Biological activity of a new Soviet natural inducer, double-stranded RNA

Antiviral activity of a two-spiral RNA (ts RNA), a new natural interferon inductor was studied. It was shown that ts RNA extracted from a phage infected E. coli culture was an active inductor of interferon and resistance to infection with the forestspring encephalitis virus experimental animals. In experiments on 10-12 g mice ts RNA administered in a dose of 50 micrograms/mouse 6 hours after the infection induced up to 1280 units/ml of the serum interferon. When the inductor was administered repeatedly, the experimental animals developed hyporeactivity resulting in a marked decrease in interferon production after the 3rd subsequent injection. The most pronounced effect with respect to the forest-spring encephalitis virus was observed when the inductor was administered intraperitoneally in a dose of 20 micrograms/mouse 4 hours before the infection. The protective effect was less pronounced when the inductor was administered 24 and 48 hours before the infection. A two-fold administration of the inductor did not increase the antiviral effect. When the inductor was administered in a dose of 100 micrograms 14 days before the infection, the animals showed an increase in the nonspecific resistance to the infection resulting in a marked antiviral effect.     

Interferon-inducing and antiviral effects of inosiplex in combination with high-molecular interferon inducers

It has been shown that the immunostimulant inosiplex (IP) is capable of inducing interferon production in mice and of stimulating interferon induction by high-molecular inducers. The combined use of IP and poly (G)-poly (C) or dsRNA (RFf2) leads to a longer interferon circulation in the blood. All the combination schemes ensuring the effect of interferon production prolongation were tested for anti-viral activity. The prophylactic administration of the drugs permitted the attainment in mice of the increased resistance to experimental influenza.     

Pharmacological analysis of the effect of natural double-helical nucleic acids on the detoxifying function of the liver

The effect of interferon inductors i.e. double stranded RNAs from S. cerevisiae and phage F6 on the liver detoxicating function was studied on noninbred albino mice. The liver detoxicating function was tested by duration of hexenal sleep. It was shown that intraperitoneal administration of the yeast and phage RNAs in doses of 1/5 LD50 for three times led to increasing of the narcotic sleep duration in the animals by 65 and 207 per cent, respectively. The effect was of the dose-dependent nature. The doses not inducing reliable inhibition of hexenal metabolism were equal to 1/10 LD50 for the yeast dsRNA and 1/27 LD50 for the phage dsRNA. The inhibitory effect of the dsRNAs was retained for 2-3 days after discontinuation of the drug use. When the dsRNAs were administered simultaneously with nembutal, an inductor of the liver microsomal enzymes, the dsRNAs eliminated its inducing effect. Simultaneous administration of alpha-tocopherol lowered the dsRNA effect on hexenal metabolism. The findings suggested that the dsRNA inhibitory effect on the liver detoxicating function was grounded on the mechanisms associated with inhibition of syntheses and activation of lipid peroxidation specific of the monooxygenase system under the action of the dsRNAs.     

Pre- and postexposure protection by passive immunoglobulin but no enhancement of infection with a flavivirus in a mouse model.

Antibody-dependent enhancement of flavivirus infection, which except for dengue virus is without clear proof in vivo, is still under debate. Recently, postexposure immunoglobulin prophylaxis against tick-borne encephalitis virus, a flavivirus, was claimed to possibly have worsened the outcome of infection due to antibody-dependent enhancement. In the present study, antibody-dependent enhancement and pre- or postexposure protection by passive administration of tick-borne encephalitis virus immunoglobulin were evaluated in a mouse model. Preexposure treatment with homologous murine or heterologous human immunoglobulin provided complete protection against lethal challenge with tick-borne encephalitis virus. For postexposure treatment with antibody, the degree of protection correlated with the amount of immunoglobulin administered and was inversely related to the time interval between infection and treatment. Indications of enhancement of infection would have been increased lethality or reduced mean survival time, but neither was observed under the conditions used in our experiments despite the broad range of immunoglobulin and virus challenge doses applied. In contrast to these in vivo results, antibody-dependent enhancement of tick-borne encephalitis virus infection of murine peritoneal macrophages was readily demonstrable in vitro. Thus, antibody-dependent enhancement of viral infection in vitro does not necessarily predict enhancement in vivo.     

Activity of recombinant human alpha interferon against influenza virus infection in mice

The in vivo antiviral activity of recombinant human leukocyte hybrid interferon, HuIFN-alpha AD, was examined. Results showed that this material in highly purified form did not protect mice against a lethal dose of influenza virus, although administration of natural MuIFN-alpha/beta to mice infected with a lethal dose of influenza virus had a marked protective effect. The effect of alveolar macrophages treated with IFN on influenza virus replication was examined in vitro. The antiviral activity of alveolar macrophages treated with HuIFN-alpha AD was lower than that of MuIFN-alpha/beta. It is concluded that HuIFN-alpha AD is effective in direct inhibition of influenza virus, but not in indirect inhibition mediated by alveolar macrophages or in protection of mice from influenza virus infection.     

Interferonogenic and antiviral activity of the double-stranded replicative form of phage f2 RNA in tissue culture and on animals]

Biological activity of a new natural interferon inductor, the replicative RNA form of phage f2 (RFf2) was studied. A possibility of using RFf2 for production of highly active interferon under conditions of superinduction providing an increase in the interferon yield by to 256--512 times as compared to the control samples was shown. The protective interferonogenic and antiviral effect of RFf2 in mice infected with Semliki forest virus (SFV) and tickborne encephalitis virus (TBEV) was studied on administration of the inductor by various routes. It was found that intraperitoneal administration of RFf2 in a dose of 10 gamma per a mouse protected the infected animals from death. It was accompanied by production of up to 1280 units/ml of interferon in the blood serum of the animals. Maximum protection of the animals from death under conditions of the experiment (80 per cent on infection with SFV and 65 per cent on infection with TBEV) was observed when the preparation was administered twice: 4 hours after the infection. Combined use of RFf2 with chemotherapeutics (rimantadine) increased the protective effect both in the tissue culture and in vivo.     

Relation between interferon-inducing activity of natural interferon inducers and their chemical structure]

The interferon-inducing activity of new derivatives of gossypol, a low molecular weight natural polyphenol, and its model compounds was studied in regard to their chemical structure. The active groups required for development of optimal interferon inductors on the basis of the natural polyphenols were defined. As a result of the studies the new interferon inductor AC-1 which is a condensation product of gossypol, a natural polyphenol, and a cellulose derivative was isolated. The interferon-inducing activity of AC-1 was investigated with respect to its dose and the administration route. High interferon-inducing activity of the interferon inductor after its administration by any route in doses of 10 to 100 mg/kg was demonstrated. The serum interferon titers reached 2560 units/ml.     

Study of the antiviral activity of a poly I : poly-C complex with poly-L-lysine in monkeys]

Antiviral activity of poly-I-poly-C complex with poly-L-lysine was studied on macaco rhesus. The complex bifilamentous polyribonucleotide induced active production of serum interferon and provided pronounced protection of the monkeys infected intracutaneously with the variolovaccine virus (10 LD50 for the monkeys in intracutaneous infection). The effectiveness of the protective effect depended on the scheme and route of the drug administration. The highest prophylactic and therapeutic effect was provided by local administration of the complex in a dose of I mg per I kg of the body weight, the incubation period being increased 2--3 times and the period of the skin affections being decreased approximately 2 times. The results of the studies on the effect of poly-I-poly-C complex with poly-L-lysine were evident of definite prophylactic activity of the drug against experimental vernal encephalitis in the monkeys. The animals not treated with the inductor died on the 16th or 17th day after infection because of the paralysis of the trunc and extremities muscles. The clinical evidences of the disease in the animals treated with the drug were not uniform: from complete health to death.     

Correlation of TBE Incidence with Red Deer and Roe Deer Abundance in Slovenia

Tick-borne encephalitis (TBE) is a virus infection which sometimes causes human disease. The TBE virus is found in ticks and certain vertebrate tick hosts in restricted endemic localities termed TBE foci. The formation of natural foci is a combination of several factors: the vectors, a suitable and numerous enough number of hosts and in a habitat with suitable vegetation and climate. The present study investigated the influence of deer on the incidence of tick-borne encephalitis. We were able to obtain data from deer culls. Using this data, the abundance of deer was estimated and temporal and spatial analysis was performed. The abundance of deer has increased in the past decades, as well as the incidence of tick-borne encephalitis. Temporal analysis confirmed a correlation between red deer abundance and tick-borne encephalitis occurrence. Additionally, spatial analysis established, that in areas with high incidence of tick-borne encephalitis red deer density is higher, compared to areas with no or few human cases of tick-borne encephalitis. However, such correlation could not be confirmed between roe deer density and the incidence of tick-borne encephalitis. This is presumably due to roe deer density being above a certain threshold so that availability of tick reproduction hosts has no apparent effect on ticks'' host finding and consequently may not be possible to correlate with incidence of human TBE.     

Resistance of natural and synthetic polyribonucleotide inducers of interferon to human blood ribonucleases]

The resistance of polyribonucleotide inductors of interferon to blood ribonucleases was studied. Blood resistance of larifan and ridostin in the free and shielded state as well as that of the complexes of poly(I)-poly(C) and poly(G)-poly(C) were also investigated. A protective action of polylysine against the inductors was detected which, in case it had no effect on the biological activity of the drugs, could provide its recommendation as a compound for shielding the inductors.     

Assay of interferon and viral antibodies in the cerebrospinal fluid in clinical neurology and psychiatry

Cerebrospinal fluids (CSF) of 245 neurological and 194 psychiatric patients were tested for viral antibodies and interferon. Complement dependent neutralizing antibodies to Herpesvirus hominis 1 were found in the CSF of patients with encephalitis (50.6%), meningitis (35.4%), lesions of peripheral nerves (36.9%), sclerosis multiplex (41.2%), schizophrenia (31.9%), senile dementia (51.4%), mental retardation (11.1%), ethylism (43.5%). Neutralizing antibodies to tick-borne encephalitis virus were found in the CSF of 38% patients with encephalitis, in 14% meningitis, 11% lesions of peripheral nerves and also in 5.6--11.8% of psychiatric patients. In encephalitis, meningitis and in lesions of peripheral nerves were found in the CSF frequently plaque neutralizing antobidies to the tick-borne orbivirus Lipovník, complement-fixing antibodies to lymphocytic choriomeningitis virus and hemagglutination inhibiting antibodies to measles virus. In multiple sclerosis were detected CSF antibodies to measles virus (44%), Herpesvirus hominis 1 (41.2%) and Lipovník virus (52.6%). In neurological patients were observed CSF antibodies simultaneously to two or three viruses in 16.7 to 40.6%, while in psychiatric patients in zero to 4.6%. CSF interferon was found in psychiatric patients with an equal or even higher incidence (33.7 to 57.1%) than in the neurological patients (29.6--38.6%, in multiple sclerosis only 16.7%). Non-interferon virus inhibitors were excluded. The evaluation of the ratio of serum and CSF titers of viral antibodies and of interferon indicated local synthesis of both in the central nervous system -- with the exception of antibodies to Herpesvirus hominis 1 in CSF of some patients with very high titres in serum and probable lesions of the blood brain barrier.     

Interferon-inducing activity of hydroxybenzylamine derivative]

Interferon-inducing activity of the interferon inductor savrats, an oxybenzylamine derivative of was studied. It was shown on experimental animals that along with its low toxicity, savrats had a high interferon inducing capacity. There were early and late peaks of interferon production (4-8 and 48-96 hours later, respectively) depending on the route of the inductor administration. It was noted that for optimization of the schemes for using interferon inductors, careful choosing of the drug pairs participating in the induction and employment of various routes for administration of the same drug are required.     

Dry erythrocytic diagnostic agent for the determination of antiglobulins]

Dry erythrocytic diagnostic agents were obtained under experimental conditions for determination of antiglobulins forming in the organism of man and animals under the effect of serum preparations from the blood of horses and homologoum immunoglobulins. A study was made of the sera of 100 patients with tick-borne encephalitis treated with heterologous and homologous immunoglobulins of directed action; in response to the administration of horse gamma-globulin antiglobulins (in titres below 1 : 10000) appeared in the serum; they circulated in the blood for long periods and inhibited the accumulation of hormonal antibodies to the causative agent; in the majority of cases a high level of antiglobulins to the foreign protein correlated with the presence of remote side-reactions of the serum sickness type. In patients treated with immunoglobulin of human origin antiglobulins were determined in low titres, disappeared from the blood in 15--20 days and did not hinder the accumulation of antihemmagglutinins to the tick-borne encephalitis virus.     

Role of sialic acids in the immune response formation in tick-borne encephalitis

The content of individual forms of sialic acids and total sialic acids in the lymphocytes of tick-borne encephalitis patients has been studied. The level of sialic acids has been found to depend on the clinical form of the disease and on the content of specific IgM and IgG to tick-borne encephalitis virus. Similar dependence has been established with respect to total sialic acids in lymphocytes.     

Immunomodulating properties of interferon inducers

Data on the immunomodulating activity of interferon inductors are presented. It was revealed that the inductors increased the animal vaccinal response. Schemes for combined use of the interferon inductors and immunomodulators were developed. The immunomodulators were shown to increase the host interferon response evident from synergistic increasing of the interferon titers or prolongation of interferon circulation in blood of the animals. The efficiency of the schemes for combined use of the interferon inductors and immunomodulators was obvious from stimulation of the antibody production. As a result the time of the antibody circulation in blood increased. The effect of the combined use of the immunomodulators and interferon inductors was studied. The combined use of the preparations significantly increased the average life-span of the animals and the rate of their survival.     

Experimental study of the prophylactic anti-influenza and interferon-inducing activity of epsilon-aminocaproic acid

Epsilon-aminocaproic acid, an original antifibrinolytic had a rapid and prolonged protective effect, thus lowering the death rate of experimental mice contaminated with virulent strains of the influenza virus. The protective effect of the acid was observed after its intraperitoneal administration in a dose of 20 mg/mouse 24 hours before contamination by the strain with the H3N2 antigenic formula and after intranasal application of 15 mg of epsilon-aminocaproic acid divided into 2 doses 2 days before contamination by the strain with the H1N1 antigenic formula. In the animals contaminated by the H1N1 influenza virus 5 days after subcutaneous administration of the drug in a dose of 30 mg there was detected in the lungs a much lower number of the infectious viruses 3 days after the contamination. It was shown that 3 and 6 weeks after initiation of the 5-day treatment course with subcutaneous administration of epsilon-aminocaproic acid in a daily dose of 90 mg/mouse the animal resistance to the H1N1 influenza virus increased. No interferonogenic activity after administration of epsilon-aminocaproic acid was observed in the mice.     

Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus

We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem-loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents.     

Influence of Statolon on Resistance of Mice to Influenza

Various interferon inducers are known to elicit protection against lethal or infecting doses of certain viral agents. Because of the relatively high morbidity rate of influenza and its seasonal occurrence, we wished to determine whether statolon-induced interferon might be effective in controlling this disease. Mice were treated intraperitoneally with statolon and challenged with influenza A(2) virus by the intranasal route. Although interferon was present in the serum at the time of virus administration, no change in mortality rate was observed. There was, however, a significant increase in the mean survival time of treated animals. Similar results were obtained when Newcastle disease virus was used as the interferon inducer. To determine the effect of the route of challenge, other mice were treated with statolon or Newcastle disease virus and inoculated with mengovirus by the intranasal or intraperitoneal route. The results demonstrated that the treated mice were protected to similar degree against challenge by either route. It is suggested that the relative ineffectiveness of interferon in protecting mice against influenza is due to an intrinsic characteristic of the virus itself rather than the type of interferon induced or the route of virus challenge.     

Effect of a natural interferon inducer (RFf2) on the formation of vaccinal immunity to tick-borne encephalitis]

The effect of the interferon inductor of the natural origin (RFf2) on formation of vaccinal immunity to vernal encephalitis was studied. A single intraperitoneal administration of the preparation in a dose of 10 gamma per a mouse 2 hours after the first injection of the vaccine resulted in increased resistance of the mice to the lethal dose of the infecting virus which was introduced 14 days after the vaccination completion. The production dynamics of interferon induced by RFf2 and its level in the serum of the immunized mice were the same as those in the non-vaccinated animals. An increased number of the vaccine injections, up to 3 did not result in a significant increase in the immunity with respect to either the level of the antiviral resistance or the level of the virus-neutralizing antibody accumulation.     

Activation of interferon regulatory factor 3 is inhibited by the influenza A virus NS1 protein

We present a novel mechanism by which viruses may inhibit the alpha/beta interferon (IFN-alpha/beta) cascade. The double-stranded RNA (dsRNA) binding protein NS1 of influenza virus is shown to prevent the potent antiviral interferon response by inhibiting the activation of interferon regulatory factor 3 (IRF-3), a key regulator of IFN-alpha/beta gene expression. IRF-3 activation and, as a consequence, IFN-beta mRNA induction are inhibited in wild-type (PR8) influenza virus-infected cells but not in cells infected with an isogenic virus lacking the NS1 gene (delNS1 virus). Furthermore, NS1 is shown to be a general inhibitor of the interferon signaling pathway. Inhibition of IRF-3 activation can be achieved by the expression of wild-type NS1 in trans, not only in delNS1 virus-infected cells but also in cells infected with a heterologous RNA virus (Newcastle disease virus). We propose that inhibition of IRF-3 activation by a dsRNA binding protein significantly contributes to the virulence of influenza A viruses and possibly to that of other viruses.