Suppression of BK virus replication and cytopathic effect by inhibitors of prokaryotic DNA gyrase.

Nalidixic acid and oxolinic acid, two antibacterial agents known to inhibit bacterial DNA gyrase, are shown to suppress the replication, as well as the cytopathic effect, of BK virus in Vero cell cultures. The inhibition of virus replication was detectable at day 4 post infection in cultures which had been continuously exposed to drugs at concentrations as low as 0.02 to 0.04 mM of nalidixic acid and 0.2 mM of oxolinic acid. These active concentrations are inferior to plasma levels attained in the course of clinical use of the drugs for antibacterial chemotherapy. Also, under these circumstances, no cytotoxicity occurred. The inhibition of development of cytopathology and of virus-induced cell death was demonstrable in cultures treated for 12 days with the drugs. Under these circumstances of prolonged action, oxolinic acid proved to be slightly cytotoxic in that virus inhibitory doses reduced the viability of normal cells. No alterations in the topological conformation of the viral genome or accumulation of end products of viral DNA replication were detected. However, accumulation of viral DNA form I at 48 h post infection suggests that the drugs act through a mechanism involving DNA topoisomerase.

Passive immune protection by herpes simplex virus-specific monoclonal antibodies with different plaque development inhibition activity.

A series of herpes simplex viruses type 1 (HSV-1) neutralizing monoclonal antibodies (mAbs) with different plaque development inhibition (PDI) activity were tested for passive protection in mice. When virus was inoculated intracranially, mAbs with PDI activity were not more protective than mAbs without PDI activity. However, when virus was inoculated percutaneously, there was a trend indicating that neutralizing mAbs with PDI power were more active in protecting mice from the cutaneous lesion than mAbs without PDI power. The results are discussed in relation to the possible involvement of PDI activity in in vivo protection and to the fact that this mechanism of protection might operate in cutaneous, but not in nervous tissue.

Cell-type dependent sensitivity of herpes simplex virus 1 mutants to plaque development inhibition by an anti-gD monoclonal antibody.

Herpes simplex virus 1 (HSV-1) mutants selected in Vero cells either for resistance to plaque development inhibition (PDI) (P1, P2 and P3) or for resistance to neutralization (N1, N2, and N3) against an anti-glycoprotein D (gD) monoclonal antibody (mAb) were characterized both in Vero and BHK cells. In Vero cells P mutants were completely resistant to PDI, while N mutants showed from moderate to good resistance. In BHK cells P mutants lost their resistance to PDI, while N mutants became fully resistant. Cell type influenced the plaque size of the mutants as well. In Vero cells P mutant plaques were larger, and N mutant plaques smaller than wild type virus plaques. In BHK cells all plaques were comparable. With one exception (N2 in BHK) resistance to neutralization could be clearly appreciated at high but not at low mAb:virus particles ratio. For most of the mutants the neutralization values remained approximately the same in Vero and BHK cells. P2 and N2 mutants were more resistant to neutralization in BHK than in Vero cells. However, only for N2 mutant did the change in neutralization resistance go in the same direction as the change in PDI resistance. The results show that it is possible to dissociate the neutralizing and the PDI activities of a mAb and that the sensitivity of a virus to plaque inhibition by an anti-gD mAb is cell-type dependent.

Growth, spread, and extracellular localization of herpes simplex virus 1 in Vero cells in the presence of an anti-gD plaque inhibiting monoclonal antibody.

Evidence for a direct cell-to-cell virus transfer could be provided by an agent that inhibits plaque formation without interfering with the processes that determine plaque growth in the exit and reinfection pathway of virus transfer. We studied the process of Vero cell infection by herpes simplex virus type 1 laboratory strain F [HSV-1 (F)] in the presence of monoclonal antibody (mAb) F10, an anti gD mAb that inhibits plaque development but does not neutralize virus infectivity in the absence of complement. In virus growth curves, cell associated virus was inhibited at low (0.01) but not at high (10) MOI when all cells are simultaneously infected, showing that the target of the mAb is the process of progressive cells recruitment and not the rate of virus replication. The mAb slightly inhibited virus exit and delayed virus entry. However these two additional inhibitory activities were not responsible for inhibition of virus spread, at least at early time of infection. In fact inhibition of virus spread, as measured by reduction of infectious centers (IC) from infected monolayers, could be appreciated before the appearance of extracellular virus in control cultures. We obtained electron microscope evidence that, both in the absence and in the presence of mAb, extracellular virus was initially concentrated at the interspaces between adjacent cell membranes, with little or no virus present at free cell surfaces. At more advanced stages of infection, only virus at free cell surfaces was found. The results of the study of virus replication in the presence of the mAb confirmed the hypothesis of the existence of a pathway of virus transfer between adjacent cells independent from extracellular virus. However, no electron microscope evidence for a direct cell-to-cell virus passage or for a modification of virus transfer brought about by the plaque inhibiting mAb was obtained. Interestingly, electron microscope studies suggested a targeting of the virions to different extracellular spaces, intercellular spaces and free cell surfaces, in intact and damaged cells respectively.

[Substances with antiviral activity. XIX. Amidinohydrazones of formylesters and formylthioesters with aromatic N-heterocyclic substrates]. / Richerche su sostanze ad attività antivirale. Nota XIX--Amidinoidrazoni di formilesteri e formiltioesteri consubstrati N-eterociclici aromatici.

Some formylthioester and formylester amidinohydrazones with aromatic N-heterocyclic moieties, were synthesized and screened in vitro against the MP mutant of herpes simplex virus type 1 [HSV-1 (MP)] and parainfluenza type 3 virus, HA-1/CR-8 strain. The amidinohydrazone of 1-phenyl-4-formyl-3-(ethylthio)carbonylpyrrole (I) was the most active compound.

[Substances with antiviral. XVI. Thiosemicarbazones containing benzofuran and 1H-indene moieties]. / Ricerche su sostanze ad attività antivirale. Nota XVI--Tiosemicarbazoni contenenti unità benzofuranica e 1H-indenica.

A few hydrazones with benzofuran and 1H-indene moieties were synthesized and screened in vitro against vaccinia virus strain IHD, parainfluenza type 3 virus strain HA-1/CR-8, and the MP mutant of herpes simplex virus type 1 [HSV-1 (MP)]. Only the thiosemicarbazones were active. The thiosemicarbazone of 3-chloro-2-formyl-1H-indene inhibited vaccinia virus to a greater extent than methisazone, used as reference standard. Furthermore, this compound was active also against parainfluenza and herpes viruses.

[Research on substances with antiviral activity. II. Derivatives of N-substituted 2-chloro-3-formylindoles]. / Ricerche su sostanze ad attività antivirale. II.- Derivati di 2-cloro-3-formilindoli N-sostituiti

Thiosemicarbazones and amidinohydrazones of N-substituted 2-chloroindole-3-aldehydes were synthesized and investigated for antiviral activity against RNA virus (parainfluenza type 3, HA-I/CR-8 stock) and DNA virus (vaccinia virus, HID stock). The thiosemicarbazones of 1-(o,m,p,-chlorobenzoyl)-2-chloroindole-3-aldehydes were more active than methisazone against vaccinia virus.

Selective inhibition of herpes simplex virus glycoprotein synthesis by a benz-amidinohydrazone derivative.

1H-benz[f]indene-1.3(2H)dione-bis-amidinohydrazone (benzhydrazone) inhibited incorporation of 14C-glucosamine, 14C-fucose and 14C-mannose into glycoproteins of HEp-2 cells infected with various strains of herpes simplex virus 1 (HSV-1) and impaired RNA and protein synthesis to a low extent. These biochemical effects are very similar to those induced by glycosylation inhibitors such as tunicamycin, D-glucosamine and 2-deoxy-D-glucose. In contrast to these inhibitors, benzhydrazone reduced HSV glycoprotein synthesis selectively since it did not significantly modify i) the saccharide uptake into glycoproteins of uninfected and of Sindbis virus-infected cells, ii) viral growth and cell fusion in paramyxovirus-infected cells, two activities which depend on viral glycoprotein synthesis. Benzhydrazone had only minor effects on the overall metabolism of uninfected cells, since it did not alter cell growth rate, and amino acid, uridine, and hexose incorporations were about 80% those of untreated cells.

[Research on substances with antiviral activity. X. 1-substituted-2-(methylamino) naphtho/2,1-b/furan hydrochlorides]. / Ricerche su sostanze ad attività antivirale. Nota X - Cloridrati di 2-(amminometil)nafto [2,1-b] furani-1-sostituiti.

Some 2-(methylamino)naphtho [2,1-b] furans hydrochlorides 1-substituted were synthetized and investigates for antiviral activity in vitro against Herpes simplex virus type I (VHS-I) and parainfluenza virus type 3 HA-I/CR-8 stock.

Lack of correlation between the expression of herpes simplex virus type 1 (HSV-1) glycoprotein C and both the maintenance of persistence in vitro and the capacity to establish latency in vivo.

The possible involvement of herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) in the maintenance of persistence in vitro and the establishment of latency in the mouse was investigated by the use of virus mutans which do not express glycoprotein C (gC-). A persistent infection of MDBK cells could be established with 3 of 3 gC- mutans derived from HSV-1 (MP) and with 1 of 2 gC- mutans derived from HSV-1 (HFEM)H6; cells infected with the other gC- mutant derived from HSV-1(HFEM)H6 survived for 25 days only. Of the 4 lines of persistence obtained, only one showed the appearance of revertants expressing gC (gC+). gC+ revertants, which appeared also in the "short term infection", rapidly became the majority population once they had arisen. gC- mutans could establish latency in mice, and the virus recovered from the latently infected ganglia maintained its gC- phenotype. The results show that expression of gC is not essential for the maintenance of persistence in MDBK cells and for the establishment of latency in the mouse. In addition, the results indicate that MDBK cells could offer a model to study the role of gC in the virus replication cycle in vitro.

Characterization of virus obtained from MDBK cells persistently infected with a variant of herpes simplex virus type 1 strain MP [HSV-1(MP)].

Virus clones which express glycoprotein gC (gC+) were obtained from two persistently infected (p.i.) MDBK cell lines which had been independently established by infection with HSV-1(MP)10311, a gC- syncytial (syn) variant of herpes simplex virus type 1 strain MP [HSV-1(MP)]. The gC+ revertants were syn in MDBK, HEp-2, and Vero cell lines and in primary human fibroblasts; this offers further evidence that glycoprotein gC does not inhibit cell fusion. The gC+ revertants represented from 70 to 100 percent of the virions present in the virus populations examined, thus suggesting a possible selective advantage of the gC+ revertants in this system of persistent infection.

Inhibition of herpes simplex virus type 1 (HSV-1) plaque enlargement by neutralizing sera.

The inhibitory activity of neutralizing sera on Herpes simplex virus type 1 (HSV-1) plaque enlargement (PE) is easily detected using the S variant of low passage clinical isolates (Mannini-Palenzona et al., 1985b; Costanzo et al., 1986). The same sera show little or no activity on PE of the product of the S variant rapid in vitro conversion, the L variant, and laboratory strains HSV-1 (F) and (MP). No significant difference was found in the inhibitory activity of sera from healthy individuals with no history of recurrence and patients with recurrences.

Impairment of host cell ribonucleic acid polymerase II after infection with frog virus 3.

Evidence is presented that, in frog virus 3-infected BHK cells, inhibition of ribonucleic acid (RNA) synthesis is paralleled by a decreased activity of host cell RNA polymerase II, while the template ability of host cell nuclei and chromatin is unaffected.

[Studies of substances with antiviral activity. VI. Activity on Herpes simplex virus of bis-homoanalogs of nucleosides and of hydrazone derivatives of various aromatic substrates]. / Ricerche su sostanze ad attività antivirale. Nota VI - attività su virus dell'herpes simplex di bis-omoanaloghi di nucleosidi e di derivati idrazonici di alcuni substrati aromatici.

Derivatives of different chemical structures, namely hydrazones of aromatic compounds and bis-homoanalogues of pyrimidine and purine nucleosides, have been investigated for inhibiting activity on Herpes simplex virus infection of human HEp-2 cell cultures. Some thiosemicarbazones, nucleoside analogues and guanylhydrazones proved active [compounds (I), (II b), (II c), (III), (IV a), (IV b), (V a), (V b); Fig. 1]. An increase in the number of guanylhydrazone chains linked to a fluorene structure [from 1 to 3: compounds (II a), (II b) and (II c)] increased the "activity index" (i.e., toxic versus active concentrations: Table II). Further in vitro investigations have been carried out on bis-guanylhydrazone of 1-phenyl-2-chloro-3,7-diformylindole (I) and it was found that its activity is of the order of 1 microgram per ml (Fig. 3). The compound completely blocks low multiplicity infections while it only blocks cytopathogenicity, not viral replication, in high multiplicity infections.

Ribonucleic acid polymerase induced by vaccinia virus: lack of inhibition by rifampicin and alpha-amanitin.

The RNA polymerase activity present in the cytoplasm of BHK cells infected with vaccinia virus is not affected by rifampicin or by alpha-amanitin.

[Substances with antiviral activity. XX. Thiosemicarbazones of 1-substituted 3-formyl-2-ethoxycarbonyl(carboxy)indoles]. / Ricerche su sostanze ad attività antivirale. Nota XX- Tiosemicarbazoni di 3-formil-2-etossicarbonil(carbossi)indoli 1-sostituiti.

A series of 3-formyl-2-ethoxycarbonyl(carboxy)indole thiosemicarbazones 1-substituted were synthesized and evaluated for in vitro antiviral activity against vaccinia virus strain IHD. The thiosemicarbazone of 1-(m-chlorobenzoyl)-3-formyl-2-ethoxycarbonylindole [(II g); Table II] was the most active compound. Some structure activity relationships are discussed.

[Research on substances with antiviral activity. XVIII. Bis-amidinohydrazones of N-heterocyclic aromatic dialdehydes]. / Richerche su sostanze ad attività antivirale. Nota XVIII - bis-amidinoidrazoni di dialdeidi aromatiche N-eterocicliche.

Some bis-amidinohydrazones of N-heterocyclic aromatic dialdehydes were synthesised and tested in vitro against the MP mutant of the herpes simplex virus type 1 [HSV - I (MP)]. Of the compounds tested, only those with and indole substrate showed some measure of biological activity.

[Homoanalogues of pyrimidine nucleosides. XII. 3,6-anhydro-1,2-bis-desoxy-1-(pyrimidin- and 5-halopyrimidin-1-yl)-D-arabinohexitols; 3,6-anhydro-1-desoxy-1(5-halopyrimidin-1-yl)-D-glucitols]. / Homoanalogues des nucléosides pyrimidiques. XII - 3,6-anhydro-1,2-bis-désoxy-1-(pyrimidin- et 5-halopyrimidin-1-yl)-D-arabino-hexitols; 3,6-anhydro-1-désoxy-1-(5-halopyrimidin-1-yl)-D-glucitols.

Lithium aluminium hydride reduction of 3,6-anhydro-1-o-benzoyl-4,5-o-isopropylidene-2-o-p-tolylsulfonyl-D-mannitol (II) gives with 74% yield 3,6-anhydro-2-deoxy-4,5-o-isopropylidene-D-arabino-hexitol (III). This compound is used as its corresponding 1-o-p-toluene-sulfonate (IV) for the N-1 alkylation of uracil, 5-fluorouracil, thymine and N-acetylcytosine. Acidic cleavage of the acetal protecting group gives the expected bis-homoanalogues of the pyrimidine- and halopyrimidine-nucleosides (V-VIII) which are characterized mainly through their mass spectra and U.V. absorption. 3,6-Anhydro-1,2-dideoxy-1-(5-bromo- and 5-iodouracil-1-yl)-D-arabino-hexitols (XIII, XIV), 3,6-anhydro-1-deoxy-1-(5-bromo- and 5-iodouracil-1-yl)-D-glucitols (XV, XVI) are similarly prepared by direct halogenation of the corresponding uracil derivatives. Results of cytotoxic, cytostatic and antiviral tests are described.

Bam HI characterization of 15 HSV-DNAs isolated from recrudescent lesions of three individuals.

A molecular epidemiology study has been conduced in a virology unit where Herpes simplex viruses (HSV) are studied. Fifteen HSVs have been isolated from recurrences in three individuals during a two-year period and their DNAs analized with Bam HI restriction endonuclease.

[Substances with antiviral activity. XV. Preparation and antiherpes activity of alkyl- and acylamino-dihalogenated pyrimidines and pyridines]. / Ricerche su sostanze ad attività antivirale. Nota XV - Preparazione e attività antierpetica di alchil- e acilammino- -dialogeno-pirimide e -piridine.

Some dihalo-amino-pyrimidines and -pyridines were alkylated and acylated at the amino group. The resulting twenty-four compounds were then tested for their action on Herpes simplex virus infection in human HEp-2 cell cultures. Five compounds were active and 2-benzamido-3,5-dichloropyridine [(III B); Table I] showed the highest antiviral activity.