. The majority occurred in 1970 in conjunction with a rodent plague, but retrospective diagnosis of occasional similar mortalities in previous years was possible. Mortality rates in pigs aged 3 to 16 weeks ranged from a few percent to over 50% and the rate was generally highest in younger animals. It is likely that a considerable proportion of cases was sub-clinical. Focal or diffuse pale areas occurred in the myocardium, the wall of the conus arteriosus being most severely affected. In a few animals, resolving lesions were present. Encephalomyocarditis virus was consistently recovered from pigs with acute heart lesions, but not from those in which myocardial fibrosis had commenced. The virus was demonstrated in rodents, although the dependence of pig disease on rodent-derived virus could not be established. Because of similarities between EMC and FMD myocarditides in pigs, specific diagnosis by virus isolation and identification should be obtained whenever possible.

Synthesis of proinsulin and large glucagon immunoreactivity in isolated langerhans islets from EMC-virus infected mice

The protein synthesis in normal and in EMC-virus infected mouse islets of Langerhans was investigated. Mouse large glucagon immunoreactivity was determined by an immunoassay after chromatographic separation. It was characterized as a peptide of 16 000 MW with in intracellular half-life of 35-45 min. The proportional reduction of void volume proteins, large glucagon immunoreactivity and proinsulin synthesis after infection shows, that both alpha- and beta-cells are damaged by the virus. A reduction in the synthesis of the three protein fractions was already found 6 hrs after inoculation of the virus and remained nearly constant for 48 hrs. An almost complete breakdown of protein synthesis occurred 60 to 70 hrs after infection and was paralleled by the first light microscopic changes in the islets. The stimulation of proinsulin synthesis by glucose was preserved for 48 hrs after EMC-virus infection.

Effect of Receptor-Destroying Enzyme on the Growth of EMC Virus in Tissue Culture

Summary and Conclusions Receptor-destroying enzyme (RDE), extracted from cultures of Vibrio cholerae, protected HeLa cell cultures against infection with EMC tissue culture virus. No such protection was observed against infection with Coxsackie Group B (types 3 and 5) or poliomyelitis (types 1–3) tissue culture virus. Further analysis of the protective reaction showed that contact between HeLa cells and RDE results in adsorption, with subsequent elution, of the enzyme and that enzyme-modified cells are temporarily incapable of adsorbing an infectious dose of EMC virus. RDE was not adsorbed on L cells and did not protect these cells in tissue culture against EMC virus infection.