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The combined protective effect of a novel naturally glycosylated Cu/Zn-containing superoxide dismutase, produced by the fungus Humicula lutea (HL-SOD) strain 103, and the selective anti-influenza drug rimantadine hydrochloride (Rim) was evaluated in experimental virus infection in mice, induced with influenza virus A/Aichi/2/68 (H3N2). A combined application of HL-SOD and Rim in doses, which by themselves did not protect significantly mice against the infection, resulted in a synergistically increased protection, determined on the basis of protective indices. Lung virus titers, lung weights and consolidation and mortality rates were all decreased significantly, while survival times were prolonged.

The protective effect of an immunostimulatory bacterial preparation, cytoplasmic membranes of Escherichia coli WF stable protoplast type L-forms (CM) alone and in combination with the selective antiviral drug rimantadine was evaluated in experimental influenza A/Aichi/2/68 (H3N2) virus infection in mice. In sublethal infection, CM administered intraperitoneally (i.p.) 7 days before virus exposure in a single dose of 25 mg/kg did not reduce significantly the virus lung titers. In lethal infection, CM applied in the same way weakly reduced the mortality rate. The combined application of CM with rimantadine resulted in synergistically increased protection, determined on the basis of virus lung titers, lung consolidation, mortality rates, protective indices, and survival times.

Using 14 monoclonal antibodies (MoAbs) in solid-phase ELISA it was found that influenza virus A/Hong Kong/1/68 (H3N2) mutants resistant to the antiviral compound mopyridone as compared to the mopyridone-sensitive mutant manifested significant changes in the antigenic structure (sites 1A, 2 and 3) of M1 protein. No differences in M1 were found between rimantadine-resistant and rimantadine-sensitive mutants of influenza virus A(H3N2).

The results of the study on the combined antiviral activity of the SHS-174 preparation (a lyophylized infusion from three higher plants) and three amantadine derivatives (rimantadine, amantadine glucuronide and its derivative) are presented. The antiviral effect of the drugs on the reproduction of influenza virus strains A/H1N1 and A/H3N2 in vitro was studied. The combined antiviral effect was evaluated on the basis of viral yields and in many cases a synergism was found. The most synergistic effect was shown for the combination of SHS-174 with the derivative of amantadine glucuronide.

Rimantadine in the concentration of 50 micrograms/ml did not affect the distribution in subcellular fractions of MDCK cells of the parent influenza virus A/Khabarovsk/74/77(H1N1) labelled with 14C-amino acids, 3H-nucleosides or 32P-orthophosphoric acid. In the presence of rimantadine viral structures of heterogenous composition were detected in the nuclei of infected cells. Their buoyant densities of 1.34-1.36, 1.41-1.42 and 1.28-1.32 g/cm3 in the CsCl gradient were characteristic for RNP and "nucleoids" (complex of RNP and M protein), respectively. Similar structures were also detected in the nuclei of cells infected in the absence of rimantadine. No primary transcription of the viral genome was registered in the presence of rimantadine, although the genetic material in the nuclei was present in a form suitable for transcription.

The sensitivity to rimantadine of the reproduction of influenza virus A/Texas/1/77 (H3N2) and A (H1N1) isolates Nos 897, 778 and 782, isolated from patients during an epidemic influenza outbreak in Moscow in the winter of 1977/1978 was compared. A (H3N2) virus proved to be more sensitive than the A (H1N1) viruses tested, as manifested by decreases of the infectious titre in ovo and of the haemagglutinin titre in MDCK cells.

The haemadsorption reduction test with Ehrlich mouse ascites tumour cells proved to be a simple method for testing the sensitivity of influenza A virus strains to antiviral substances. Ribavirin and 2-deoxy-D-glucose were poorly effective in this system while all virus strains except A/PR/8/34 (H0N1) were highly sensitive to amantadine and rimantadine, the latter being the strongest inhibitor.

The plaque inhibition method was modified in order to evaluate the effectiveness of various combinations of antiviral substances. One substance (A) diffuses from the centre of cell culture, the other (B) is incorporated into the agar overlay at subinhibitory concentration. The inhibitory effect of the combination (A + B) is demonstrated by the increase in size of the inhibitory zone in comparison with the control inhibitory zone produced by the substance A alone. The ratio of the diameter of the inhibitory zone with substance combination (A + B) to the diameter of single drug control zone (substance A) serves as index DI (degree of interaction). Quantitative evaluation of the degree of potentiation using isobolograms showed that DI greater than 1.5 indicate a synergistic effect of the respective combinations. This inexpensive method can serve for rapid selection of suitable combinations out of number of substances. Model experiments were performed with combinations of selected inhibitors of virus replication.

Rimantadine prevents the conformational changes of influenza virus haemagglutinin (HA) caused by acid pH and the acquisition of sensitivity to trypsin, protects the haemolytic activity from inactivation and prevents the morphological changes of HA spikes on the virus surface.

All rimantadine-resistant variants of influenza virus prepared by consecutive passages in the presence of rimantadine had increased virion transcriptase activity as compared to the original strains. The increased virion transcriptase activity of rimantadine-resistant strains was unrelated to the possible role of M1 protein, since RNPs isolated from the virions of these variants also revealed higher transcriptase activity as compared to RNPs isolated from rimantadine-sensitive virus. The study of rimantadine-resistant recombinant X-4 which inherited from the resistant fowl plague virus (FPV) only the gene 7 coding for M proteins provided additional evidence for the suggestion that the increased virion transcriptase activity of rimantadine-resistant influenza virus variants is coincidental rather than directly associated with such resistance.

Virion deproteinization and viral RNA transport to the isolated cell nuclei have been studied in the presence of rimantadine with rimantadine-sensitive influenza viruses fowl plague (H7N7), A/Krasnodar/101/59 (H2N2) and rimantadine-resistant influenza strains (WSN/H1N1 and A/Krasnodar/101/59-R). Rimantadine failed to affect deproteinization during incubation with the isolated cellular plasma membranes as well as the transport to isolated cell nuclei of the viral RNA of either sensitive or resistant strains of influenza virus. Using photosensitive viruses (labelled with neutral red) rimantadine exerted dissimilar effects on deproteinization of the sensitive and resistant influenza virus strains. The possible effects of rimantadine on influenza virus deproteinization is discussed.

The sensitivities to amantadine and rimantadine of influenza A virus epidemic strains were assayed by the haem-adsorption reduction test in mouse Ehrlich ascites cells in comparison with prototype strains and a rimantadine-resistant mutant. Besides a majority of sensitive strains, two relatively resistant epidemic strains were identified. The possible origin of resistant strains and their importance for medical practice are discussed.