The mechanism of the antiviral activity of an antiviral factor of a non-interferon nature]

Antiviral factor (AF) of protein nature has been isolated from chick embryo fibroblasts infected with Venezuelan equine encephalitis virus. The suppression of virus reproduction has been observed both in homologous and heterologous cell cultures when the preparation was introduced immediately after the adsorption of the virus after pretreatment of the cell monolayer. The study has demonstrated that the antiviral effect of AF is not linked with its IFN-alpha and TNF-alpha activity. Analysis of the results obtained in this study and earlier data contained in literature suggests that infected chick embryo fibroblasts release original cytokine of non-interferon nature with antiviral activity.     

Nonspecificity of antiviral protein action]

The authors studied the antiviral activity of the informative RNA of antiviral protein (M-RNA AVP) isolated from the cells following superinduction of interferon in them for the purpose of ascertaining the action specificity of the product of their translation--AVP. Following the administration of M-RAN AVP a marked (from 1 to 5 lg PFU/mil) reduction of the infectious titres was observed in the homologous and heterologous cells. RNA preparations from control (noninduced) cells possessed a weak (0.4--0.1 lg PFU/mil) antiviral activity.     

Characterization of the Antiviral Activity for Influenza Viruses M1 Zinc Finger Peptides

We sought to investigate the cellular uptake and antiviral activity for the M1 zinc finger peptides derived from influenza A and influenza B viruses in vitro. No cellular uptake was detected by fluorescent microscopy for the synthetic zinc finger peptides. When flanked to a cell permeable peptide Tp10, the zinc finger recombinant proteins were efficiently internalized by MDCK cells. However, no antiviral activity was detected against homologous or heterologous virus infections for the synthetic peptides or the Tp10-flanked recombinant proteins, regardless treated with or without Zn²⁺. Nevertheless, MDCK cell constitutively expressing the M1 zinc finger peptides in cell nuclei potently inhibited replication of homologous, but not heterologous influenza viruses. Adenoviral vector delivered M1 zinc finger peptides also exhibited potent antiviral activity against homologous viruses challenge. Transduction at 100 PFU dose of recombinant adenovirus efficiently protected 99% of the cells from 100 TCID₅₀ of different virus infections for both peptides. These results brought new insight to the antiviral researches against influenza virus infections.     

Induction of antiviral state in fish cells by Japanese flounder, Paralichthys olivaceus, interferon regulatory factor-1

Interferon regulatory factor-1 (IRF-1) mediates an antiviral state in cells by regulating the expression of the interferon (IFN-alpha/beta) system. To elucidate the role of IRF-1 in fish during virus infections, we constructed a recombinant plasmid of the Japanese flounder, Paralichthys olivaceus IRF-1 (JF IRF-1) under the control of the cytomegalovirus (CMV) immediate/early enhancer promoter. The antiviral mechanism of JF IRF-1 was studied using transfection experiments in a homologous cell line. Here, we show that cell supernatants obtained from transiently transfected cells enhanced cell viability of a heterologous cell line upon incubation, reduced the titers of hirame rhabdovirus (HIRRV) and viral hemorrhagic septicemia virus (VHSV), and possessed cytokine-like activity, as shown by their ability to protect cells against virus infections. The supernatants also inhibited the replication of the rhabdoviruses during the early stages of infection as indicated by the reduction of viral titers in the presence of the supernatants obtained from the transfected cells. Further analysis showed that the cell culture supernatants contain cytokine-like substances that possess acid-labile and temperature-resistant properties. These results indicate that JF IRF-1 induces an antiviral state in cells by mediating the production of cytokine-like substances. Thus, JF IRF-1 might be useful as an adjuvant in the development of DNA vaccines against commercially important viral pathogens in Japanese flounder aquaculture.     

Production of monoclonal antibodies against nucleocapsid proteins of herpes simplex virus types 1 and 2.

We prepared mouse hybrid cell lines which produced antibodies against herpes simplex virus type 1 and 2 nucleocapsids. Cell lines 1D4 and 3E1, respectively, secreted immunoglobulin G1 herpes simplex virus type 1 and immunoglobulin G1 herpes simplex virus type 2 antibodies which immunoprecipitated proteins designated p40 and p45 from homologous nucleocapsid preparations but precipitated no proteins from heterologous preparations. In contrast, guinea pig antisera prepared against either herpes simplex virus type 1 or 2 p40 precipitated p40 and p45 from both homologous and heterologous preparations. These findings suggest that p40 and p45 possess similar antigenic determinants and that the monoclonal antibodies that were tested reacted preferentially with the homologous determinants.     

Triple combination antiviral drug (TCAD) composed of amantadine, oseltamivir, and ribavirin impedes the selection of drug-resistant influenza A virus

Widespread resistance among circulating influenza A strains to at least one of the anti-influenza drugs is a major public health concern. A triple combination antiviral drug (TCAD) regimen comprised of amantadine, oseltamivir, and ribavirin has been shown to have synergistic and broad spectrum activity against influenza A strains, including drug resistant strains. Here, we used mathematical modeling along with three different experimental approaches to understand the effects of single agents, double combinations, and the TCAD regimen on resistance in influenza in vitro, including: 1) serial passage at constant drug concentrations, 2) serial passage at escalating drug concentrations, and 3) evaluation of the contribution of each component of the TCAD regimen to the suppression of resistance. Consistent with the modeling which demonstrated that three drugs were required to suppress the emergence of resistance in influenza A, treatment with the TCAD regimen resulted in the sustained suppression of drug resistant viruses, whereas treatment with amantadine alone or the amantadine-oseltamivir double combination led to the rapid selection of resistant variants which comprised ～100% of the population. Furthermore, the TCAD regimen imposed a high genetic barrier to resistance, requiring multiple mutations in order to escape the effects of all the drugs in the regimen. Finally, we demonstrate that each drug in the TCAD regimen made a significant contribution to the suppression of virus breakthrough and resistance at clinically achievable concentrations. Taken together, these data demonstrate that the TCAD regimen was superior to double combinations and single agents at suppressing resistance, and that three drugs at a minimum were required to impede the selection of drug resistant variants in influenza A virus. The use of mathematical modeling with multiple experimental designs and molecular readouts to evaluate and optimize combination drug regimens for the suppression of resistance may be broadly applicable to other infectious diseases.     

Experimental vaccine protection against homologous and heterologous strains of feline immunodeficiency virus.

More than 90% of cats immunized with inactivated whole infected-cell or cell-free feline immunodeficiency virus (FIV) vaccines were protected against intraperitoneal infection with 10 50% animal infectious doses of either homologous FIV Petaluma (28 of 30 cats) or heterologous FIV Dixon strain (27 of 28 cats). All 15 control cats were readily infected with either strain of FIV. Protection appears to correlate with antiviral envelope antibody levels by a mechanism yet to be determined.     

DNA-binding and antiviral activities of bis-netropsins

The DNA-binding and antiviral activitus of bis-netropsins in which two monomers are attached covalently via three glycin residue were studied. These compounds have the same C-end groups but contain clusters with different numbers of lysine residues at the N-end of the molecule. In the homologous series of these compounds, bis-neropsins containing 15 and 31 branched lysine residues at the N-end of the molecule appear to be the most effective inhibitors of reproduction of the simplex herpes virus of type I in the Vero cell culture, including the virus versions resistant to aciclovir, ganciclovir, and other medicinal preparations. It was shown that the cytotoxicity of all the compounds studied is much lower than that of netropsin. The antiviral activity of the compounds correlates with their ability to selectively interact with the expanded clusters of the AT-pairs of DNA bases in the form of a monomer or a dimer, stabilized by interaction between the C-end halves of two bis-netropsin molecules bound at the neighboring overlapping binding sites on the DNA. The possible sites of their binding are the expanded clusters of AT-pairs at the origin of replication of OriS and OriL of the herpes virus.     

The alpha(1,2)-mannosidase I inhibitor 1-deoxymannojirimycin potentiates the antiviral activity of carbohydrate-binding agents against wild-type and mutant HIV-1 strains containing glycan deletions in gp120

Exposure of carbohydrate-binding agents (CBAs) (i.e. the mannose-specific plant lectins Hippeastrum hybrid agglutinin and Galanthus nivalis agglutinin) to HIV-1 progressively select for mutant HIV-1 strains that contain N-glycan deletions in their envelope gp120. This results in resistance of the mutant virus strains to the CBAs. Exposure of such mutant virus strains to the alpha(1,2)-mannosidase I inhibitor 1-deoxymannojirimycin (DMJ) results in an enhanced suppression of mutant virus-induced cytopathicity in CEM cell cultures. Moreover, when combined with CBAs at concentrations that showed poor if any suppression of mutant virus replication as single drugs, a synergistic antiviral activity of DMJ was observed. These observations argue for a combined exposure of CBAs and glycosylation inhibitors such as DMJ to HIV to afford a more pronounced suppression of virus replication, prior to, or during, CBA resistance development.     

The influence of homologous vs. heterologous challenge virus strains on the serological test results of rabies virus neutralizing assays

The effect that the relatedness of the viral seed strain used to produce rabies vaccines has to the strain of challenge virus used to measure rabies virus neutralizing antibodies after vaccination was evaluated. Serum samples from 173 subjects vaccinated with either purified Vero cell rabies vaccine (PVRV), produced from the Pittman Moore (PM) seed strain of rabies virus, or purified chick embryo cell rabies vaccine (PCECV), produced from the Flury low egg passage (Flury-LEP) seed strain of rabies virus, were tested in parallel assays by RFFIT using a homologous and a heterologous testing system. In the homologous system, CVS-11 was used as the challenge virus in the assay to evaluate the humoral immune response in subjects vaccinated with PVRV and Flury-LEP was used for subjects vaccinated with PCECV. In the heterologous system, CVS-11 was used as the challenge virus in the assay to evaluate subjects vaccinated with PCECV and Flury-LEP was used for subjects vaccinated with PVRV. Although the difference in G protein homology between the CVS-11 and Flury-LEP rabies virus strains has been reported to be only 5.8%, the use of a homologous testing system resulted in approximately 30% higher titers for nearly two-thirds of the samples from both vaccine groups compared to a heterologous testing system. The evaluation of equivalence of the immune response after vaccination with the two different vaccines was dependent upon the type of testing system, homologous or heterologous, used to evaluate the level of rabies virus neutralizing antibodies. Equivalence between the vaccines was achieved when a homologous testing system was used but not when a heterologous testing system was used. The results of this study indicate that the strain of virus used in the biological assays to measure the level of rabies virus neutralizing antibodies after vaccination could profoundly influence the evaluation of rabies vaccines.     

An antiviral mechanism investigated with ribavirin as an RNA virus mutagen for foot-and-mouth disease virus

To prove whether error catastrophe/lethal mutagenesis is the primary antiviral mechanism of action of ribavirin against foot-and-mouth disease virus (FMDV). Ribavirin passage experiments were performed and supernatants of Rp1 to Rp5 were harvested. Morphological alterations as well as the levels of viral RNAs, proteins, and infectious particles in the BHK-21 cells infected using the supernatants of Rp1 to Rp5 and control were measured by microscope, real-time RT-PCR, western-blotting and plaque assays, respectively. The mutation frequency was measured by sequencing the complete P1- and 3D-encoding region of FMDV after a single round of virus infection from ribavirin-treated or untreated FMDV-infected cells. Ribavirin treatment for FMDV caused dramatically inhibition of multiplication in cell cultures. The levels of viral RNAs, proteins, and infectious particles in the BHK-21 cells infected were more greatly reduced along with the passage from Rp1 to Rp5, moreover, nucleocapsid protein could not be detected and no recovery of infectious virus in the supernatant or detection of intracellular viral RNA was observed at the Rp5-infected cells. A high mutation rate, giving rise to an 8-and 11-fold increase in mutagenesis and resulting in some amino acid substitutions, was found in viral RNA synthesized at a single round of virus infection in the presence of ribavirin of 1000 microM and caused a 99.7% loss in viral infectivity in contrast with parallel untreated control virus. These results suggest that the antiviral molecular mechanism of ribavirin is based on the lethal mutagenesis/error catastrophe, that is, the ribavirin is not merely an antiviral reagent but also an effective mutagen.     

Viral class 1 RNase III involved in suppression of RNA silencing

Double-stranded RNA (dsRNA)-specific endonucleases belonging to RNase III classes 3 and 2 process dsRNA precursors to small interfering RNA (siRNA) or microRNA, respectively, thereby initiating and amplifying RNA silencing-based antiviral defense and gene regulation in eukaryotic cells. However, we now provide evidence that a class 1 RNase III is involved in suppression of RNA silencing. The single-stranded RNA genome of sweet potato chlorotic stunt virus (SPCSV) encodes an RNase III (RNase3) homologous to putative class 1 RNase IIIs of unknown function in rice and Arabidopsis. We show that RNase3 has dsRNA-specific endonuclease activity that enhances the RNA-silencing suppression activity of another protein (p22) encoded by SPCSV. RNase3 and p22 coexpression reduced siRNA accumulation more efficiently than p22 alone in Nicotiana benthamiana leaves expressing a strong silencing inducer (i.e., dsRNA). RNase3 did not cause intracellular silencing suppression or reduce accumulation of siRNA in the absence of p22 or enhance silencing suppression activity of a protein encoded by a heterologous virus. No other known RNA virus encodes an RNase III or uses two independent proteins cooperatively for RNA silencing suppression.     

Antiviral action of mouse interferon in heterologous cells

Buckler, Charles E. (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), and Samuel Baron. Antiviral action of mouse interferon in heterologous cells. J. Bacteriol. 91:231-235. 1966.-The antiviral action of mouse interferon in cell cultures of mouse, hamster, rat, chicken, and monkey origin was investigated. Using a vesicular stomatitis virus (VSV) plaque reduction test, we found that mouse serum interferon, assayed on closely related rat or hamster cells, exerted 5% of its homologous antiviral activity. This activity was characterized as interferon by its temperature of inactivation, trypsin sensitivity, nonsedimentability, stability at pH 2, lack of inactivation by antibody to virus, and inability to be washed off cells. In the more distantly related chicken and monkey cells, mouse interferon had less than 0.1% of its homologous activity. Conflicting reports of heterologous activity of chicken and mouse interferon preparations may result in part from the observed action of noninterferon inhibitors of vaccinia virus. These inhibitors, like interferon, are stable at pH 2. They are present in mouse serum, mouse lung extracts, and allantoic fluid, and they prevent the development of vaccinia plaques when allowed to remain in contact with cells during virus growth. Unlike interferon the inhibitors are removed by adequate washing of cells prior to virus challenge, and they are not active in the VSV assay system. These findings reemphasize the need for thorough characterization of interferon preparations.     

Monoclonal antibody, but not synthetic peptide, targeting the ectodomain of influenza B virus M2 proton channel has antiviral activity

The proton channels of influenza A virus (A/M2) and influenza B virus (BM2) are essential for viral replication. Previously we have shown that monoclonal antibodies targeting the ectodomain of the A/M2 proton channel have antiviral activity in vitro. In this study, we generated both monoclonal antibody and phage displayed peptide against the eight amino acids comprising the ectodomain of the BM2 proton channel and investigated their antiviral activities in vitro. A cytopathic assay showed that the monoclonal antibody potently protected MDCK cells from homologous, but not heterologous, virus infections. A plaque forming assay showed that viral replication was not completely neutralized, but greatly inhibited, by the monoclonal antibody. In contrast, no antiviral activity was observed for the synthetic native or engineered peptides. These results indicate that antibody targeting the M2 proton channel is a promising therapeutic candidate for treating influenza virus infections, and that antibody structure is important for antiviral activity.     

Human T cell leukemia virus type 1 Tax inhibits innate antiviral signaling via NF-kappaB-dependent induction of SOCS1

Human T cell leukemia virus type 1 (HTLV-1) inhibits host antiviral signaling pathways although the underlying mechanisms are unclear. Here we found that the HTLV-1 Tax oncoprotein induced the expression of SOCS1, an inhibitor of interferon signaling. Tax required NF-κB, but not CREB, to induce the expression of SOCS1 in T cells. Furthermore, Tax interacted with SOCS1 in both transfected cells and in HTLV-1-transformed cell lines. Although SOCS1 is normally a short-lived protein, in the presence of Tax, the stability of SOCS1 was greatly increased. Accordingly, Tax enhanced the replication of a heterologous virus, vesicular stomatitis virus (VSV), in a SOCS1-dependent manner. Surprisingly, Tax required SOCS1 to inhibit RIG-I-dependent antiviral signaling, but not the interferon-induced JAK/STAT pathway. Inhibition of SOCS1 by RNA-mediated interference in the HTLV-1-transformed cell line MT-2 resulted in increased IFN-β expression accompanied by reduced HTLV-1 replication and p19(Gag) levels. Taken together, our results reveal that Tax inhibits antiviral signaling, in part, by hijacking an interferon regulatory protein.     

Mechanism of interferon action. Kinetics of induction of the antiviral state and protein phosphorylation in mouse fibroblasts treated with natural and cloned interferons

The induction of phosphorylation of both protein P1 and protein synthesis initiation factor eIF-2 alpha and the inhibition of virus replication were examined in mouse L929 fibroblasts treated with either natural mouse or individual cloned human interferons (IFN). Natural mouse IFN synthesized in Newcastle disease virus-induced L929 cells and two cloned human leukocyte IFN subspecies synthesized in Escherichia coli, IFN-alpha D and IFN-alpha A/D, possessed antiviral activity in L929 cells as measured by single cycle virus yield reduction with both vesicular stomatitis virus and reovirus. Natural L929 IFN and cloned IFNs, alpha D and alpha A/D, also induced the protein kinase that catalyzed the phosphorylation of endogenous ribosome-associated protein P1 and the alpha subunit of purified initiation factor eIF-2. Two other cloned human IFNs, alpha A and alpha D/A, were poor inducers of both the antiviral state and the phosphorylation of P1 and eIF-2 alpha in mouse L929 cells. The ability of individual human IFN-alpha subspecies to induce P1 and eIF-2 alpha phosphorylation in mouse L929 cells correlated with their ability to induce an antiviral state. Furthermore, the detailed kinetics of induction, in mouse L929 cells, of P1 and eIF-2 alpha phosphorylation and of the antiviral state by the heterologous cloned human IFN-alpha A/D were equivalent to the kinetics of induction by the homologous natural mouse L929 IFN. These results suggest that different subspecies of biologically active IFN induce equivalent antiviral activities and biochemical changes in mouse L929 cells, and that protein phosphorylation may play a major role in the antiviral mechanism of IFN action in mouse L929 fibroblasts.     

Comparison of the antiviral action of different human interferons against DNA and RNA viruses

Abstract Five different interferon preparations were compared for their antiviral activity against Herpes simplex virus type 1 (HSV-1) and several RNA viruses. The interferons used were: interferon α from human buffy coats, interferon β from human fibroblasts, interferon γ from human lymphocytes after stimulation with phytohemagglutinin (PHA), lymphoblastoid interferon from Namalva cells IFN-α (Ly) and cloned α ₂ interferon produced by Escherichia coli containing the human gene for interferon α ₂. All preparations were able to protect monolayers of HeLa cells against HSV-1 infection when low multiplicities were used. The five IFN preparations were also tested against encephalomyocarditis (EMC) virus, poliovirus and vesicular stomatitis virus (VSV).     

Cross-reactive and serotype-specific antibodies against foot-and-mouth disease virus generated by different regions of the same synthetic peptide.

Synthetic peptides based on the VP1 proteins of two serotypes of foot-and-mouth disease virus (FMDV) and having the general formula C-C-(200-213)-P-P-S-(141-158)-P-C-G induce heterologous as well as homologous protection against challenge. Substitution of the sequence consisting of residues 200 to 213 (200-213 sequence) with a second copy of the homologous 141-158 sequence (i.e., homodimers) resulted in failure of either serotype peptide to protect heterologously. The antiviral and antipeptide titers of sera from guinea pigs immunized with the homodimeric 141-158 peptides showed serotype specificity and, with the data from the heterodimeric peptide vaccines, suggested that the C-terminal 141-158 sequence was more effectively recognized by the immune system than the N-terminal sequence. Whereas heterologous antiviral titers as measured by enzyme-linked immunosorbent assay and virus neutralization tests have not been observed with sera from cross-protected animals, epitope-mapping studies established that there was heterologous recognition of an octapeptide within the 200-213 sequence. That the 200-213 sequence was required for the induction of heterologous protection was also confirmed with a number of peptides, including hybrids based on the 200-213 sequence of one virus and the 141-158 sequence of a second virus. Thus, peptides of the general formula given above induce serotype-specific and serotype-cross-reactive protective antibodies and are unique in their induction of significant levels of heterologous protection, a property which has never been reported for whole FMDV vaccines.     

Differentiation of the immunosuppressive and antiviral effects of interferon.

Virus-induced (virus-type) interferon suppression of the in vitro antibody response of mouse (C57B1/6) spleen cells to sheep red blood cells was blocked by 5 × 10^?5M 2-mercaptoethanol (2-ME). The blockade was not due to a direct effect on interferon since 2-ME was capable of blocking the suppression when added to cultures up to 48 hr after interferon. 2-ME blockade of virus-type interferon immunosuppression was not due to the immunoenhancing property of 2-ME. Similar protective effects of 2-ME were observed during immunosuppression by virus-type interferon inducers, but not T-cell mitogen inducers of interferon (immune interferon). The data suggest that the immunosuppressive properties of virus-type and immune interferon preparations involve different mechanisms. Virus-type interferon inhibited DNA synthesis in unstimulated spleen cell cultures and in 2-ME stimulated cultures, and the degree of inhibition of DNA synthesis appeared to be related to the immunosuppressive property of interferon in the absence or presence of 2-ME. 2-ME did not affect the antiviral properties of either virus-type or immune interferon in nonlymphoid cells. Further, the induction of virustype interferon in spleen cells was neither inhibited nor enhanced by 2-ME, while the induction of immune interferon was enhanced. This enhancement is consistent with 2-ME enhancement of the immunosuppressive effects of immune interferon inducers.There are two possibilities for 2-ME blockade of the immunosuppressive effect of virus-type interferon, while not affecting the antiviral property. Firstly, the immunosuppressive and antiviral properties of virus-type interferon may involve different mechanisms at the subcellular level. Secondly, the selectivity of the blockade by 2-ME could be due to the fact that spleen cells are the target cells in immunosuppression, while L cells are the target cells in inhibition of virus replication. Thus, virus-type interferon may suppress the immune response at the level of the macrophage and 2-ME may reverse this effect by replacing a blocked macrophage function.     

Multiple molecular forms of interferon display different specific activities in the induction of the antiviral state and 2'5' oligoadenylate synthetase

Both Hu IFN-alpha A and Hu IFN-alpha D, produced by two independent recombinant bacterial clones, are mixtures of monomers, dimers and trimers. These forms, when assayed individually in heterologous MDBK cells, induced different degree of antiviral and 2'5' oligoadenylate synthetase (2'5' A synthetase) activities: the antiviral activity of the monomer is greater than that of the dimer and the trimer, whereas the activity of 2'5' A synthetase induction is lower with the monomer than with the dimer or the trimer. Similar differences are also observed on human cells. Compared to the mononeric form, the dimeric and the trimeric forms of Hu IFN-alpha A show higher antiviral inducing activity on heterologous MDBK cells than on homologous WISH cells, whereas the 2'5' A synthetase inducing activity in these two cell lines is about the same. Thus for the same antiviral activity, the trimer or the dimer compared to the monomer are much better inducers of the 2'5' A synthetase on human than on MDBK cells.