Effects of chlorophyllin on replication of poliovirus and bovine herpesvirus in vitro

Aims:  Chlorophyllin (CHLN), a synthetic derivative of chlorophyll, was assayed in the replication of poliovirus (PV-1) and bovine herpesvirus (BoHV-1) in HEp-2 cell cultures.
Methods and Results:  Virucidal activity of CHLN was evaluated and the time-of-addition assay was performed as follows: before the infection (−1 and −2 h), at the time of the infection (0 h) and after the infection (1 and 2 h). Plaque reduction assay (PRA) showed that CHLN inhibited BoHV-1 and PV-1 infection and the 50% inhibitory concentrations (IC₅₀) against BoHV-1 and PV-1 infection were 8·6 and 19·8 μg ml⁻¹, respectively. The time-of-addition study demonstrated that the CHLN was effective inhibiting viral replication in 51% and 66·5% for PV-1 and BoHV-1, respectively, at the highest concentration of 20·0 μg ml⁻¹, when added during the infection. The directed effect of CHLN on viral strains demonstrated an inhibition of 62% and 66·4% for PV-1 and BoHV-1, respectively, by PRA.
Conclusions:  These results demonstrated that CHLN could be used as an antiviral suggesting directed activity on virus particles and on virus-receptor sites to BoHV. For poliovirus, CHLN also demonstrated virucide activity, moreover, showed to inhibit early steps of the replication cycle.
Significance and Impact of the Study:  CHLN demonstrated promising selectivity index for both virus strains; therefore, it can be used for the development of an antiviral agent.     

Antiviral activity of aqueous and ethanol extracts and of an isolated polysaccharide from Agaricus brasiliensis against poliovirus type 1

AIMS: Agaricus brasiliensis (previously named Agaricus blazei ss. Heinem), also known as the sun mushroom is native of Southeast Brazil, and is widely consumed, mainly in the form of tea, due to its nutritional and pharmacological properties. In this study, we tested aqueous (AqE) and ethanol (EtOHE) extracts and an isolated polysaccharide (PLS) from the fruiting body of A. brasiliensis, for antiviral activity against poliovirus type 1 in HEp-2 cells. METHODS AND RESULTS: The evaluation of the time of addition by plaque assay showed that when AqE, PLS and EtOHE were added, just after the virus inoculation (time 0 h), there was a concentration-dependent reduction in the number of plaques up to 50%, 67% and 88%, respectively, with a selectivity index (SI) of 5.4, 9.9 and 12.3, respectively. CONCLUSIONS: The test substances showed antiviral activity and were more effective when added during the poliovirus infection. As they had little effect on reducing viral adsorption and did not show any virucidal effect, we suggest that they act at the initial stage of the replication of poliovirus. SIGNIFICANCE AND IMPACT OF THE STUDY: These results corroborate that basidiomycetes can be a rich source of potential antiviral compounds.     

Poliovirus and polio antibody assay in HEp-2 and Vero cell cultures

HEp-2 cell cultures were about three to 30 times more sensitive for poliovirus titration than Vero cells. Attenuated strains induced a complete cytopathic effect in HEp-2 but not in Vero cells. For polio antibody titration, HEp-2 and Vero cells were equally suitable. A high degree of sensitivity and reproducibility of virus neutralization was achieved in tests utilizing a low virus dose and serum-virus incubation overnight at 36 degrees C. Staining of infected trays with crystal violet obviated reading of viral CPE under the microscope and expedited the evaluation of larger-scale tests.     

Hydrolytic enzymes in KB cells infected with poliovirus and herpes simplex virus

Flanagan, John F. (Duke University School of Medicine, Durham. N.C.). Hydrolytic enzymes in KB cells infected with poliovirus and herpes simplex virus. J. Bacteriol. 91:789-797. 1966.-The effect of poliovirus and herpes simplex virus infection on the activity of five hydrolytic enzymes was studied in tissue culture cells of KB type. During the course of poliovirus infection, the activity of beta-glucuronidase, acid protease, acid ribonuclease, acid deoxyribonuclease, and acid phosphatase in the cytoplasm rose to levels two- to fourfold greater than the activity present in the cytoplasm of uninfected cells. The rise in cytoplasmic activity was accompanied by a concomitant decrease in enzymatic activity bound to cell particles. Shift of enzymatic activity from the particulate to soluble state was first detected at 6 hr after poliovirus infection, coinciding with the appearance of new infectious particles and virus cytopathic effect. No net synthesis of these enzymes after poliovirus infection was found. Hydrocortisone added to the culture medium failed to affect either the titer of virus produced in the cells or the release of hydrolytic enzymes from the particulate state. Herpes simplex infection produced minimal alterations in the state of these enzymes in KB cells. It is hypothesized that the breakdown of lysosomes and release of hydrolytic enzymes accompanying poliovirus infection is produced by alterations in cell membrane permeability during the course of virus replication and by the consequent change in the ionic content of the cell sap.     

The in vitro antiviral activity of an aliphatic nitro compound from Heteropteris aphrodisiaca

We investigated the antiviral activity of an aliphatic nitro compound (NC) isolated from Heteropteris aphrodisiaca O. Mach. (Malpighiaceae), a Brazilian medicinal plant. The NC was tested for its antiviral activity against poliovirus type 1 (PV-1) and bovine herpes virus type 1 (BHV-1) by plaque reduction assay in cell culture. The NC showed a moderate antiviral activity against PV-1 and BHV-1 in HEp-2 cells, and the 50% inhibitory concentration (IC50) were 22.01 microg/ml (selectivity index (SI)=2.83) and 21.10 microg/ml (SI=2.95), respectively. At the highest concentration of the drug (40 microg/ml) a reduction of approximately 80% in plaque assay was observed for both viruses. The treatment of cells or virus prior to infection did not inhibit the replication of virus strains.     

Delay of Poliovirus Plaque Formation with Use of Fresh Bovine Serum in the Growth Medium of Host Cellsd

The plaque formation of poliovirus in HeLa cell monolayers was decreased upto 95% when the host cells were subcultured several times in a growth medium containing fresh bovine serum prior to their inoculation with virus. Extended incubation of infected cell monolayers indicated that the inhibition was in the form of a delay in plaque formation rather than a permanent inhibition of it. The inhibitory factor, which was found in most bovine sera, was very unstable and disappeared after storage of the serum at -20 C for a few weeks or at 36 C within a few days. In HeLa-cell monolayers, the fresh serum brought about a decrease in the plaque formation of all three poliovirus types as well as that of poliovirus ribonucleic acid. The plaque formation of poliovirus in monkey heart and HEp-2-cell monolayers was decreased irregularly by the use of fresh serum in the growth medium of these cells. Speculations were made as to the possible mode of action of the bovine serum inhibitor.     

Differential cell membrane labilizing effect of adenovirus type 5 and 12 observed during productive and abortive infection.

Culture fluid of human epitheloid (HEp-2) cells was examined for extracellular lactate dehydrogenase activity as an indicator of cell damage during a 48 h period in which virus replication and changes in cell morphology occurred. Uninfected and adenovirus type 5-infected cells had the same levels of extracellular enzyme activity both before and after the appearance of morphological changes in cells due to virus infection, whereas adenovirus type 12-infected cells showed increased extracellular enzyme activity. Cells infected with either adenovirus type 5 or type 12 had the same total cellular and extracellular lactate dehydrogenase activity. Hydrocortisone, a membrane stabilizing agent, prevented abnormal leakage of lactate dehydrogenase from adenovirus type 12-infected cells, but had no effect on virus replication or total enzyme activity of infected cells. After inoculation of monkey kidney (Vero) cells the yield of progeny adenovirus type 5 virions was greatly reduced and there was no production of adenovirus type 12 virions. The pattern of extracellular lactate dehydrogenase activity of uninfected and adenovirus type 5- and type 12-infected Vero cells was like that with HEp-2 cells. Therefore, production of adenovirus type 12 virions is not necessary for the virus-cell interaction causing cell membrane labilization.     

Intestinal trypsin can significantly modify antigenic properties of polioviruses: implications for the use of inactivated poliovirus vaccine.

It was recently reported that the intestinal protease trypsin cleaves in vitro the VP1 protein of type 3 poliovirus at antigenic site 1 (J. P. Icenogle, P. D. Minor, M. Ferguson, and J. M. Hogle, J. Virol. 60:297-301, 1986). We found that incubation of purified or crude type 3 poliovirus preparations with specimens of human intestinal fluid brings about a similar change in the virion structure. Sera from children immunized solely with the regular inactivated poliovirus vaccine (IPV) neutralized trypsin-cleaved Sabin 3 virus poorly, if at all, despite moderate levels of antibodies to the corresponding intact virus. Sera containing very high titers of the intact virus also neutralized the trypsin-cleaved virus but at a relatively weaker capacity. Most sera from older persons who may have been exposed to a natural poliovirus infection before the introduction of the poliovirus vaccines as well as sera from children infected with type 3 poliovirus during the recent outbreak in Finland were able to neutralize the trypsin-cleaved type 3 polioviruses. Serum specimens collected 1 month after a single dose of live poliovirus vaccine from children previously immunized with IPV were able to neutralize the trypsin-cleaved virus as well. During natural infection and after live poliovirus vaccine administration polioviruses are exposed to proteolytic enzymes in the gut. Our results may offer an alternative explanation for the relatively weak mucosal immunity obtained with IPV. Improvement of IPV preparations by incorporation of trypsin-treated type 3 polioviruses in the vaccine should be studied.     

Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase Activity in Cells Infected with Influenza Virus

Deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase activity was assayed on nuclear preparations of chick embryo fibroblast cells at various times after infection with an influenza A virus (fowl plague virus) and was compared with the activity of uninfected cells. Polymerase activity was increased by about 60% by 2 hr after infection, and this increase coincided with an increase in RNA synthesis in infected cells, as determined by pulse-labeling with uridine. No difference could be detected between the polymerases of infected and uninfected cells as to their requirements for DNA primer, divalent cations, and nucleoside triphosphates, and they were equally sensitive to addition of actinomycin D to the reaction mixture. It is possible that host cell DNA-dependent RNA polymerase is involved in the replication of influenza virus RNA.     

Monensin and nigericin prevent the inhibition of host translation by poliovirus, without affecting p220 cleavage.

Addition of monensin or nigericin after poliovirus entry into HeLa cells prevents the inhibition of host protein synthesis by poliovirus. The infected cells continue to synthesize cellular proteins at control levels for at least 8 h after infection in the presence of the ionophore. Cleavage of p220 (gamma subunit of eukaryotic initiation factor 4 [eIF-4 gamma]), a component of the translation initiation factor eIF-4F, occurs to the same extent in poliovirus-infected cells whether or not they are treated with monensin. Two hours after infection there is no detectable intact p220, but the cells continue to translate cellular mRNAs for several hours at levels similar to those in uninfected cells. Nigericin or monensin prevented the arrest of host translation at all the multiplicities of poliovirus infection tested. At high multiplicities of infection, an unprecedented situation was found: cells synthesized poliovirus and cellular proteins simultaneously. Superinfection of vesicular stomatitis virus-infected HeLa cells with poliovirus led to a profound inhibition of vesicular stomatitis virus protein synthesis, while nigericin partially prevented this blockade. Drastic inhibition of translation also took place in influenza virus-infected Vero cells treated with nigericin and infected with poliovirus. These findings suggest that the translation of newly synthesized mRNAs is dependent on the integrity of p220, while ongoing cellular protein synthesis does not require an intact p220. The target of ionophore action during the poliovirus life cycle was also investigated. Addition of nigericin at any time postinfection profoundly blocked the synthesis of virus RNA, whereas viral protein synthesis was not affected if nigericin was added at 4 h postinfection. These results agree well with previous findings indicating that inhibitors of phospholipid synthesis or vesicular traffic interfere with poliovirus genome replication. Therefore, the action of nigericin on the vesicular system may affect poliovirus RNA synthesis. In conclusion, monensin and nigericin are potent inhibitors of poliovirus genome replication that prevent the shutoff of host translation by poliovirus while still permitting cleavage of p220.     

Growth of poliovirus in HeLa cells persistently infected with HVJ (Sendai virus)

The growth of poliovirus in a HeLa cell culture persistently infected with the hemagglutinating virus of Japan (HVJ, the Sendai strain of parainfluenza 1 virus) (HeLaHVJ) was studied. Plaques produced by poliovirus on HeLaHVJ cell monolayers were hazier, smaller and fewer than those on HeLa cells. HeLaHVJ cells were indistinguishable from normal HeLa cells with respect to adsorption rate and penetration efficiency of poliovirus. Extracellular yields of poliovirus in HeLaHVJ cells were lower, and the cytopathic changes were less than those in normal HeLa cells, while cell-associated virus growth in HeLaHVJ cells was nearly equal to that in HeLa cells. HeLaHVJ cells responded more effectively to the action of magnesium chloride, which facilitates virus release from infected cells, resulting in an cytopathic effects. No reduction in poliovirus yield could be detected in HeLa cells acutely infected with HVJ. The relationship between the inhibition of the release of poliovirus from HeLaHVJ cells and the persistent infection of the cells with HVJ is discussed.     

Effect of viral infection on host protein synthesis and mRNA association with the cytoplasmic cytoskeletal structure

We studied the association of several eucaryotic viral and cellular mRNAs with cytoskeletal fractions derived from normal and virus-infected cells. We found that all mRNAs appear to associate with the cytoskeletal structure during protein synthesis, irrespective of their 5' and 3' terminal structures: e.g., poliovirus that lacks a 5' cap structure or reovirus and histone mRNAs that lack a 3' poly A tail associated with the cytoskeletal framework to the same extent as capped, polyadenylated actin mRNA. Cellular (actin) and viral (vesicular stomatitis virus and reovirus) mRNAs were released from the cytoskeletal framework and their translation was inhibited when cells were infected with poliovirus. In contrast, actin mRNA was not released from the cytoskeleton during vesicular stomatitis virus infection although actin synthesis was inhibited. In addition, several other conditions under which protein synthesis is inhibited did not result in the release of mRNAs from the cytoskeletal framework. We conclude that the association of mRNA with the cytoskeletal framework is required but is not sufficient for protein synthesis in eucaryotes. Furthermore, the shut-off of host protein synthesis during poliovirus infection and not vesicular stomatitis virus infection occurs by a unique mechanism that leads to the release of host mRNAs from the cytoskeleton.     

Nucleocytoplasmic traffic disorder induced by cardioviruses

Some picornaviruses, for example, poliovirus, increase bidirectional permeability of the nuclear envelope and suppress active nucleocytoplasmic transport. These activities require the viral protease 2A(pro). Here, we studied nucleocytoplasmic traffic in cells infected with encephalomyocarditis virus (EMCV; a cardiovirus), which lacks the poliovirus 2A(pro)-related protein. EMCV similarly enhanced bidirectional nucleocytoplasmic traffic. By using the fluorescent "Timer" protein, which contains a nuclear localization signal, we showed that the cytoplasmic accumulation of nuclear proteins in infected cells was largely due to the nuclear efflux of "old" proteins rather than impaired active nuclear import of newly synthesized molecules. The nuclear envelope of digitonin-treated EMCV-infected cells permitted rapid efflux of a nuclear marker protein. Inhibitors of poliovirus 2A(pro) did not prevent the EMCV-induced efflux. Extracts from EMCV-infected cells and products of in vitro translation of viral RNAs contained an activity increasing permeability of the nuclear envelope of uninfected cells. This activity depended on the expression of the viral leader protein. Mutations disrupting the zinc finger motif of this protein abolished its efflux-inducing ability. Inactivation of the L protein phosphorylation site (Thr47-->Ala) resulted in a delayed efflux, while a phosphorylation-mimicking (Thr47-->Asp) replacement did not significantly impair the efflux-inducing ability. Such activity of extracts from EMCV-infected cells was suppressed by the protein kinase inhibitor staurosporine. As evidenced by electron microscopy, cardiovirus infection resulted in alteration of the nuclear pores, but it did not trigger degradation of the nucleoporins known to be degraded in the poliovirus-infected cells. Thus, two groups of picornaviruses, enteroviruses and cardioviruses, similarly alter the nucleocytoplasmic traffic but achieve this by strikingly different mechanisms.     

Comparative study of rabies virus persistence in human and hamster cell lines.

Persistent infections by rabies virus in BHK-21/13S and HEp-2 cells were studied comparatively. No evidence of interferon production, selection of virus-resistant cells, or integration of the viral genome could be found. Persisting viruses replicated efficiently at 34, 36, and 40 degrees C. Both persistently infected cultures released defective interfering virus particles. A cyclical pattern of infection, which was not characteristic of the persistently infected HEp-2 system, was observed in persistently infected BHK cultures. The virus from persistently infected BHK cultures lost its virulence for mice, whereas the virus from persistently infected HEp-2 cultures retained mouse-killing capacity for more than 3 years.     

Accumulation of herpes simplex virus type 1 early and leaky-late proteins correlates with apoptosis prevention in infected human HEp-2 cells

We previously reported that a recombinant ICP27-null virus stimulated, but did not prevent, apoptosis in human HEp-2 cells during infection (M. Aubert and J. A. Blaho, J. Virol. 73:2803-2813, 1999). In the present study, we used a panel of 15 recombinant ICP27 mutant viruses to determine which features of herpes simplex virus type 1 (HSV-1) replication are required for the apoptosis-inhibitory activity. Each virus was defined experimentally as either apoptotic, partially apoptotic, or nonapoptotic based on infected HEp-2 cell morphologies, percentages of infected cells with condensed chromatin, and patterns of specific cellular death factor processing. Viruses d27-1, d1-5, d1-2, M11, M15, M16, n504R, n406R, n263R, and n59R are apoptotic or partially apoptotic in HEp-2 cells and severely defective for growth in Vero cells. Viruses d2-3, d3-4, d4-5, d5-6, and d6-7 are nonapoptotic, demonstrating that ICP27 contains a large amino-terminal region, including its RGG box RNA binding domain, which is not essential for apoptosis prevention. Accumulations of viral TK, VP16, and gD but not gC, ICP22, or ICP4 proteins correlated with prevention of apoptosis during the replication of these viruses. Of the nonapoptotic viruses, d4-5 did not produce gC, indicating that accumulation of true late gene products is not necessary for the prevention process. Analyses of viral DNA synthesis in HEp-2 cells indicated that apoptosis prevention by HSV-1 requires that the infection proceeds to the stage in which viral DNA replication takes place. Infections performed in the presence of the drug phosphonoacetic acid confirmed that the process of viral DNA synthesis and the accumulation of true late (gamma(2)) proteins are not required for apoptosis prevention. Based on our results, we conclude that the accumulation of HSV-1 early (beta) and leaky-late (gamma(1)) proteins correlates with the prevention of apoptosis in infected HEp-2 cells.     

A fungal tRNA of Aspergillus niger induces IFN-beta synthesis in HEp-2 cells

In this work, we evaluated the capacity of a fungal transfer RNA (F-tRNA) from Aspergillus niger to protect HEp-2 cells against a viral infection, and as an inducer of IFN-beta synthesis. HEp-2 cells previously incubated with F-tRNA, polyI:polyC, or IFN-alpha, at different concentrations for 24 h were infected with 200 pfu of adenovirus type 6 (AdV-6); after 5 days, we determined cellular viability, cytopathic effect of the virus, optimal concentration necessary to inhibit the cytopathic effect, and IFN-beta expression by RT-PCR. Results showed that HEp-2 cells treated with F-tRNA were less susceptible to the cytopathic effect of AdV-6 infection than those incubated with polyI:polyC (p < 0.05). On the other hand, F-tRNA- treated HEp-2 cells expressed IFN-beta mRNA, whereas monolayers incubated with polyI:polyC or IFN-alpha did not. Our results suggest that F-tRNA protected HEp-2 cells against AdV-6 infection, due to its capacity to induce IFN-beta synthesis.     

Cytotoxic activity of clinical Stenotrophomonas maltophilia

AIMS: To determine the potential virulence factors produced by culture supernatants of clinical isolates of Stenotrophomonas maltophilia. METHODS AND RESULTS: Culture supernatants of clinical isolates of S. maltophilia were assayed for haemolytic, enzymatic (lipase, protease and phospholipase) and cytotoxic activity. Cytotoxic activity was assayed in Vero (African green monkey), HeLa (human cervix) and HEp-2 (human larynx epidermoid carcinoma) cells. Microscopic analyses revealed intensive rounding, loss of intercellular junctions and membrane alterations (blebbing) followed by death of HEp-2 cells. In Vero and HeLa cells, the cytotoxic effects were characterized by vigorous endocytosis and cell aggregation. The viability of cultured mammalian cells was determined with neutral red and demonstrated that the sensitivity among the cells was different. This activity was inactivated by heating at 56 degrees C for 15 min and protease inhibitors did not inhibit cytotoxic activity. The clinical S. maltophilia presented a cell-free haemolytic activity similar to the 'hot-cold' haemolysins. CONCLUSIONS: S. maltophilia culture supernatants caused vigorous endocytosis and cell aggregation in HeLa and Vero cells, produced haemolytic and enzymatic activities. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed the presence of putative virulence factors that could be associated with human infections involving Stenotrophomonas maltophilia strains.     

Wild-type herpes simplex virus 1 blocks programmed cell death and release of cytochrome c but not the translocation of mitochondrial apoptosis-inducing factor to the nuclei of human embryonic lung fibroblasts

Programmed cell death activated by herpes simplex virus 1 mutants can be caspase dependent or independent depending on the nature of the infected cell. The recently discovered mitochondrial apoptosis-inducing factor (AIF) on activation is translocated to the nucleus and induces programmed cell death that is caspase independent. To assess the role of AIF and also to assay apoptosis-related events in primary human embryonic lung (HEL) fibroblasts, cells were mock infected or infected with wild-type virus previously shown not to induce apoptosis in continuous lines of primate cells or with the d120 mutant lacking infected cell protein no. 4 (ICP4) and were shown to induce apoptosis in all cell lines tested. Cells exposed to dexamethasone or osmotic shock induced by sorbitol were the positive controls. The results were as follows: (i) AIF was translocated to the nucleus in all infected cell cultures and in cells treated with dexamethasone or sorbitol, but cells infected with the wild type-virus showed no evidence of undergoing programmed death. (ii) Cytochrome c was released from mitochondria of cells infected with the d120 mutant or exposed to dexamethasone or sorbitol but not from mitochondria in cells treated with sorbitol and infected with the wild-type virus. (iii) Poly(ADP-ribose) polymerase was cleaved in mock-infected cells exposed to sorbitol or dexamethasone and in cells infected with the d120 mutant but not in either untreated cells infected with wild-type virus or cells exposed to sorbitol and then infected with wild-type virus. In contrast to HEp-2 cells, neither d120 infection nor treatment with dexamethasone or sorbitol caused fragmentation of DNA in HEL fibroblasts. Electron microscopic examination showed chromatin condensation and vacuolization in a fraction of cells infected with d120 but not in wild-type virus-infected cells or cells treated with dexamethasone or sorbitol. We conclude that AIF is translocated to the nucleus in infected cells but apoptosis does not ensue in wild-type-infected cells. HEL fibroblasts infected with the d120 virus exhibit symptoms of classical apoptosis, such as cytochrome c release and cleavage of poly(ADP-ribose) polymerase observed also in cells undergoing caspase 3-dependent programmed cell death in which AIF is either not involved or not a contributory factor.