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The threat of smallpox virus as a bioterrorist weapon is raising international concerns again since the anthrax attacks in the USA in 2001. The medical readiness of treating patients suffering from such infections is a prerequisite of an effective civil defense system. Currently the only therapeutic option for the treatment of poxvirus infections relies on the virostatic nulceosid analog cidofovir, although severe side effects and drug resistant strains have been described. A growing understanding of poxvirus pathogenesis raises the possibility to explore other appropriate targets involved in the viral replication cycle. Poxvirus encoded growth factors such as the Vaccinia Growth Factor (VGF) stimulate host cells via the Epidermal Growth Factor Receptor (EGFR) and thereby facilitate viral spreading. In this study we could visualize for the first time the paracrine priming of uninfected cells for subsequent infection by orthopoxviruses directly linked to EGFR phosphorylation. Since EGFR is a well known target for anti-tumor therapy small molecules for inhibition of its tyrosine kinase (TK) activity are readily available and clinically evaluated. In this study we analyzed three different EGFR receptor tyrosine kinase inhibitors for inhibition of orthopoxvirus infection in epithelial cells. The inhibitor shown to be most effective was Gefitinib (Iressa) which is already approved as a drug for anti-tumor medication in the USA and in Europe. Thus Gefitnib may provide a new therapeutic option for single or combination therapy of acute poxvirus infections, acting on a cellular target and thus reducing the risk of viral resistance to treatment., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Vaccinia virus (VacV) enters mammalian cells, replicates extranuclearly, and produces virions that move to the cell surface along microtubules, fuse with the plasma membrane, and move from infected cells toward apposing cells on actin-filled membranous protrusions or actin tails. To form actin tails, cell-associated enveloped virions (CEV) require Abl and Src family tyrosine kinases. Furthermore, release of CEV from the cell requires Abl but not Src family tyrosine kinases and is blocked by imatinib mesylate (STI-571; Gleevec), an Abl family kinase inhibitor used to treat chronic myelogenous leukemia in humans. Here we demonstrate that the Poxviridae family members monkeypox virus (MPX) and variola virus (VarV) use conserved mechanisms for actin motility and extracellular enveloped virion (EEV) release. Furthermore, we show that imatinib mesylate is effective in a mouse model of infection with VacV, whether delivered prophylactically or postinfection, and restricts spread of virions from the site of inoculation. While inhibitors of both Src and Abl family kinases, such as dasatinib (BMS-354825; Sprycel), are effective in limiting dissemination of VacV, VarV, and MPX in vitro, members of this class of drugs appear to have immunosuppressive effects in vivo that preclude their use as anti-infectives. Together, these data suggest a possible utility for imatinib mesylate in treating smallpox or MPX infections or complications associated with vaccination.

An adenovirus 5 vector encoding for mouse interferon alpha, subtype 5 (mDEF201) was evaluated for efficacy against lethal vaccinia virus (WR strain) respiratory infections in mice. mDEF201 was administered as a single intranasal treatment either prophylactically or therapeutically at doses of 10(6) to 10(8) plaque forming units/mouse. When the prophylactic treatment was given at 56 days prior to infection, it protected 90% of animals from death (100% protection for treatments given between 1-49 days pre-infection), with minimal weight loss occurring during infection. Surviving animals re-challenged with virus 22 days after the primary infection were protected from death, indicating that mDEF201 did not compromise the immune response against the initial infection. Post-exposure therapy was given between 6-24 h after vaccinia virus exposure and protection was afforded by a 10(8) dose of mDEF201 given at 24 h, whereas a 10(7) dose was effective up to 12 h. Comparisons were made of the ability of mDEF201, given either 28 or 1 day prior to infection, to inhibit tissue virus titers and lung infection parameters. Lung, liver, and spleen virus titers were inhibited to nearly the same extent by either treatment, as were lung weights and lung hemorrhage scores (indicators of pneumonitis). Lung virus titers were significantly (>100-fold) lower than in the placebo group, and the other infection parameters in mDEF201 treated mice were nearly at baseline. In contrast, viral titers and lung infection parameters were high in the placebo group on day 5 of the infection. These results demonstrate the long-acting prophylactic and treatment capacity of mDEF201 to combat vaccinia virus infections.

Although a highly effective vaccine against smallpox, vaccinia virus (VV) is not without adverse events, some of which can be life-threatening, particularly in immunocompromised individuals. We have recently demonstrated that the immunogenicity and protective efficacy of Dryvax(®) in immunocompetent mice is preserved even when co-administered with ST-246, an orally bioavailable small-molecule inhibitor of orthopoxvirus egress and dissemination. In addition, ST-246 markedly reduced the reactogenicity of the smallpox vaccine ACAM2000 and the highly neurovirulent VV strain Western Reserve (VV-WR). Here, we evaluated the impact of ST-246 co-administration on ACAM2000 reactogenicity, immunogenicity, and protective efficacy in seven murine models of varying degrees of humoral and cellular immunodeficiency: BALB/c and B-cell deficient (JH-KO) mice depleted of CD4(+) or CD8(+) or both subsets of T cells. We observed that ST-246 reduced vaccine lesion severity and time to complete resolution in all of the immunodeficient models examined, except in those lacking both CD4(+) and CD8(+) T cells. Although VV-specific humoral responses were moderately reduced by ST-246 treatment, cellular responses were generally comparable or slightly enhanced at both 1 and 6 months post-vaccination. Most importantly, in those models in which vaccination given alone conferred protection against lethal VV challenge, similar levels of protection were observed at both time points when vaccination was given with ST-246. These data suggest that, with the exception of individuals with irreversible, combined CD4(+) and CD8(+) T-cell deficiency, ST-246 co-administered at the time of vaccination may help reduce vaccine reactogenicity--even in those lacking humoral immunity--without impeding the induction of protective immunity., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The synthesis and antiviral activity of a series of novel polycyclic analogues of the orthopoxvirus egress inhibitor tecovirimat (ST-246) is presented. Several of these compounds display sub-micromolar activity against vaccinia virus, and were more potent than cidofovir (CDV). The more active compounds were about 10-fold more active than CDV, with minimum cytotoxic concentrations above 100 μM. Chemical manipulations of the two carbon-carbon double bonds present in the compounds were carried out to further explore the structure-activity relationships of these new polycyclic imides. Hydrogenation of the two carbon-carbon double bonds decreases antiviral activity, whereas either cyclopropanation or epoxidation of the double bonds fully eliminates the antiviral activity.

Terameprocol (TMP) is a methylated derivative of nordihydroguaiaretic acid, a phenolic antioxidant originally derived from creosote bush extracts. TMP has previously been shown to have antiviral and anti-inflammatory activities, and has been proven safe in phase I clinical trials conducted to evaluate TMP as both a topical and parenteral therapeutic. In the current study, we examined the ability of TMP to inhibit poxvirus growth in vitro, and found that TMP potently inhibited the growth of both cowpox virus and vaccinia virus in a variety of cell lines. TMP treatment was highly effective at reducing infectious virus yield in multi-step virus growth assays, but it did not substantially inhibit the synthesis of infectious progeny viruses in individual infected cells. These contrasting results showed that TMP inhibits poxvirus growth in vitro by preventing the efficient spread of virus particles from cell to cell. The canonical mechanism of poxvirus cell-to-cell spread requires morphogenesis of cell-associated, enveloped virions. The virions then trigger the formation of actin tails to project them from the cell surface. The number of actin tails present at the surface of poxvirus-infected cells was reduced dramatically by treatment with TMP. Whether TMP inhibits poxvirus morphogenesis, or subsequent events required for actin tail formation, remains to be determined. The results of this study, together with the clinical safety record of TMP, support further evaluation of TMP as a poxvirus therapeutic., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Poxviruses remain a significant public health concern due to their potential use as bioterrorist agents and the spread of animal borne poxviruses, such as monkeypox virus, to humans. Thus, the identification of small molecule inhibitors of poxvirus replication is warranted. Vaccinia virus is the prototypic member of the Orthopoxvirus genus, which also includes variola and monkeypox virus. In this study, we demonstrate that the carboxylic ionophore nigericin is a potent inhibitor of vaccinia virus replication in several human cell lines. In HeLa cells, we found that the 50% inhibitory concentration of nigericin against vaccinia virus was 7.9 nM, with a selectivity index of 1038. We present data demonstrating that nigericin targets vaccinia virus replication at a post-entry stage. While nigericin moderately inhibits both early vaccinia gene transcription and translation, viral DNA replication and intermediate and late gene expression are severely compromised in the presence of nigericin. Our results demonstrate that nigericin has the potential to be further developed into an effective antiviral to treat poxvirus infections., (Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.)

The antiherpesvirus activity of newly synthesized DNA-binding compounds for cultured Vero E6 cells was examined. The compounds were found to have selective antiherpesviral activity. Their antiviral activity was shown against the virus strains isolated from clinical specimens. The test compounds were ascertained to have also a high activity against herpes simplex virus type 1 (HSV-1/L2 TC-) that was very resistant to acyclovir. The authors' data demonstrated an obvious dose-dependent antiviral effect, which was representatively seen when Pt-bis-Dst and Lys-bis-Nt were used. The findings have offered the challenge to test some of these compounds in experiments on laboratory animals.

Antimicrobial peptides have activity against a wide variety of biological membranes and are an important component of innate immunity in vertebrate as well as invertebrate systems. The mechanisms of action of these peptides are incompletely understood and a number of competing but not necessarily mutually exclusive models exist. In this study we examined the virucidal activity of four peptides, the human cathelicidin derived LL37, Xenopus alanine-substituted Magainin-2 amide, uperin-3.1, and a cecropin-LL37 hybrid against vaccinia virus. The peptides were shown to be differentially virucidal but all were shown to attack the viral envelope, with LL37 being the most effective and uperin-3.1 the least. Density gradient analysis of the treated virions indicated the virus outer membrane was efficiently removed by peptide action and suggests a mechanism of direct virus inactivation that is consistent with the carpet model for peptide-mediated membrane disruption. Interestingly, the least effective peptide uperin-3.1 was equally effective as the others at inducing susceptibility to neutralizing antibody. This suggests that in addition to direct killing by a carpet-based mechanism, the peptides may simultaneously operate a different mechanism that exposes sequestered antigen without membrane removal., (Copyright © 2010. Published by Elsevier Inc.)

Aims: To evaluate the antiviral activity of Bignoniaceae species occurring in the state of Minas Gerais, Brazil., Methods and Results: Ethanol extracts of different anatomical parts of bignoniaceous plant species have been evaluated in vitro against human herpesvirus type 1 (HSV-1), vaccinia virus (VACV) and murine encephalomyocarditis virus (EMCV) by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. A total of 34 extracts from 18 plant species selected according to ethnopharmacological and taxonomic criteria were screened. Fifteen of the 34 extracts (44.1%) have disclosed antiviral activity against one or more of the viruses assayed with EC(50) values in the range of 23.2 ± 2.5-422.7 ± 10.9 μg ml(-1)., Conclusions: Twelve of the 34 extracts (35.3%) might be considered promising sources of antiviral natural products, as they have shown EC50 ≤ 100 μg ml(-1). The present screening discloses the high potential of the Bignoniaceae family as source of antiviral agents., Significance and Impact of the Study: Active extracts were identified and deserve bioguided studies for the isolation of antiviral compounds and studies on mechanism of action., (© 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.)

Suppressors of cytokine signaling (SOCSs) are negative regulators of both innate and adaptive immunity via inhibition of signaling by cytokines such as type I and type II IFNs. We have developed a small peptide antagonist of SOCS-1 that corresponds to the activation loop of JAK2. SOCS-1 inhibits both type I and type II IFN activities by binding to the kinase activation loop via the kinase inhibitory region of the SOCS. The antagonist, pJAK2(1001-1013), inhibited the replication of vaccinia virus and encephalomyocarditis virus in cell culture, suggesting that it possesses broad antiviral activity. In addition, pJAK2(1001-1013) protected mice against lethal vaccinia and encephalomyocarditis virus infection. pJAK2(1001-1013) increased the intracellular level of the constitutive IFN-beta, which may play a role in the antagonist antiviral effect at the cellular level. Ab neutralization suggests that constitutive IFN-beta may act intracellularly, consistent with recent findings on IFN-gamma intracellular signaling. pJAK2(1001-1013) also synergizes with IFNs as per IFN-gamma mimetic to exert a multiplicative antiviral effect at the level of transcription, the cell, and protection of mice against lethal viral infection. pJAK2(1001-1013) binds to the kinase inhibitory region of both SOCS-1 and SOCS-3 and blocks their inhibitory effects on the IFN-gamma activation site promoter. In addition to a direct antiviral effect and synergism with IFN, the SOCS antagonist also exhibits adjuvant effects on humoral and cellular immunity as well as an enhancement of polyinosinic-polycytidylic acid activation of TLR3. The SOCS antagonist thus presents a novel and effective approach to enhancement of host defense against viruses.

Background: Vaccinia virus strain Lister Elstree (VACV) is a test virus in the DVV/RKI guidelines as representative of the stable enveloped viruses. Since the potential risk of laboratory-acquired infections with VACV persists and since the adverse effects of vaccination with VACV are described, the replacement of VACV by the modified vaccinia Ankara strain (MVA) was studied by testing the activity of different chemical biocides in three German laboratories., Methods: The inactivating properties of different chemical biocides (peracetic acid, aldehydes and alcohols) were tested in a quantitative suspension test according to the DVV/RKI guideline. All tests were performed with a protein load of 10% fetal calf serum with both viruses in parallel using different concentrations and contact times. Residual virus was determined by endpoint dilution method., Results: The chemical biocides exhibited similar virucidal activity against VACV and MVA. In three cases intra-laboratory differences were determined between VACV and MVA - 40% (v/v) ethanol and 30% (v/v) isopropanol are more active against MVA, whereas MVA seems more stable than VACV when testing with 0.05% glutardialdehyde. Test accuracy across the three participating laboratories was high. Remarkably inter-laboratory differences in the reduction factor were only observed in two cases., Conclusions: Our data provide valuable information for the replacement of VACV by MVA for testing chemical biocides and disinfectants. Because MVA does not replicate in humans this would eliminate the potential risk of inadvertent inoculation with vaccinia virus and disease in non-vaccinated laboratory workers.

Antimicrobial peptides (AMPs) are gaining importance as effective therapeutic alternatives to conventional antibiotics. Recently we have shown that a set of nine synthetic antimicrobial peptides, four originating from thrombin-induced human platelet-derived antimicrobial proteins named PD1-PD4 and five synthetic repeats of arginine-tryptophan (RW) repeats (RW1-5) demonstrate antibacterial activity in plasma and platelets. Using WR strain of vaccinia virus (VV) as a model virus for enveloped virus in the present study, we tested the same nine synthetic peptides for their antiviral activity. A cell culture-based standard plaque reduction assay was utilized to estimate antiviral effectiveness of the peptides. Our analysis revealed that peptides PD3, PD4, and RW3 were virucidal against VV with PD3 demonstrating the highest antiviral activity of 100-fold reduction in viral titers, whereas, PD4 and RW3 peptide treatments resulted in 10-30-fold reduction. The EC(50) values of PD3, PD4 and RW3 were found to be 40 microg/ml, 50 microg/ml and 6.5 microM, respectively. In VV-spiked plasma samples, the virucidal activity of PD3, PD4 and RW3 was close to 100% (90-100-fold reduction). Overall, the present study constitutes a new proof-of-concept in developing peptide therapeutics for vaccinia virus infections in biothreat scenarios and as in vitro viral reduction agents., (Published by Elsevier B.V.)

Poxvirus morphogenesis is a complex process that involves the successive wrapping of the virus in host cell membranes. We screened by plaque assay a focused library of kinase inhibitors for those that caused a reduction in viral growth and identified several compounds that selectively inhibit phosphatidylinositol 3-kinase (PI3K). Previous studies demonstrated that PI3Ks mediate poxviral entry. Using growth curves and electron microscopy in conjunction with inhibitors, we show that that PI3Ks additionally regulate morphogenesis at two distinct steps: immature to mature virion (IMV) transition, and IMV envelopment to form intracellular enveloped virions (IEV). Cells derived from animals lacking the p85 regulatory subunit of Type I PI3Ks (p85alpha(-/-)beta(-/-)) presented phenotypes similar to those observed with PI3K inhibitors. In addition, VV appear to redundantly use PI3Ks, as PI3K inhibitors further reduce plaque size and number in p85alpha(-/-)beta(-/-) cells. Together, these data provide evidence for a novel regulatory mechanism for virion morphogenesis involving phosphatidylinositol dynamics and may represent a new therapeutic target to contain poxviruses.

The 14 kDa homodimeric N1L protein is a potent vaccinia and variola (smallpox) virulence factor. It is not essential for viral replication, but it causes a strong attenuation of viral production in culture when deleted. The N1L protein is predicted to contain the BH3-like binding domain characteristic of Bcl-2 family proteins, and it is able to bind the BH3 peptides. Its overexpression has been reported to prevent infected cells from committing apoptosis. Therefore, interfering with the N1L apoptotic blockade may be a legitimate therapeutic strategy affecting the viral growth. By using in silico ligand docking and an array of in vitro assays, we have identified submicromolar (600 nM) N1L antagonists belonging to the family of polyphenols. Their affinity is comparable to that of the BH3 peptides (70-1000 nM). We have also identified the natural polyphenol resveratrol as a moderate N1L inhibitor. Finally, we show that our ligands efficiently inhibit growth of vaccinia virus.

Background: Aurintricarboxylic acid (ATA) and ethacrynic acid (ECA) have been reported to exhibit antiviral activity against vaccinia virus infections in cell culture by inhibiting early and late gene transcription, respectively. The purpose of this work was to determine if these inhibitors would effectively treat vaccinia virus infections in mice, which has not previously been studied., Methods: ECA was investigated by cell culture plaque reduction assay for the inhibition of cowpox and vaccinia virus infections to clarify issues regarding its potency and selectivity. Mice infected intranasally with vaccinia virus were treated by intraperitoneal route twice daily for 5 days with ATA (10 and 30 mg/kg/day) and ECA (15 and 30 mg/kg/day) or once daily for 2 days with cidofovir (100 mg/kg/day)., Results: ECA caused 50% inhibition of virus plaque formation at 20-79 muM in four cultured cell lines, with 50% cytotoxicity at 84-173 muM, giving low (1.3-4.2) selectivity index values. Preliminary toxicity tests in uninfected mice indicated that ATA and ECA were both overtly toxic at 100 mg/kg/day. No protection from mortality was afforded by treatment of vaccinia virus infections with ATA or ECA, but 100% survival was achieved in the cidofovir group. ATA- and ECA-treated mice died significantly sooner than placebo-treated animals, indicating that these compounds exacerbated the infection., Conclusions: Both ATA and ECA lack antiviral potency and selectivity in cell culture. The compounds were ineffective in treating mice at intraperitoneal doses of

The type III interferon (IFN) family elicits an antiviral response that is nearly identical to that evoked by IFN-alpha/beta. However, these cytokines (known as IFN-lambda1, 2, and 3) signal through a distinct receptor, and thus may be resistant to the evasion strategies used by some viruses to avoid the IFN-alpha/beta response. Orthopoxviruses are highly resistant to IFN-alpha/beta because they encode well-characterized immunomodulatory proteins that inhibit IFN activity. These include a secreted receptor (B18R) that neutralizes IFN-alpha/beta, and a cytoplasmic protein (E3L) that blocks IFN-alpha/beta effector functions in infected cells. We therefore determined the ability of these immunomodulators to abrogate the IFN-lambda-induced antiviral response. We found that (i) vaccinia virus (VACV) replication is resistant to IFN-lambda antiviral activity; (ii) neither VACV B18R nor the variola virus homolog B20R neutralizes IFN-lambda; (iii) VACV E3L inhibits the IFN-lambda-mediated antiviral response through a PKR-dependent pathway; (iv) VACV infection inhibits IFN-lambdaR-mediated signal transduction and gene expression. These results demonstrate differential sensitivity of IFN-lambda to multiple distinct evasion mechanisms employed by a single virus.

Orthopoxvirus infections, such as smallpox, can lead to severe systemic disease and result in considerable morbidity and mortality in immunologically naïve individuals. Treatment with ST-246, a small-molecule inhibitor of virus egress, has been shown to provide protection against severe disease and death induced by several members of the poxvirus family, including vaccinia, variola, and monkeypox viruses. Here, we show that ST-246 treatment not only results in the significant inhibition of vaccinia virus dissemination from the site of inoculation to distal organs, such as the spleen and liver, but also reduces the viral load in organs targeted by the dissemination. In mice intranasally infected with vaccinia virus, virus shedding from the nasal and lung mucosa was significantly lower (approximately 22- and 528-fold, respectively) upon ST-246 treatment. Consequently, virus dissemination from the nasal site of replication to the lung also was dramatically reduced, as evidenced by a 179-fold difference in virus levels in nasal versus bronchoalveolar lavage. Furthermore, in ACAM2000-immunized mice, vaccination site swabs showed that ST-246 treatment results in a major (approximately 3,900-fold by day 21) reduction in virus detected at the outside surfaces of lesions. Taken together, these data suggest that ST-246 would play a dual protective role if used during a smallpox bioterrorist attack. First, ST-246 would provide therapeutic benefit by reducing the disease burden and lethality in infected individuals. Second, by reducing virus shedding from those prophylactically immunized with a smallpox vaccine or harboring variola virus infection, ST-246 could reduce the risk of virus transmission to susceptible contacts.

Solanum species are traditionally employed as antiherpes and anticancer agents in different countries. S. paniculatum has widespread ethnomedical uses in Brazil, including the treatment of viral infections. This paper reports on the isolation of neotigogenin (1) and the new compound delta25(27)-tigogenin-3-O-beta-D-glucopyranoside (2), obtained as a mixture of R and S diastereoisomers at C22 from an ethanol extract of S. paniculatum leaves, along with the determination of their cytotoxicity against Vero cells and antiviral effect against human herpes virus type 1 (HHV-1), murine encephalomyocarditis virus (EMCv), and vaccinia virus strain Western Reserve (VACV-WR). The extract of S. paniculatum inhibited HHV-1 replication [EC50 = (298.0 +/- 11.2) microg/ml] and showed no effect on EMCv and VACV-WR. On its turn, 1 was inactive against the assayed strains but presented high cytotoxicity [CC50 = (2.03 +/- 0.03) microg/ml], whereas 2 exhibited significant antiherpes [EC50 = (170.8 +/- 1.7) microg/ml] and antivaccinia virus effects [EC50 = (177.0 +/- 3.3) microg/ml], with low cytotoxicity (CC50 > 400 microg/ml). The results corroborate Solanum paniculatum as a source of cytotoxic and antiviral compounds.

Eczema vaccinatum is a potentially fatal, disseminated viral skin infection that develops in individuals with atopic dermatitis after exposure to the vaccinia virus (VV). Despite advances in modern medicine, there are few options for those suffering from disseminated VV infections. Ceragenins (CSAs) are synthetic antimicrobial compounds designed to mimic the structure and function of endogenous antimicrobial peptides (AMPs). We show that CSA-13 exhibits potent antiviral activity against VV by (1) direct antiviral effects against VV; and (2) stimulating the expression of endogenous AMPs with known antiviral activity against VV. In addition, we show that a topical application of CSA-13 penetrates the skin and reduces subsequent satellite lesion formation. This suggests that treatment with CSA-13 may be an intervention for individuals with a disseminated VV skin infection.

Cidofovir (CDV) is one of the most effective antiorthopoxvirus drugs, and it is widely accepted that viral DNA replication is the main target of its activity. In the present study, we report a detailed analysis of CDV effects on the replicative cycles of distinct vaccinia virus (VACV) strains: Cantagalo virus, VACV-IOC, and VACV-WR. We show that despite the approximately 90% inhibition of production of virus progeny, virus DNA accumulation was reduced only 30%, and late gene expression and genome resolution were unaltered. The level of proteolytic cleavage of the major core proteins was diminished in CDV-treated cells. Electron microscopic analysis of virus-infected cells in the presence of CDV revealed reductions as great as 3.5-fold in the number of mature forms of virus particles, along with a 3.2-fold increase in the number of spherical immature particles. A detailed analysis of purified virions recovered from CDV-treated cells demonstrated the accumulation of unprocessed p4a and p4b and nearly 67% inhibition of DNA encapsidation. However, these effects of CDV on virus morphogenesis resulted from a primary effect on virus DNA synthesis, which led to later defects in genome encapsidation and virus assembly. Analysis of virus DNA by atomic force microscopy revealed that viral cytoplasmic DNA synthesized in the presence of CDV had an altered structure, forming aggregates with increased strand overlapping not observed in the absence of the drug. These aberrant DNA aggregations were not encapsidated into virus particles.

Entry of vaccinia virus (VACV) into cells occurs by fusion with the plasma membrane and via a low pH-dependent endosomal pathway, presumably involving unidentified cellular receptors. In addition to approximately 25 viral proteins, the membrane of VACV mature virions contains several phospholipids including phosphatidylserine (PS). A recent model posits that PS flags virions as apoptotic debris to activate a common cellular uptake pathway to gain cell entry, perhaps through an interaction with a PS-specific cell surface receptor. To evaluate the apoptotic mimicry model, we reconstituted the membrane of detergent-extracted virions with several different phospholipids. Although the ability of the L-stereoisomer of PS to reconstitute infectivity was confirmed, the nonbiologically relevant D-stereoisomer of PS, and phosphatidylglycerol, which are not normally present in the virion membrane, functioned as well. Regardless of which phospholipid reconstituted infectivity, virus entry was inhibited by a neutralizing monoclonal antibody to a virion surface protein and by the drugs blebbistatin and bafilomycin A1, suggesting that in each case virus uptake was specific and occurred by a similar mechanism involving macropinocytosis and a low-pH endocytic pathway. Lipid-reconstituted and nonreconstituted, membrane-extracted virions were equally capable of binding to cells. However, the physical association of phospholipids with virus particles during membrane reconstitution correlated directly with rescue of particle infectivity and cell entry capability. Our results support a role for PS in poxvirus entry, but demonstrate that other phospholipids, not known to signal uptake of apoptotic debris, can function similarly.

Cellular tropism of vaccinia virus (VACV) is regulated by host range genes, including K1L, C7L, and E3L. While E3L is known to support viral replication by antagonizing interferon (IFN) effectors, including PKR, the exact functions of K1L and C7L are unclear. Here, we show that K1L and C7L can also inhibit antiviral effectors induced by type I IFN. In human Huh7 and MCF-7 cells, a VACV mutant lacking both K1L and C7L (vK1L-C7L-) replicated as efficiently as wild-type (WT) VACV, even in the presence of IFN. However, pretreating the cells with type I IFN, while having very little effect on WT VACV, blocked the replication of vK1L-C7L- at the step of intermediate viral gene translation. Restoring either K1L or C7L to vK1L(-)C7L(-) fully restored the IFN resistance phenotype. The deletion of K1L and C7L from VACV did not affect the ability of the virus to inhibit IFN signaling or its ability to inhibit the phosphorylation of PKR and the alpha subunit of eukaryotic initiation factor 2, indicating that K1L and C7L function by antagonizing an IFN effector(s) but with a mechanism that is different from those of IFN antagonists previously identified for VACV. Mutations of K1L that inactivate the host range function also rendered K1L unable to antagonize IFN, suggesting that K1L supports VACV replication in mammalian cells by antagonizing the same antiviral factor(s) that is induced by IFN in Huh7 cells.

This paper describes the antiviral evaluation of new N-amino-1,2,3-triazole derivatives, 1-(substituted-phenylamino)-5-methyl-1H-[1,2,3]-triazole-4-carboxylic acid ethyl esters, 3 and 1-(4-substituted-phenylamino)-5-methyl-1H-[1,2,3]-triazole-4-carboxylic acid hydrazides, 4, on Cantagalo virus replication. 1-(4-Fluoro-phenylamino)-5-methyl-1H-[1,2,3]-triazole-4-carboxylic acid hydrazide, 4e, exhibited a significant antiviral effect. Characterization of all compounds was confirmed by IR, (1)H and (13)C spectroscopies and elemental analysis. In addition, molecular structure of 4e was also reported.

Some virus species can resist harsh environmental conditions, surviving on surfaces for long periods with the possibility of being transmitted to susceptible hosts. Studies are limited on the efficacy of disinfectants against viruses dried onto surfaces, in particular, with the identification of new pathogenic non-enveloped viruses that are expected to have high resistance to disinfection, such as parvoviruses. In this study a range of commonly used biocides, including heat, was tested against porcine parvovirus (PPV), minute virus of mice (a parvovirus), poliovirus type 1, adenovirus type 5, and vaccinia virus dried onto surfaces. PPV was the most resistant species identified, since many biocides generally considered as effective against non-enveloped viruses and used for high level disinfection demonstrated limited activity. Ethanol had poor activity against all non-enveloped viruses. Effectiveness against these viruses may be important in preventing nosocomial transmission of emerging pathogenic species such as bocavirus and other parvoviruses. This work confirms the need to validate disinfection products against viruses dried onto surfaces and demonstrates that PPV is a particularly resistant surrogate.

F1L is a functional Bcl-2 homologue that inhibits apoptosis at the mitochondria during vaccinia infection. However, the extent and timing of cell death during DeltaF1L virus infection suggest that additional viral effectors cooperate with F1L to limit apoptosis. Here we report that vaccinia growth factor (VGF), a secreted virulence factor, promotes cell survival independently of its role in virus multiplication. Analysis of single and double knockout viruses reveals that VGF acts synergistically with F1L to protect against cell death during infection. Cell survival in the absence of F1L is dependent on VGF activation of the epidermal growth factor receptor. Furthermore, signalling through MEK kinases is necessary and sufficient for VGF-dependent survival. We conclude that VGF stimulates an epidermal growth factor receptor-MEK-dependent pro-survival pathway that synergizes with F1L to counteract an infection-induced apoptotic pathway that predominantly involves the BH3-only protein Bad.

We report a 1-step assay to screen antiviral substances combining the simultaneous application of cells, virus sample, and drug to 96-well plates. The results were obtained within 26 h when vaccinia virus plaques were counted or virus-induced cytopathic effect was measured. This fast cost-effective procedure may be used for other cytopathic viruses and is suitable to 1st screening tests.

Viral manipulation of the transduction pathways associated with key cellular functions such as actin remodeling, microtubule stabilization, and survival may favor a productive viral infection. Here we show that consistent with the vaccinia virus (VACV) and cowpox virus (CPXV) requirement for cytoskeleton alterations early during the infection cycle, PBK/Akt was phosphorylated at S473 [Akt(S473-P)], a modification associated with the mammalian target of rapamycin complex 2 (mTORC2), which was paralleled by phosphorylation at T308 [Akt(T308-P)] by PI3K/PDK1, which is required for host survival. Notably, while VACV stimulated Akt(S473-P/T308-P) at early (1 h postinfection [p.i.]) and late (24 h p.i.) times during the infective cycle, CPXV stimulated Akt at early times only. Pharmacological and genetic inhibition of PI3K (LY294002) or Akt (Akt-X and a dominant-negative form of Akt-K179M) resulted in a significant decline in virus yield (from 80% to >/=90%). This decline was secondary to the inhibition of late viral gene expression, which in turn led to an arrest of virion morphogenesis at the immature-virion stage of the viral growth cycle. Furthermore, the cleavage of both caspase-3 and poly(ADP-ribose) polymerase and terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end labeling assays confirmed that permissive, spontaneously immortalized cells such as A31 cells and mouse embryonic fibroblasts (MEFs) underwent apoptosis upon orthopoxvirus infection plus LY294002 treatment. Thus, in A31 cells and MEFs, early viral receptor-mediated signals transmitted via the PI3K/Akt pathway are required and precede the expression of viral antiapoptotic genes. Additionally, the inhibition of these signals resulted in the apoptosis of the infected cells and a significant decline in viral titers.

Concerns about the possible use of Variola virus, the causative agent of smallpox, as a weapon for bioterrorism have led to renewed efforts to identify new antivirals against orthopoxviruses. We identified a peptide, EB, which inhibited infection by Vaccinia virus with an EC(50) of 15 microM. A control peptide, EBX, identical in composition to EB but differing in sequence, was inactive (EC50>200 microM), indicating sequence specificity. The inhibition was reversed upon removal of the peptide, and EB treatment had no effect on the physical integrity of virus particles as determined by electron microscopy. Viral adsorption was unaffected by the presence of EB, and the addition of EB post-entry had no effect on viral titers or on early gene expression. The addition of EB post-adsorption resulted in the inhibition of beta-galactosidase expression from an early viral promoter with an EC(50) of 45 microM. A significant reduction in virus entry was detected in the presence of the peptide when the number of viral cores released into the cytoplasm was quantified. Electron microscopy indicated that 88% of the virions remained on the surface of cells in the presence of EB, compared to 37% in the control (p<0.001). EB also blocked fusion-from-within, suggesting that virus infection is inhibited at the fusion step. Analysis of EB derivatives suggested that peptide length may be important for the activity of EB. The EB peptide is, to our knowledge, the first known small molecule inhibitor of Vaccinia virus entry.

In view of the threat of the potential use of variola virus in a terrorist attack, considerable efforts have been performed to develop new antiviral strategies against orthopoxviruses. Here we report on the use of RNA interference, either alone or in combination with cidofovir, as an approach to inhibit orthopoxvirus replication. Two selected small interfering RNAs (siRNAs), named siB1R-2 and siG7L-1, and a previously reported siRNA, i.e., siD5R-2 (which targets the viral D5R mRNA), were evaluated for antiviral activity against vaccinia virus (VACV) by plaque reduction and virus yield assays. siB1R-2 and siG7L-1, administered before or after viral infection, reduced VACV replication by more than 90%. Also, these two siRNAs decreased monkeypox virus replication by 95% at a concentration of 1 nM. siB1R-2 and siG7L-1 were demonstrated to specifically silence their corresponding transcripts, i.e., B1R and G7L mRNAs, without induction of a beta interferon response. Strong synergistic effects were observed when siB1R-2, siG7L-1, or siD5R-2 was combined with cidofovir. In addition, the antiviral activities of these three siRNAs were evaluated against VACV resistant to cidofovir and other acyclic nucleoside phosphonates. siG7L-1 and siD5R-2 remained active against four of five VACV mutants, while siB1R-2 showed activity against only one of the mutants. Our results showed that siRNAs are potent inhibitory agents in vitro, not only against wild-type VACV but also against several cidofovir-resistant VACV. Furthermore, we showed that a combined therapy using siRNA and cidofovir may be useful in the treatment of poxvirus infections.

A20 protein is a major component of the vaccinia virus replication complex. It binds to the DNA polymerase E9, the uracil DNA glycosylase D4 and the primase/helicase D5, three proteins that are essential for viral DNA synthesis. The identification of molecules able to interact with the replication complex and inhibit its activity is a promising strategy for the design of new anti-orthopoxvirus drugs. In this study, we used a yeast genetic approach to select, from combinatorial libraries, 8-mers peptide aptamers that specifically interact with A20. From this screen, we isolated five peptide aptamers whose binding to A20 was confirmed by a glutathione S-transferase (GST) pull-down assay. Among those, we determined that peptide aptamer 72 binds to a central domain on A20. Interestingly, this region of A20 was previously shown to be important for its function in DNA replication. We next showed that vaccinia virus DNA synthesis was impaired in cells constitutively expressing peptide aptamer 72 and that virus production was inhibited in those cells. Thus, peptide aptamer 72 may be a useful tool for the development of new compounds specifically targeting poxvirus replication.

The synthesis of the dideoxy fluoro ketopyranonucleoside analogues, 1-(2,3-dideoxy-3-fluoro-6-O-trityl-beta-d-glycero-hexopyranosyl-4-ulose)-N(4)-benzoyl cytosine (7a), 1-(3,4-dideoxy-3-fluoro-6-O-trityl-beta-d-glycero-hexopyranosyl-2-ulose)-N(4)-benzoyl cytosine (13a) and their detritylated analogues 8a and 14a, respectively, is described. Condensation of peracetylated 3-deoxy-3-fluoro-D-glucopyranose (1) with silylated N(4)-benzoyl cytosine, followed by selective deprotection and isopropylidenation afforded compound 2. Routine deoxygenation at position 2', followed by a deprotection-selective reprotection sequence afforded the partially tritylated dideoxy nucleoside of cytosine 6, which upon oxidation of the free hydroxyl group at the 4'-position, furnished the desired tritylated 2,3-dideoxy-3-fluoro ketonucleoside 7a in equilibrium with its hydrated form 7b. Compound 2 was the starting material for the synthesis of the dideoxy fluoro ketopyranonucleoside 13a. Similarly, several subsequent protection and deprotection steps as well as routine deoxygenation at position 4', followed by oxidation of the free hydroxyl group at the 2'-position of the partially tritylated dideoxy nucleoside 12, yielded the desired carbonyl compound 13a in equilibrium with its hydrated form 13b. Finally, trityl removal from 7a/b and 13a/b provided the unprotected 2,3-dideoxy-3-fluoro-4-keto and 3,4-dideoxy-3-fluoro-2-ketopyranonucleoside analogues 8a and 14a, in equilibrium with their gem-diol forms 8b and 14b. None of the compounds showed inhibitory activity against a wide variety of DNA and RNA viruses at subtoxic concentrations, except 7a/b that was highly efficient against rotavirus infection. Nucleoside 7a/b also exhibited cytostatic activity against cells of various cancers. BrdU-cell cycle analysis revealed that the mechanism of cytostatic activity may be related to a delay in G1/S phase and initiation of programmed cell death.

A series of water-soluble dipeptide ester prodrugs of the antiviral acyclovir (ACV) were evaluated for their chemical stability, cytotoxicity, and antiviral activity against several strains of Herpes Simplex-1 and -2, vaccinia, vesicular stomatitis, cytomegalovirus and varicella zoster viruses. ACV dipeptide esters were very active against herpetic viruses, independently of the rate at which they liberate the parent drug. Their minimum cytotoxic concentrations were above 100 microM and the resulting MCC/EC(50) values were lower than those of ACV. When comparing the reactivity of Phe-Gly esters and amides (ACV, zidovudine, paracetamol, captopril and primaquine) in pH 7.4 buffer it was found that the rate of drug release increases with drug's leaving group ability. Release of the parent drug from Phe-Gly in human plasma is markedly faster than in pH 7.4 buffer, thus suggesting that the dipeptide-based prodrug approach can be successfully applied to bioactive agents containing thiol, phenol and amine functional groups.

Poxviruses are subjected to extraordinarily high levels of genetic recombination during infection, although the enzymes catalyzing these reactions have never been identified. However, it is clear that virus-encoded DNA polymerases play some unknown yet critical role in virus recombination. Using a novel, antiviral-drug-based strategy to dissect recombination and replication reactions, we now show that the 3'-to-5' proofreading exonuclease activity of the viral DNA polymerase plays a key role in promoting recombination reactions. Linear DNA substrates were prepared containing the dCMP analog cidofovir (CDV) incorporated into the 3' ends of the molecules. The drug blocked the formation of concatemeric recombinant molecules in vitro in a process that was catalyzed by the proofreading activity of vaccinia virus DNA polymerase. Recombinant formation was also blocked when CDV-containing recombination substrates were transfected into cells infected with wild-type vaccinia virus. These inhibitory effects could be overcome if CDV-containing substrates were transfected into cells infected with CDV-resistant (CDV(r)) viruses, but only when resistance was linked to an A314T substitution mutation mapping within the 3'-to-5' exonuclease domain of the viral polymerase. Viruses encoding a CDV(r) mutation in the polymerase domain still exhibited a CDV-induced recombination deficiency. The A314T substitution also enhanced the enzyme's capacity to excise CDV molecules from the 3' ends of duplex DNA and to recombine these DNAs in vitro, as judged from experiments using purified mutant DNA polymerase. The 3'-to-5' exonuclease activity appears to be an essential virus function, and our results suggest that this might be because poxviruses use it to promote genetic exchange.

The interferon-induced, double-stranded RNA-dependent protein kinase (PKR) can play critical roles in inhibiting virus replication and inducing apoptosis. To develop new agents that may inhibit viral replication or induce apoptosis in cancer cells via the PKR signaling pathway, we screened a chemical library for compounds that have differential cytotoxic effects on wild-type [mouse embryonic fibroblast (MEF)/PKR(+/+)] and PKR-knockout [MEF/PKR(-/-)] mouse embryonic fibroblast cells. We identified a synthetic compound, BEPP [1H-benzimidazole1-ethanol,2,3-dihydro-2-imino-a-(phenoxymethyl)-3-(phenylmethyl)-,monohydrochloride], that induces a cytotoxic effect more effectively in MEF/PKR(+/+) cells than in MEF/PKR(-/-) cells. BEPP also relatively effectively inhibited the growth of a human lung cancer cell line overexpressing PKR, compared with other cancer cell lines. In sensitive cells, BEPP induced apoptosis with activation of caspase-3. Treatment with BEPP led to increased phosphorylation of PKR and eIF2alpha, increased expression of BAX, and decreased expression of Bcl-2. BEPP-induced apoptosis was PKR dependent and was blocked by the adenovector expressing the dominant-negative PKR. Furthermore, pretreatment of HeLa cells at a noncytotoxic dose of BEPP effectively inhibited Vaccinia virus replication. Together, our results suggest that BEPP and its analogs may induce PKR-dependent apoptosis and inhibition of viral replication and that they can be a potential anticancer or anti-virus agent.

The suppressor of cytokine signaling 1 (SOCS-1) protein modulates cytokine signaling by binding to and inhibiting the function of Janus kinases (JAKs), ErbB, and other tyrosine kinases. We have developed a small tyrosine kinase inhibitor peptide (Tkip) that binds to the autophosphorylation site of tyrosine kinases and inhibits activation of STAT transcription factors. We have also shown that a peptide corresponding to the kinase-inhibitory region of SOCS-1, SOCS1-KIR, similarly interacts with the activation loop of JAK2 and blocks STAT activation. Poxviruses activate cellular tyrosine kinases, such as ErbB-1 and JAK2, in the infection of cells. We used the pathogenesis of vaccinia virus in C57BL/6 mice to determine the ability of the SOCS-1 mimetics to protect mice against lethal vaccinia virus infection. Injection of mice intraperitoneally with Tkip or SOCS1-KIR containing a palmitate for cell penetration, before and at the time of intranasal challenge with 2 x 10(6) PFU of vaccinia virus, resulted in complete protection at 100 microg. Initiation of treatment 1 day postinfection resulted in 80% survival. Administration of SOCS-1 mimetics by the oral route also protected mice against lethal effects of the virus. Both SOCS1-KIR and Tkip inhibited vaccinia virus transcription and replication at early and possibly later stages of infection. Vaccinia virus-induced phosphorylation of ErbB-1 and JAK2 was inhibited by the mimetics. Protected mice mounted a strong humoral and cellular response to vaccinia virus. The use of SOCS-1 mimetics in the treatment of poxvirus infections reveals an endogenous regulatory system that previously was not known to have an antiviral function.

Smallpox was declared eradicated in 1980. However recently, the need of agents effective against poxvirus infection has emerged again. In this paper, we report an original finding that two redox-modulating agents, the ethacrynic and alpha-lipoic acids (EA, LA), inhibit growth of vaccinia virus (VACV) in vitro. The effect of EA and LA was compared with those of beta-mercaptoethanol, DTT and ascorbic acid, but these agents increased VACV growth in HeLa G cells. The inhibitory effects of EA and LA on the growth of VACV were further confirmed in several cell lines of different embryonic origin, in epithelial cells, fibroblasts, macrophages and T-lymphocytes. Finally, we have analyzed the mechanism of action of the two agents. They both decreased expression of VACV late genes, as demonstrated by western blot analysis and activity of luciferase expressed under control of different VACV promoters. In contrast, they did not inhibit virus entry into the cell, expression of VACV early genes or VACV DNA synthesis. The results suggest new directions in development of drugs effective against poxvirus infection.

The antiviral effect of cidofovir was evaluated against two strains of vaccinia virus: the field strain Cantagalo virus (CTGV) and the smallpox vaccine IOC. The drug severely inhibited virus replication, revealing an EC(50) (drug concentration required to inhibit 50% of virus replication) of 7.68 microM and 9.66 microM, respectively, for CTGV and vaccine strain IOC. Similarly, other field isolates of Cantagalo-like viruses recently collected in distinct outbreaks were equally sensitive to the drug. Pre-treatment of cells prior to infection effectively established an antiviral state, inhibiting virus replication by >90% after 24h in the absence of cidofovir. CTGV infections represent an emerging zoonosis, and outbreaks have been frequently reported in several states of Brazil. Also, the possibility of resuming the manufacture of smallpox vaccine supports the need to evaluate the effect of antiviral drugs on the Brazilian vaccine strain IOC. As there is no currently approved antipoxvirus therapy, our data are extremely encouraging.

The acyclic nucleoside phosphonate (ANP) family of drugs shows promise as therapeutics for treating poxvirus infections. However, it has been questioned whether the utility of these compounds could be compromised through the intentional genetic modification of viral sequences by bioterrorists or the selection of drug resistance viruses during the course of antiviral therapy. To address these concerns, vaccinia virus (strain Lederle) was passaged 40 times in medium containing an escalating dose of (S)-1-[3-hydroxy-2-(phosphonomethoxypropyl)-2,6-diaminopurine [(S)-HPMPDAP], which selected for mutant viruses exhibiting a approximately 15-fold-increased resistance to the drug. (S)-HPMPDAP-resistant viruses were generated because this compound was shown to be one of the most highly selective and effective ANPs for the treatment of poxvirus infections. DNA sequence analysis revealed that these viruses encoded mutations in the E9L (DNA polymerase) gene, and marker rescue studies showed that the phenotype was produced by a combination of two (A684V and S851Y) substitution mutations. The effects of these mutations on drug resistance were tested against various ANPs, both separately and collectively, and compared with E9L A314T and A684V mutations previously isolated using selection for resistance to cidofovir, i.e., (S)-1-[3-hydroxy-2-(phosphonomethoxypropyl)cytosine]. These studies demonstrated a complex pattern of resistance, although as a general rule, the double-mutant viruses exhibited greater resistance to the deoxyadenosine than to deoxycytidine nucleotide analogs. The S851Y mutant virus exhibited a low level of resistance to dCMP analogues but high-level resistance to dAMP analogues and to 6-[3-hydroxy-2-(phosphonomethoxy)propoxy]-2,4-diaminopyrimidine, which is considered to mimic the purine ring system. Notably, (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]-3-deazaadenine retained marked activity against most of these mutant viruses. In vitro studies showed that the A684V mutation partially suppressed a virus growth defect and mutator phenotype created by the S851Y mutation, but all of the mutant viruses still exhibited a variable degree of reduced virulence in a mouse intranasal challenge model. Infections caused by these drug-resistant viruses in mice were still treatable with higher concentrations of the ANPs. These studies have identified a novel mechanism for the development of mutator DNA polymerases and provide further evidence that antipoxviral therapeutic strategies would not readily be undermined by selection for resistance to ANP drugs.

Nearly all DNA polymerases require processivity factors to ensure continuous incorporation of nucleotides. Processivity factors are specific for their cognate DNA polymerases. For this reason, the vaccinia DNA polymerase (E9) and the proteins associated with processivity (A20 and D4) are excellent therapeutic targets. In this study, we show the utility of stepwise rapid plate assays that (i) screen for compounds that block vaccinia DNA synthesis, (ii) eliminate trivial inhibitors, e.g. DNA intercalators, and (iii) distinguish whether inhibitors are specific for blocking DNA polymerase activity or processivity. The sequential plate screening of 2222 compounds from the NCI Diversity Set library yielded a DNA polymerase inhibitor (NSC 55636) and a processivity inhibitor (NSC 123526) that were capable of reducing vaccinia viral plaques with minimal cellular cytotoxicity. These compounds are predicted to block cellular infection by the smallpox virus, variola, based on the very high sequence identity between A20, D4 and E9 of vaccinia and the corresponding proteins of variola.

Variola virus, the causative agent of smallpox, is a potential bioweapon. The development of new antiviral compounds for smallpox prophylaxis and treatment is critical, especially because the virus can acquire resistance to the drugs that are currently available. We have identified novel small chemical inhibitors that target DNA synthesis of vaccinia, the prototypical poxvirus. Robotic high-throughput screening of 49663 compounds and follow-up studies identified very potent inhibitors of vaccinia DNA synthesis, with IC 50 values as low as 0.5 microM. Cell-based assays showed that 16 inhibitors effectively blocked vaccinia infection with minimal cytotoxicity. Three inhibitors had selectivity indexes that approximate that of cidofovir. These new non-nucleoside inhibitors are expected to interfere with components of the vaccinia DNA synthesis apparatus that are distinct from cidofovir. On the basis of the high sequence similarity between the proteins of vaccinia and variola viruses, these new inhibitors are anticipated to be equally effective against smallpox.

It is well documented in the scientific literature that ozone-oxygen mixtures inactivate microorganisms including bacteria, fungi and viruses (Hoff, J.C., 1986. Inactivation of microbial agents by chemical disinfectants. EPA 600 S2-86 067. Office of Water, U.S. Environmental Protection Agency, Washington, DC; Khadre, M.A., Yousef, A.E., Kim, J.-G., 2001. Microbiological aspects of ozone applications in food: a review. J. Food Sci. 66, 1242-1252). In the current study, delivery and absorption of precisely known concentrations of ozone (in liquid media) were used to inactivate virus infectivity. An ozone-oxygen delivery system capable of monitoring and recording ozone concentrations in real time was used to inactivate a series of enveloped and non-enveloped viruses including herpes simplex virus type-1 (HHV-1, strain McIntyre), vesicular stomatitis Indiana virus (VSIV), vaccinia virus (VACV, strain Elstree), adenovirus type-2 (HAdV-2), and the PR8 strain of influenza A virus (FLUAVA/PR/8/34/H1N1; FLUAV). The results of the study showed that ozone exposure reduced viral infectivity by lipid peroxidation and subsequent lipid envelope and protein shell damage. These data suggest that a wide range of virus types can be inactivated in an environment of known ozone exposure.

Orthopoxviruses, such as variola and monkeypox viruses, can cause severe disease in humans when delivered by the aerosol route, and thus represent significant threats to both military and civilian populations. Currently, there are no antiviral therapies approved by the U.S. Food and Drug Administration (FDA) to treat smallpox or monkeypox infection. In this study, we showed that administration of the antiviral compound ST-246 to rabbits by oral gavage, once daily for 14 days beginning 1h postexposure (p.e.), resulted in 100% survival in a lethal aerosolized rabbitpox model used as a surrogate for smallpox. Furthermore, efficacy of delayed treatment with ST-246 was evaluated by beginning treatment on days 1, 2, 3, and 4 p.e. Although a limited number of rabbits showed less severe signs of the rabbitpox disease from the day 1 and day 2 p.e. treatment groups, their illness resolved very quickly, and the survival rates for these group of rabbits were 88% and 100%, respectively. But when the treatment was started on days 3 or 4 p.e., survival was 67% and 33%, respectively. This work suggests that ST-246 is a very potent antiviral compound against aerosolized rabbitpox in rabbits and should be investigated for further development for all orthopoxvirus diseases.

Vaccinia virus was treated in a controlled manner with various combinations of nonionic detergents, reducing agents, and proteolytic enzymes, and successive products of the reactions were visualized using atomic force microscopy (AFM). Following removal of the outer lipid/protein membrane, a layer 20 to 40 nm in thickness was encountered that was composed of fibrous elements which, under reducing conditions, rapidly decomposed into individual monomers on the substrate. Beneath this layer was the virus core and its prominent lateral bodies, which could be dissociated or degraded with proteases. The core, in addition to the lateral bodies, was composed of a thick, multilayered shell of proteins of diverse sizes and shapes. The shell, which was readily etched with proteases, was thoroughly permeated with pores, or channels. Prolonged exposure to proteases and reductants produced disgorgement of the viral DNA from the remainders of the cores and also left residual, flattened, protease-resistant sacs on the imaging substrate. The DNA was readily visualized by AFM, which revealed some regions to be "soldered" by proteins, others to be heavily complexed with protein, and yet other parts to apparently exist as bundled, naked DNA. Prolonged exposure to proteases deproteinized the DNA, leaving masses of extended, free DNA. Estimates of the interior core volume suggest moderate but not extreme compaction of the genome.

Recombinant vaccinia virus (VACV), varicella zoster virus (VZV) and two human cytomegaloviruses (HCMV) expressing the green fluorescent protein (GFP) and the enhanced yellow fluorescent protein (EYFP) were used to develop a fluorescence-based assay for testing antiviral compounds. Infection of human embryonic lung fibroblasts (HEL) with the different recombinant viruses produced stable and detectable amount of GFP and EYFP signal as quantitated by automated fluorometry. The sensitivity of the recombinant viruses to a panel of antiviral drugs was measured and the fluorescence-based assay was compared to the cytopathic effect reduction assay (CPE-RA) in case of VACV and HCMV or to the plaque reduction assay (PRA) in case of VZV. The 50% inhibitory concentration (IC(50)) values for reference anti-pox and anti-herpesvirus compounds were comparable to those determined by CPE-RA or PRA assays. Furthermore the fluorimetric data could be confirmed by a flow cytometry assay. GFP- and EYFP-recombinant viruses proved to be a convenient tool for the evaluation of antiviral agents.

Vaccinia virus replication is inhibited by etoposide and mitoxantrone even though poxviruses do not encode the type II topoisomerases that are the specific targets of these drugs. Furthermore, one can isolate drug-resistant virus carrying mutations in the viral DNA ligase and yet the ligase is not known to exhibit sensitivity to these drugs. A yeast two-hybrid screen was used to search for proteins binding to vaccinia ligase, and one of the nine proteins identified comprised a portion (residue 901 to end) of human topoisomerase IIbeta. One can prevent the interaction by introducing a C(11)-to-Y substitution mutation into the N terminus of the ligase bait protein, which is one of the mutations conferring etoposide and mitoxantrone resistance. Coimmunoprecipitation methods showed that the native ligase and a Flag-tagged recombinant protein form complexes with human topoisomerase IIalpha/beta in infected cells and that this interaction can also be disrupted by mutations in the A50R (ligase) gene. Immunofluorescence microscopy showed that both topoisomerase IIalpha and IIbeta antigens are recruited to cytoplasmic sites of virus replication and that less topoisomerase was recruited to these sites in cells infected with mutant virus than in cells infected with wild-type virus. Immunoelectron microscopy confirmed the presence of topoisomerases IIalpha/beta in virosomes, but the enzyme could not be detected in mature virus particles. We propose that the genetics of etoposide and mitoxantrone resistance can be explained by vaccinia ligase binding to cellular topoisomerase II and recruiting this nuclear enzyme to sites of virus biogenesis. Although other nuclear DNA binding proteins have been detected in virosomes, this appears to be the first demonstration of an enzyme being selectively recruited to sites of poxvirus DNA synthesis and assembly.

Background: The emergence of drug resistant viruses, together with the possibility of increased virulence, is an important concern in the development of new antiviral compounds. Cidofovir (CDV) is a phosphonate nucleotide that is approved for use against cytomegalovirus retinitis and for the emergency treatment of smallpox or complications following vaccination. One mode of action for CDV has been demonstrated to be the inhibition of the viral DNA polymerase., Results: We have isolated several CDV resistant (CDVR) vaccinia viruses through a one step process, two of which have unique single mutations within the DNA polymerase. An additional resistant virus isolate provides evidence of a second site mutation within the genome involved in CDV resistance. The CDVR viruses were 3-7 fold more resistant to the drug than the parental viruses. The virulence of the CDVR viruses was tested in mice inoculated intranasally and all were found to be attenuated., Conclusion: Resistance to CDV in vaccinia virus can be conferred individually by at least two different mutations within the DNA polymerase gene. Additional genes may be involved. This one step approach for isolating resistant viruses without serial passage and in the presence of low doses of drug minimizes unintended secondary mutations and is applicable to other potential antiviral agents.

Background: The vaccinia virus (VV) F2L gene encodes a functional deoxyuridine triphosphatase (dUTPase) that catalyzes the conversion of dUTP to dUMP and is thought to minimize the incorporation of deoxyuridine residues into the viral genome. Previous studies with with a complex, multigene deletion in this virus suggested that the gene was not required for viral replication, but the impact of deleting this gene alone has not been determined in vitro or in vivo. Although the crystal structure for this enzyme has been determined, its potential as a target for antiviral therapy is unclear., Results: The F2L gene was replaced with GFP in the WR strain of VV to assess its effect on viral replication. The resulting virus replicated well in cell culture and its replication kinetics were almost indistinguishable from those of the wt virus and attained similar titers. The virus also appeared to be as pathogenic as the WR strain suggesting that it also replicated well in mice. Cells infected with the dUTPase mutant would be predicted to affect pyrimidine deoxynucleotide pools and might be expected to exhibit altered susceptibility to pyrimidine analogs. The antiviral activity of cidofovir and four thymidine analogs were evaluated both in the mutant and the parent strain of this virus. The dUTPase knockout remained fully susceptible to cidofovir and idoxuridine, but was hypersensitive to the drug (N)-methanocarbathymidine, suggesting that pyrimidine metabolism was altered in cells infected with the mutant virus. The absence of dUTPase should reduce cellular dUMP pools and may result in a reduced conversion to dTMP by thymidylate synthetase or an increased reliance on the salvage of thymidine by the viral thymidine kinase., Conclusion: We confirmed that F2L was not required for replication in cell culture and determined that it does not play a significant role on virulence of the virus in intranasally infected mice. The recombinant virus is hypersensitive to (N)-methanocarbathymidine and may reflect metabolic differences in the mutant virus.