Semliki Forest Virus Infection Research Articles

Varying role of alpha/beta interferon in the antiviral efficacy of synthetic immunomodulators against semliki forest virus infection

The question of whether interferon alpha/beta is the common mechanism of antiviral action of synthetic immunomodulators was investigated in B6C3F1 mice infected with Semliki Forest virus. Mice were treated with various concentrations of normal sheep serum or potent anti-alpha/beta interferon antiserum, inoculated with the immunomodulators, and infected 24 hours later with virus. Three patterns emerged. The antiviral action of the pyrimidinone (ABMP) and the oral interferon inducer (CL246,738) appeared to be mediated primarily by interferon alpha/beta; their protective ability was almost completely abrogated by treatment with low levels of anti-alpha/beta interferon antiserum. The antiviral action of two other immunomodulators, a mismatched polyribonucleotide (Ampligen) and a polyanionic copolymer (MVE-2) at least partially involved interferon. Activity of these compounds was reduced, but not consistently eliminated by treatments with high doses of antiserum. The antiviral activity of another polyribonucleotide, polyriboinosinic-cytidylic acid complexed with lysine carboxymethylcellulose (poly ICLC), was not affected by treatment with even the highest amount of antiserum (two injections of 100000 neutralizing units each). Almost complete protection by poly ICLC was observed despite the fact that this high concentration of antiserum, when given alone, caused a decrease in natural resistance to Semliki Forest virus infection. Taken together, these results indicate that induction of interferon alpha/beta does not appear to be the major common mechanism of antiviral activity among these diverse synthetic immunomodulators.

Identification of linear epitopes on Semliki Forest virus E2 membrane protein and their effectiveness as a synthetic peptide vaccine

Semliki Forest virus (SFV) infection of mice was used as a model to study the applicability of synthetic peptides containing only linear epitopes as viral vaccines. The identification of linear epitopes with vaccine potential on the E2 membrane protein of SFV was based on the binding of SFV-specific antibodies to a set of overlapping synthetic hexapeptides (Pepscan) representing the whole E2 amino acid sequence. The 14 available E2-specific monoclonal antibodies which were protective in vivo proved to be unsuitable for the identification of linear epitopes because they recognized only conformational epitopes, as indicated by their lack of reactivity with unfolded, reduced E2 protein on immunoblots. Three epitopes were detected with polyclonal anti-SFV serum at amino acid positions 135 to 141, 177 to 185 and 240 to 246 of the E2 protein. Synthetic peptides containing these epitopes were coupled to a carrier protein and tested as a vaccine. Mice immunized with the peptide containing amino acids 240 to 255 of protein E2 were protected against a challenge with virulent SFV but protection of mice immunized with the peptides containing amino acids 126 to 141 or 178 to 186 was only marginally better than that of controls. The prechallenge sera of most peptide-immunized mice reacted with SFV-infected cells but none of these sera neutralized the virus in vitro. However, protection of mice correlated well with SFV-specific antibody titre, suggesting antibody-mediated protection.

Cholesterol is required for infection by Semliki Forest virus.

Semliki Forest virus (SFV) and many other enveloped animal viruses enter cells by a membrane fusion reaction triggered by the low pH within the endocytic pathway. In vitro, SFV fusion requires cholesterol in the target membrane, but the role of cholesterol in vivo is unknown. In this paper, the infection pathway of SFV was studied in mammalian and inset cells substantially depleted of sterol. Cholesterol-depleted cells were unaltered in their ability to bind, internalize, and acidify virus, but were blocked in SFV fusion and subsequent virus replication. Depleted cells could be infected by the cholesterol-independent vesicular stomatitis virus, which also enters cells via endocytosis and low pH-mediated fusion. The block in SFV infection was specifically reversed by cholesterol but not by cholestenone, which lacks the critical 3 beta-hydroxyl group. Cholesterol thus is central in the infection pathway of SFV, and may act in vivo to modulate infection by SFV and other pathogens.

Changes in fast axonal transport of proteins prior to demyelination in optic nerves of mice following Semliki Forest virus infection

Changes in fast axonal transport of proteins prior to demyelination in optic nerves of mice following Semliki Forest virus infection

CHANGES IN FAST AXONAL TRANSPORT OF GLYCOPROTEINS IN OPTIC NERVES OF MICE INFECTED WITH SEMLIKI FOREST VIRUS

[3H]fucose incorporation into mouse retinae and subsequent axonal transport of glycoproteins to optic nerve terminals was studied before and during optic nerve demyelination induced by Semliki Forest virus (SFV) infection. As was previously found for [3H]proline-labelled protein, axonally transported glycoprotein was increased before demyelination. Fluorographic analysis of the increased glycoproteins and proteins, after separation by gel electrophoresis, showed particularly large increases in labelling of 2 glycoproteins (38.1 kDa and 45.0 kDa) and 3 protein (15.9 kDa, 23.8 kDa and 27.7 kDa) bands. These increases were due to host-cell rather than viral components. At the time of demyelination, however, [3H]fucose incorporation into retinae of SFV-infected mice was significantly depressed, resulting in reduced label incorporation into axonally transported glycoproteins. Since glycoproteins play a role in axon-glia recognition and adhesion, the early changes in their axonal transport may contribute to the mechanism of demyelination.

A protective monoclonal anti-idiotypic vaccine to lethal Semliki Forest virus infection in BALB/c mice

Two monoclonal anti-idiotypic antibodies (ab2 MAbs), designated 1.13A112 (immunoglobulin G type 2a [IgG2a]) and 1.13A321 (IgG1), were prepared against Semliki Forest virus (SFV)-neutralizing ab1 MAb UM 1.13. They were identified in hybridoma supernatant fluid by their capacity to block UM 1.13-mediated neutralization of SFV. Although the neutralization-blocking capacities of the ab2 MAbs did not differ, only 1.13A321 evoked SFV-neutralizing ab3 antibodies upon intracutaneous and subcutaneous immunization of BALB/c mice with 1.13A321 chemically cross-linked to keyhole limpet hemocyanin and combined with the adjuvant Quil A. SFV-neutralizing ab3 antibodies appeared in serum within 10 days after primary immunization, and neutralizing antibody titers could be as high as 1/1,000 at day 35. All mice who had developed SFV-neutralizing antibodies upon anti-idiotypic immunization survived an otherwise lethal challenge with virulent SFV. However, induction of SFV-neutralizing ab3 antibodies by ab2 MAb 1.13A321 proved to be genetically restricted to BALB/c mice; even haplotype-identical (H-2d) DBA/2 mice did not respond, and consequently those animals died after infection with virulent SFV.

Direct C-glycosylation of guanine analogs: the synthesis and antiviral activity of certain 7- and 9-deazaguanine C-nucleosides

C-Glycosylation of two guanine analogues, 9-deaza- and 7-deazaguanine, has been achieved under Friedel-Crafts conditions, providing a direct synthetic route to 9-deazaguanosine (4; 2-amino-7-beta-D-ribofuranosyl-5H-pyrrolo[3,2-d]pyrimidin-4(3H)-one) and 8-beta-D-ribofuranosyl-7-deazaguanine (16), respectively. This electrophilic C-glycosylation was applied successfully to six guanine and substituted-guanine analogues resulting in yields of approximately 50%. This represents the first reported C-ribosylation of preformed nitrogen heterocycles isosteric with guanine. These C-nucleosides were evaluated for their ability to provide protection against a lethal Semliki Forest virus infection in mice, relative to 7-thia-8-oxoguanosine which was used as a positive control. Two of the C-nucleosides, 2-amino-6-chloro-5-methyl-7-beta-D-ribofuranosyl-5H-pyrrolo [3,2-d]pyrimidin-4(3H)-one (12) and the corresponding 6-bromo derivative (13), showed good prophylactic activity in this virus model system.

Induction of membrane proliferation in mouse CNS by gold sodium thiomalate with reference to increased virulence of the avirulent Semliki Forest virus

Separation of smooth membrane vesicles from whole mouse brain by isopycnic centrifugation in discontinuous sucrose density gradients show an increased membrane proliferation in gold sodium thiomalate (GSTM) treated mice. Induction of membrane proliferation by GSTM seems to be an important factor in converting the avirulent Semliki Forest virus infection into a lethal one.

Immunoelectron microscopical labelling of a glycolipid in the envelopes of brain cell-derived budding viruses, Semliki Forest, influenza and measles, using a monoclonal antibody directed chiefly against galactocerebroside resulting from Semliki Forest virus infection

Neurotropic RNA budding viruses such as Semliki Forest virus (SFV), influenza and measles were each grown in identical mouse brain cell cultures. Positive immunoelectron microscopical labelling with gold was seen in the envelope of these viruses using an anti-SFV derived glycolipid monoclonal antibody (MAb), 373 shown to be directed chiefly against galactocerebroside. The results indicate that each enveloped virus grown from the same cell type contains the same glycolipid in its envelope. The presence of common glycolipids derived from the host cell in the envelopes of various enveloped budding viruses may play a significant role in the pathogenesis of virus induced, immune mediated CNS autoimmunity and demyelination, particularly in multiple sclerosis (MS).

An Outbreak of Human Semliki Forest Virus Infections in Central African Republic

Semliki Forest (SF) virus was responsible for an outbreak of febrile illnesses in Bangui, Central African Republic (CAR), during October-December 1987. The virus was isolated at first from mosquitoes, mainly Aedes africanus, collected August-October in a gallery forest 100 km from Bangui. During October-December, 22 isolations of SF virus were made from serum samples collected from patients in Bangui presenting with fever, severe persistent headache, myalgia, arthralgia, and a convalescence marked by asthenia. During the same period, 8 SF virus isolates were obtained from mosquitoes collected in Bangui, mainly from Ae. aegypti. Europeans, particularly soldiers who had recently arrived from France, were affected. Antibodies to alphaviruses had been previously detected in a high proportion of resident human populations in CAR. We conclude that SF virus is a human pathogen.

Roles of interferon and natural killer cells in the antiviral activity of 7-thia-8-oxoguanosine against Semliki Forest virus infections in mice

7-Thia-8-oxoguanosine is a novel biological response modifier with broad-spectrum antiviral activity against many DNA and RNA viruses in vivo. Since two of its properties are to induce interferon and to activate natural killer (NK) cells, we investigated the roles of the lymphokine and NK cells in the antiviral activity of the compound against Semliki Forest virus. Antibody to interferon α/β could completely abolish the protective activity of the nucleoside against virus infection in mice, whereas antibodies to interferons β and γ could not, indicating that interferon α was of major importance to confer protection to the animals. Reduced activation of NK cells was also observed in mice treated with 7-thia-8-oxoguanosine and antibody to interferon α/β. The role of NK cells in the protective activity of the compound was directly assessed in beige mice or in Swiss Webster mice treated with asialo GM1 antibody. In both experiments, the animals were protected from lethal virus infection by treatment with nucleoside. Spleen cells primed by 7-thia-8-oxoguanosine and adoptively transferred to untreated mice could not save them from virus-induced mortality. These three results provide evidence that natural killer cells activated by 7-thia-8-oxoguanosine play a minimal role in protection from acute Semliki Forest virus infections in mice.

Postinfection therapy of arbovirus infections in mice

Most antiviral agents are efficacious prophylactically in vivo, and a few are efficacious for postinfection (p.i.) therapy. To explore possibilities for p.i. therapy of encephalogenic Banzi virus (BZV) and Semliki Forest virus infections in mice, we evaluated candidate antiviral therapies after development of the first clinical signs of infection. The earliest clinical indication of BZV viremia in mice is a rise in core body temperature beginning on day 3 p.i. BZV-infected mice showing elevated core body temperatures (greater than or equal to 37.3 degrees C) on days 3 and 4 p.i. were treated intraperitoneally with the interferon inducer poly(ICLC) (80 micrograms per mouse) and/or specific antiserum. Combined therapy on day 3 of a BZV infection protected over 75% of mice showing clinical evidence of viral disease before treatment. Protection against early brain infection must occur on day 4 p.i., since by that day BZV has started multiplying in the brains of the mice. Significant protection occurred with antiserum alone and increased with poly(ICLC). Similar protection was obtained during Semliki Forest virus viremia, but this infection is so rapid that the first clinical signs are reliably detectable only after viremia.

Immunological and cytological studies of autoimmune demyelination and multiple sclerosis

The early history of experimental allergic encephalomyelitis is reviewed from the point of view of the characterization and recognition of myelin basic protein and the active agent in acid-fast bacilli, namely muramyl dipeptide. Protocols effective in inducing demyelination are pinpointed. Special attention is paid to the protocol which depends on pretreating guinea pigs with muramyl dipeptide and foreign protein followed by a second injection of foreign protein and then the animals are injected with myelin basic protein and Freund's complete adjuvant. Variations in the timing and amounts of muramyl dipeptide are described as are their effects on the demyelination. The myelin breakdown has been studied with a monoclonal antibody reactive to P2. Similar pretreatment enhances the demyelination in Semliki Forest virus infection in mice. The changes in the blood brain barrier are found at 7 days after the myelin basic protein is injected and show grossly increased uptake by the cerebral vascular endothelium of IgG and perivascular uptake of IgG. The changes in the cerebrospinal fluid (CSF) proteins are described (IgG and albumin). Studies of P2 protein in the CSF by means of a new ELISA technique have been performed on human CSF in multiple sclerosis.

Enhancement of demyelination in the central nervous system of Semliki Forest virus infected mice after pretreatment with muramyl dipeptide.

Semliki Forest virus infections in mice produce an encephalitis with demyelination. If before giving the virus the mice are treated with muramyl dipeptide in Freund's incomplete adjuvant, there is a significant increase in demyelination. If ovalbumin is added to the above and then followed after an interval by a second dose of ovalbumin and finally by the virus, the demyelination is further, but only marginally increased. The addition of ovalbumin without muramyl dipeptide in the schedule appears to increase the number of infiltrating cells and to a lesser extent the perivascular cuffing, but does not increase the demyelination as compared to that obtained when Semliki Forest virus is given on its own.

Semliki Forest virus (A7[74]) infection of adult mice induces an immune-mediated demyelinating encephalomyelitis.

Semliki Forest virus (A7[74]) infection of adult mice induces an immune-mediated demyelinating encephalomyelitis.

Impaired interleukin 1 and interleukin 2 production following in vitro abortive infection of murine spleen mononuclear cells by semliki forest virus

The effect of Semliki Forest virus (SFV) infection of murine spleen mononuclear cells was investigated in vitro. A small percentage of spleen macrophages expressed viral antigens, but no infectious virus particles were released, indicating an abortive-type infection. Wild-type SFV infected a higher percentage of macrophages than the attenuated, demyelinating mutant A7. The proliferation of spleen mononuclear cells under Con A stimulation was inhibited by the viral infection. The supernatant (SN) harvested from infected and Con A-stimulated spleen adherent cells could not stimulate thymocytes in an interleukin 1 (IL-1) assay and indomethacin treatment of infected cultures had no effect. The stimulatory effect of SN from noninfected cultures in the IL-1 assay was reduced when SN from infected cultures was added, suggesting the presence of an IL-1 inhibitor. Interleukin 2 (IL-2) production by splenocytes also decreased after viral infection, but exogenous IL-2 restored the response to Con A stimulation of infected spleen cells. This study demonstrates that abortive SFV infection of spleen macrophages has an immunosuppressive effect which may lead to an aberrant immune regulation.

Semliki Forest Virus-specific Non-structural Protein nsP3 Is a Phosphoprotein

Antisera were raised in rabbits against fusion proteins consisting of beta-galactosidase and partial amino acid sequences of Semliki Forest virus (SFV)-specific non-structural proteins nsP1, nsP2, nsP3 and nsP4. The antisera were specific since each of them precipitated only one labelled protein of a size expected for nsP1, nsP2, nsP3 or nsP4 from lysates of [35S]methionine-labelled SFV-infected BHK-21 cells. The specific antisera also precipitated p220 (with sequences of nsP1, nsP2 and nsP3), p155 (nsP1 and nsP2) and p135 (nsP3 and nsP4) which have been previously shown to be cleavage products of the polyprotein precursor of the non-structural proteins. nsP1, nsP4 and most of nsP3, together with the virus-specific RNA polymerase activity, were in the mitochondrial pellet (P15) fraction of infected BHK-21 cells whereas nsP2 was evenly distributed between P15 and the supernatant fraction (S15). Only antisera directed against nsP3 sequences precipitated a labelled protein from cells incubated with [32P]orthophosphate during SFV infection. Treatment of the immunoprecipitate with calf alkaline intestinal phosphatase reduced the amount of labelled nsP3 considerably. Immunoprecipitated 32P-labelled nsP3, isolated by SDS-PAGE, was subjected to acid hydrolysis. Both phosphoserine and phosphothreonine but not phosphotyrosine could be identified in the hydrolysate. Approximately twice as much [32P]serine as [32P]threonine was detected in nsP3. P15 and S15 fractions were prepared from [35S]methionine- and 32P-labelled SFV-infected cells and the 35S/32P ratio of nsP3 was determined after immunoprecipitation and SDS-PAGE. The nsP3 in S15 was less heavily phosphorylated (about 50%) than P15-associated nsP3. Anti-nsP3 serum revealed large cytoplasmic vesicles in SFV-infected cells in indirect immunofluorescence microscopy.

Evidence for a T-cell related factor as the cause of demyelination in mice following Semliki Forest virus infection

Patchy demyelination throughout the central nervous system, including the optic nerve, is known to occur following infection with Semliki Forest virus (SFV) in Swiss/A 2G mice. An increase in the fast axonal transport of protein in optic nerves occurred before they showed signs of demyelination in Swiss/A 2G mice, heterozygous nude mice and nude mice reconstituted with T-cells. SFV infection, however, caused neither an increase in the fast axonal transport of protein, nor optic nerve demyelination in T-cell-deficient nude mice. Thus, the increase in both axonal transport and demyelination following SFV infection appear to be T-cell-mediated events, rather than direct effects of the virus.

Immunocytochemical evidence for Semliki Forest virus antigen persistence in mouse brain

Semliki Forest virus (SFV) is neurotropic in mice. Mature virulent virus (strain L10) can be identified within the CNS by electron microscopy in adult mice. Inspite of high virus titres, avirulent SFV A7(74) cannot be visualised in the brain of adult mice. Immunocytochemical studies using monoclonal antibodies (MAbs) to A7(74) E 1 and E 2 proteins and viral envelope glycolipids, showed viral E 1 to be labelled in the cerebral capillaries, the E 2 and the putative envelope glycolipids were labelled in the cytoplasm of neurons, particularly in the hippocampal areas and glia in the cerebellum. By double labelling the presence of viral antigens in astrocytes and oligodendrocytes was demonstrated. Viral antigens were identified in the brain up to 183 days after infection. Paraffin sections from Bouin-fixed tissue were found to be the most suitable material for immunocytochemistry of SFV. The presence of life-long anti SFV antibody in the sera of animals after SFV infection, could be due to the persistence of viral antigens acting as constant stimuli to the immune system.

The effect of gold sodium thiomalate in adult Swiss/A2G mice infected with togaviruses and flaviviruses.

Treatment of adult mice with gold sodium thiomalate made the normally non-lethal Semliki Forest virus and Sindbis virus infections lethal and increased the virulence of Langat and West Nile viruses. These changes were associated with an enhanced virus invasion of the brain. Depression of the humoral antibody response was not observed.