Host-mediated antiviral activity ofLactobacillus casei against cytomegalovirus infection in mice

The protective effect of heat-killedLactobacillus casei (LC) against murine cytomegalovirus (MCMV) infection was examined. ICR mice treated once with LC 1 day or 2 days before challenge survived lethal infection, but untreated orLactobacillus fermentum (LF)-treated mice did not. The protective effect was evidenced by an increase in plaque-forming units (PFU) per 50% lethal dose (LD₅₀) and a decrease in titers of infectious viruses replicated in the target organs. This was further confirmed by severity of histopathological damage to the target organs, especially the liver. LC neither inactivated MCMV nor inhibited its replication in mouse embryonic fibroblasts (MEF). The spleen cells from LC-treated mice inhibited its replication in MEF on co-cultivation. Augmentation by LC of splenic natural killer (NK) cell activity correlated with survival of mice from otherwise lethal MCMV infection. Cytotoxic activity of peritoneal cells and level of serum interferon (IFN) were elevated after MCMV infection, but they were not associated with survival of mice nor with treatment of LC. The protective effect of LC was not clear in NK-deficient beige mutant (bg^J/bg^J) mice, when compared with that in their littermate (bg^J/+) mice. Poor protection of bg^J/bg^J mice by LC treatment correlated with failure to induce NK cell activity by LC treatment in the mutant mice. Thus, it is likely that LC protects mice from MCMV infection by augmentation of NK cell activity.     

Host-mediated antiviral activity of Lactobacillus casei against cytomegalovirus infection in mice

The protective effect of heat-killed Lactobacillus casei (LC) against murine cytomegalovirus (MCMV) infection was examined. ICR mice treated once with LC 1 day or 2 days before challenge survived lethal infection, but untreated or Lactobacillus fermentum (LF)-treated mice did not. The protective effect was evidenced by an increase in plaque-forming units (PFU) per 50% lethal dose (LD50) and a decrease in titers of infectious viruses replicated in the target organs. This was further confirmed by severity of histopathological damage to the target organs, especially the liver. LC neither inactivated MCMV nor inhibited its replication in mouse embryonic fibroblasts (MEF). The spleen cells from LC-treated mice inhibited its replication in MEF on co-cultivation. Augmentation by LC of splenic natural killer (NK) cell activity correlated with survival of mice from otherwise lethal MCMV infection. Cytotoxic activity of peritoneal cells and level of serum interferon (IFN) were elevated after MCMV infection, but they were not associated with survival of mice nor with treatment of LC. The protective effect of LC was not clear in NK-deficient beige mutant (bgJ/bgJ) mice, when compared with that in their littermate (bgJ/+) mice. Poor protection of bgJ/bgJ mice by LC treatment correlated with failure to induce NK cell activity by LC treatment in the mutant mice. Thus, it is likely that LC protects mice from MCMV infection by augmentation of NK cell activity.     

Effect of acute rat cytomegalovirus infection on the antiviral activity of peritoneal macrophages

The effect of rat cytomegalovirus (RCMV) infection on the extrinsic antiviral activity of peritoneal rat macrophages was investigated. The presence of a non-interferon-like antiviral substance in the supernatant of the culture of peritoneal macrophages of normal rats was shown. This antiviral activity was altered during an RCMV infection in vitro resulting in the absence of activity in the supernatant of macrophages harvested at 4 days post RCMV infection. The antiviral activity was also present in vivo, namely in the peritoneal cavity of normal rats. RCMV infection altered this antiviral effect resulting in an enhancement at days 2 to 5 post infection.     

In vitro antiviral activity of fullerene amino acid derivatives in cytomegalovirus infection]

The in vitro effect of 5 water soluble fullerene C60 amino acid derivatives (FAD) on the development of cytomegalovirus infection was studied in the schemes of the therapeutic, prophylactic and virucidal action. The following compounds as FAD were used: fullerene conjugated with Na salt of gamma-aminobutyric acid (C60-ABA-Na), 2 derivatives based on Na salts of fullerene-gamma-aminobutyric acid and fullerene-omega-caproic acid (C60-ABA-OH-Na and C60-ACA-OH-Na respectively) and 2 derivatives based on methyl ethers of the above mentioned fullerene amino acids (C60-ABA-OH-CH3 and C60-ACA-OH-CH3). All the FAD were able to inhibit the development of the virus cytopathogenic action in the cell culture. However, the compounds had different antiviral properties. C60-ABA-OH-Na, C60-ABA-CH3 and C60-ACA-CH3 showed marked antiviral activity in the prophylactic scheme. 50-Percent inhibition of the virus cytopathogenic action (ID50) was observed when concentrations of the compounds were 0.31, 5 and 25 mcg/ml respectively. C60-ACA-OH-Na inhibited the development of cytomegalovirus infection in the cell culture only in the scheme of the therapeutic action (ID50 4 mcg/ml). C60-ABA-Na had the highest antiviral effect. In a concentration of 0.22 mcg/ml it inhibited the cytomegalovirus plague-forming capacity by 50% in both the prophylactic and the virucidal schemes. The chemotherapeutic index of the compound was within the limits of 2500 to 5450.     

Synthesis and antiviral activity of monobactams inhibiting the human cytomegalovirus protease.

A series of monobactam inhibitors of HCMV (N(o)) protease bearing a heterocycle linked by a methylene group at C-4 is described. Inhibitors containing a heterocycle such as a 2-furyl, 2-thiophenyl, 4-methyl-2-tetrazole and 2-benzothiazole were found to be active in a plaque reduction assay. Furthermore, 2-benzothiazole derivatives were shown to inhibit the HCMV protease activity inside cells by using a cell transfection assay, indicating that their antiviral activity in the plaque reduction assay could be attributed to protease inhibition.     

Thymidine kinase not required for antiviral activity of acyclovir against mouse cytomegalovirus.

Previous studies of herpesvirus infections have indicated that a virus-specified thymidine kinase is required for the initial phosphorylation of acyclovir [acycloguanosine or 9-(2-hydroxyethoxymethyl)guanine] in the formation of acycloguanosine triphosphate. The latter compound accumulates in infected cells and competitively inhibits the viral DNA polymerase. We found that mouse cytomegalovirus, which does not express a thymidine kinase, was sensitive to the antiviral effects of acyclovir at a 50% inhibitory dose of approximately 0.23 microM. Acyclovir was equally effective against mouse cytomegalovirus in normal 3T3 cells and in 3T3 cells deficient in cellular thymidine kinase. Furthermore, the activity of acyclovir could not be reversed by excess thymidine, which easily reversed the antiviral activity of acyclovir against herpes simplex virus. Using a high-pressure liquid chromatography technique that easily detected acycloguanosine triphosphate in cells infected with herpes simplex virus, we could not detect acycloguanosine triphosphate in mouse cytomegalovirus-infected cells. These experiments demonstrated that the activity of acyclovir against mouse cytomegalovirus is not dependent on a thymidine phosphorylation pathway. Additional experiments are underway to determine whether acycloguanosine triphosphate is produced by another pathway in concentrations sufficient to inhibit mouse cytomegalovirus DNA polymerase.     

Experimental study of Ingavirin antiviral activity against human adenovirus

Antiviral properties of Ingavirin were investigated in the Hep-2 cell culture with respect to the human respiratory tract virus (type 5 adenovirus). In concentrations of Ingavirin of 1000, 100 and 10 mcg/ml the generated posterity showed lower infective capacity (by 250, 100 and 10 times respectively). The electron microscopy of the infected cells confirmed the Ingavirin ability to disturb the adenovirus normal morphogenesis.     

Experimental investigation of Ingavirin antiviral activity against human parainfluenza virus

The Ingavirin antiviral properties with respect to the parainfluenza virus, as an actual human respiratory tract pathogen, were investigated by two methods, i.e. immunoenzymatic analysis and microtetrazolium test. The results showed that along with the immediate antiviral activity Ingavirin had nonspecific cytoprotective properties. While affecting the virus proteins synthesis, Ingavirin lowered the virus cytopathogenic action. The drug significantly decreased the portion of the bronchial epithelium cells killed at the stage of acute infection.     

Antiviral activity of berberine and related compounds against human cytomegalovirus

Berberine chloride (1) and the structurally related compounds were assessed for the anti-human cytomegalovirus (HCMV) activity using the plaque assay. The anti-HCMV activity (IC(50) 0.68 microM) of 1 was equivalent to that (IC(50) 0.91 microM) of ganciclovir (GCV). The mechanism of action by which 1 inhibits the replication of HCMV is presumed to be different from that of GCV; 1 would interfere with intracellular events after virus penetration into the host cells and before viral DNA synthesis.     

Effect of oxygen on the antiviral activity of human interferon in cell culture

Antiviral activity of human lymphocytic interferon under conditions of increased oxygen levels in the cell culture was studied. It was found that oxygen had a capacity for increasing the antiviral effect of human interferon in homologous cells. When 20-80% air was replaced by oxygen the interferon titers on an average amounted to 1:113.4-1:124.8 against 1:29.1 in the control. This means that the average titer of interferon in the experiments with oxygen was 4 times higher than that in the control. On the basis of these data it is recommended using interferon in the form of aerosols in conjunction with oxygen for the treatment of viral respiratory infections.     

Identification of acetylated, tetrahalogenated benzimidazole D-ribonucleosides with enhanced activity against human cytomegalovirus

DNA packaging is the key step in viral maturation and involves binding and cleavage of viral DNA containing specific DNA-packaging motifs. This process is mediated by a group of specific enzymes called terminases. We previously demonstrated that the human cytomegalovirus (HCMV) terminase is composed of the large subunit pUL56 and the small subunit pUL89. While the large subunit mediates sequence-specific DNA binding and ATP hydrolysis, pUL89 is required only for duplex nicking. An excellent inhibitor targeting HCMV terminase is 2-bromo-5,6-dichloro-1-(beta-d-ribofuranosyl)benzimidazole (BDCRB), but it was not developed as an antiviral drug due to its metabolic cleavage in experimental animals. We now have tested several new benzimidazole d-ribonucleosides in order to determine whether these compounds represent new, potent inhibitors. Analysis by bioluminometric ATPase activity assays identified two of the new compounds with a high inhibitory effect, 2-bromo-4,5,6-trichloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl) benzimidazole (BTCRB) and 2,4,5,6-tetrachloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl benzimidazole (Cl(4)RB). By using viral plaque formation, viral yield, and viral growth kinetics, we demonstrated that the two compounds BTCRB and Cl(4)RB had antiviral activities similar to that of BDCRB. Interestingly, BTCRB retained its inhibitory activity after preincubation with HFF cells. By use of electron microscopy, we observed an increase of B capsids and a lack of cytoplasmic capsids in the presence of the compounds that correlated with the virus yield. Furthermore, cleavage of concatenated DNA was inhibited by both compounds, and inhibition by BTCRB was shown to be dose dependent. These results demonstrate that the new compounds are highly active against HCMV and act by mechanisms similar but not identical to those of BDCRB.     

In vitro activity evaluation of Parkia pendula seed lectin against human cytomegalovirus and herpes virus 6.

Human cytomegalovirus (HCMV) in vitro infectivity was inhibited by Parkia pendula seed lectin (PpeL) in contrast to human herpes virus 6 (HHV-6) which was not affected. The antiviral activity was detected for HCMV in human embryo lung (HEL) cells using a microtechnique in culture plates. The assay showed a reduction of cellular infectivity from approximately 95%, at a concentration of 150microg/mL with minimal cytotoxicity (25%). Also, a reduction of 75% was observed in HEL cells at a concentration of 75microg/mL without toxic effect. The reduction on infectivity was observed even after virus pre-adsorption to cells suggesting that this action should occur after virus penetration, in the intracellular replication phase. MT4 lymphocytes and cord blood mononuclear cells (CBMC) were used to evaluate the lectin effect on HHV-6 following the same technique. Lectin concentrations with few or no toxic effects on lymphocytes did not show inhibitory action of HHV-6 cytopathic effect. The results obtained with PpeL demonstrate that it may have an impact in the design of pharmacological strategies to infection of cytomegalovirus.     

Virucidal activity and the antiviral mechanism of acidic polysaccharides against Enterovirus 71 infection in vitro

Enterovirus 71 (EV71) is the predominant pathogen for severe hand, foot, and mouth disease (HFMD) in children younger than 5 years, and currently no effective drugs are available for EV71. Thus, there is an urgent need to develop new drugs for the control of EV71 infection. In this study, LJ04 was extracted from Laminaria japonica using diethylaminoethyl cellulose-52 with 0.4 mol/l NaCl as the eluent, and its virucidal activity was evaluated based on its cytopathic effects on a microplate. LJ04 is composed of fucose, galactose, and mannose and mainly showed good virucidal activity against EV71. The antiviral mechanisms of LJ04 were the direct inactivation of the virus, the blockage of virus binding, disruptions to viral entry, and weak inhibitory activity against the nonstructural protein 3C. The two most important findings from this study were that LJ04 inhibited EV71 proliferation in HM1900 cells, which are a human microglia cell line, and that LJ04 can directly inactivate EV71 within 2 hr at 37°C. This study demonstrates for the first time the ability of a polysaccharide from L. japonica to inhibit viral and 3C activity; importantly, the inhibition of 3C might have a minor effect on the antiviral effect of LJ04. Consequently, our results identify LJ04 as a potential drug candidate for the control of severe EV71 infection in clinical settings.     

Bacopasaponins with cytotoxic activity against human breast cancer cells in vitro

Molecular Biology Reports - Globally, breast cancer is a serious concern that exhibits a persistent rise in its incidence and related mortality even after significant advancement in the field of...     

Novel functions of tyrosine kinase 2 in the antiviral defense against murine cytomegalovirus

We have recently reported that tyrosine kinase 2 (Tyk2)-deficient mice have a selective defect in the in vivo defense against certain viruses. In our current study we show that Tyk2 is essential for the defense against murine CMV (MCMV). In vivo challenges with MCMV revealed impaired clearance of virus from organs and decreased survival of mice in the absence of Tyk2. Our in vitro studies demonstrate that MCMV replicates to dramatically higher titers in Tyk2-deficient macrophages compared with wild-type cells. We show an essential role of type I IFN (IFN-alphabeta) in the control of MCMV replication, with a prominent role of IFN-beta. MCMV infection leads to the activation of STAT1 and STAT2 in an IFN-alphabeta receptor 1-dependent manner. Consistent with the role of Tyk2 in IFN-alphabeta signaling, activation of STAT1 and STAT2 is reduced in Tyk2-deficient cells. However, lack of Tyk2 results in impaired MCMV-mediated gene induction of only a subset of MCMV-induced IFN-alphabeta-responsive genes. Taken together, our data demonstrate a requirement for Tyk2 in the in vitro and in vivo antiviral defense against MCMV infection. In addition to the established role of Tyk2 as an amplifier of Jak/Stat signaling upon IFN-alphabeta stimulation, we provide evidence for a novel role of Tyk2 as a modifier of host responses.     

Effect of human nasal secretions on the antiviral activity of human fibroblast and leukocyte interferon.

Many normal human nasal secretions contain an inhibitor of human fibroblast IF. This inhibitor had no effect on human leukocyte IF. The amount of inhibition of fibroblast IF increased with increasing quantities of nasal secretions. Also, the inhibition could be overcome with increasing concentrations of IF.     

Phosphorylation of ganciclovir phosphonate by cellular GMP kinase determines the stereoselectivity of anti-human cytomegalovirus activity

A racemic mixture of ganciclovir phosphonate was resolved by stereoselective phosphorylation using GMP kinase. The R-enantiomer of ganciclovir phosphonate was active against human cytomegalovirus but the S-enantiomer was less active. We show that enantiomeric selectivity of antiviral for ganciclovir phosphonate was conferred by stereoselective phosphorylations by mammalian enzymes, not by stereoselective inhibition of DNA polymerase from human cytomegalovirus.