Endogenous metabolism in endothelial and immune cells generates most of the tissue vitamin B3 (nicotinamide).

In mammals, nicotinamide (NAM) is the primary NAD precursor available in circulation, a signaling molecule, and a precursor for methyl-nicotinamide (M-NAM) synthesis. However, our knowledge about how the body regulates tissue NAM levels is still limited. Here we demonstrate that dietary vitamin B3 partially regulates plasma NAM and NAM-derived metabolites, but not their tissue levels. We found that NAD de novo synthesis from tryptophan contributes to plasma and tissue NAM, likely by providing substrates for NAD-degrading enzymes. We also demonstrate that tissue NAM is mainly generated by endogenous metabolism and that the NADase CD38 is the main enzyme that produces tissue NAM. Tissue-specific CD38-floxed mice revealed that CD38 activity on endothelial and immune cells is the major contributor to tissue steady-state levels of NAM in tissues like spleen and heart. Our findings uncover the presence of different pools of NAM in the body and a central role for CD38 in regulating tissue NAM levels.

Low NAD+ Levels Are Associated With a Decline of Spermatogenesis in Transgenic ANDY and Aging Mice.

Advanced paternal age has increasingly been recognized as a risk factor for male fertility and progeny health. While underlying causes are not well understood, aging is associated with a continuous decline of blood and tissue NAD+ levels, as well as a decline of testicular functions. The important basic question to what extent ageing-related NAD+ decline is functionally linked to decreased male fertility has been difficult to address due to the pleiotropic effects of aging, and the lack of a suitable animal model in which NAD+ levels can be lowered experimentally in chronologically young adult males. We therefore developed a transgenic mouse model of acquired niacin dependency (ANDY), in which NAD+ levels can be experimentally lowered using a niacin-deficient, chemically defined diet. Using ANDY mice, this report demonstrates for the first time that decreasing body-wide NAD+ levels in young adult mice, including in the testes, to levels that match or exceed the natural NAD+ decline observed in old mice, results in the disruption of spermatogenesis with small testis sizes and reduced sperm counts. ANDY mice are dependent on dietary vitamin B3 (niacin) for NAD+ synthesis, similar to humans. NAD+-deficiency the animals develop on a niacin-free diet is reversed by niacin supplementation. Providing niacin to NAD+-depleted ANDY mice fully rescued spermatogenesis and restored normal testis weight in the animals. The results suggest that NAD+ is important for proper spermatogenesis and that its declining levels during aging are functionally linked to declining spermatogenesis and male fertility. Functions of NAD+ in retinoic acid synthesis, which is an essential testicular signaling pathway regulating spermatogonial proliferation and differentiation, may offer a plausible mechanism for the hypospermatogenesis observed in NAD+-deficient mice.

Alpha-Amino-Beta-Carboxy-Muconate-Semialdehyde Decarboxylase Controls Dietary Niacin Requirements for NAD+ Synthesis.

NAD+ is essential for redox reactions in energy metabolism and necessary for DNA repair and epigenetic modification. Humans require sufficient amounts of dietary niacin (nicotinic acid, nicotinamide, and nicotinamide riboside) for adequate NAD+ synthesis. In contrast, mice easily generate sufficient NAD+ solely from tryptophan through the kynurenine pathway. We show that transgenic mice with inducible expression of human alpha-amino-beta-carboxy-muconate-semialdehyde decarboxylase (ACMSD) become niacin dependent similar to humans when ACMSD expression is high. On niacin-free diets, these acquired niacin dependency (ANDY) mice developed reversible, mild-to-severe NAD+ deficiency, depending on the nutrient composition of the diet. NAD deficiency in mice contributed to behavioral and health changes that are reminiscent of human niacin deficiency. This study shows that ACMSD is a key regulator of mammalian dietary niacin requirements and NAD+ metabolism and that the ANDY mouse represents a versatile platform for investigating pathologies linked to low NAD+ levels in aging and neurodegenerative diseases.

Prophylactic and therapeutic intranasal administration with an immunomodulator, Hiltonol® (Poly IC:LC), in a lethal SARS-CoV-infected BALB/c mouse model.

Hiltonol®, (Poly IC:LC), a potent immunomodulator, is a synthetic, double-stranded polyriboinosinic-polyribocytidylic acid (poly IC) stabilized with Poly-L-lysine and carboxymethyl cellulose (LC). Hiltonol® was tested for efficacy in a lethal SARS-CoV-infected BALB/c mouse model. Hiltonol® at 5, 1, 0.5 or 0.25 mg/kg/day by intranasal (i.n.) route resulted in significant survival benefit when administered at selected times 24 h prior to challenge with a lethal dose of mouse-adapted severe acute respiratory syndrome coronavirus (SARS-CoV). The infected BALB/c mice receiving the Hiltonol® treatments were also significantly effective in protecting mice against weight loss due to infection (p < 0.001). Groups of 20 mice were dosed with Hiltonol® at 2.5 or 0.75 mg/kg by intranasal instillation 7, 14, and 21 days before virus exposure and a second dose was given 24 h later, prophylactic Hiltonol® treatments (2.5 mg/kg/day) were completely protective in preventing death, and in causing significant reduction in lung hemorrhage scores, lung weights and lung virus titers. Hiltonol® was also effective as a therapeutic when give up to 8 h post virus exposure; 100% of the-infected mice were protected against death when Hiltonol® was administered at 5 mg/kg/day 8 h after infection. Our data suggest that Hiltonol® treatment of SARS-CoV infection in mice leads to substantial prophylactic and therapeutic effects and could be used for treatment of other virus disease such as those caused by MERS-CoV a related coronavirus. These properties might be therapeutically advantageous if Hiltonol® is considered for possible clinical use.

Inhibition of severe acute respiratory syndrome coronavirus replication in a lethal SARS-CoV BALB/c mouse model by stinging nettle lectin, Urtica dioica agglutinin.

Urtica dioica agglutinin (UDA) is a small plant monomeric lectin, 8.7 kDa in size, with an N-acetylglucosamine specificity that inhibits viruses from Nidovirales in vitro. In the current study, we first examined the efficacy of UDA on the replication of different SARS-CoV strains in Vero 76 cells. UDA inhibited virus replication in a dose-dependent manner and reduced virus yields of the Urbani strain by 90% at 1.1 ± 0.4 µg/ml in Vero 76 cells. Then, UDA was tested for efficacy in a lethal SARS-CoV-infected BALB/c mouse model. BALB/c mice were infected with two LD50 (575 PFU) of virus for 4 h before the mice were treated intraperitoneally with UDA at 20, 10, 5 or 0 mg/kg/day for 4 days. Treatment with UDA at 5 mg/kg significantly protected the mice against a lethal infection with mouse-adapted SARS-CoV (p < 0.001), but did not significantly reduce virus lung titers. All virus-infected mice receiving UDA treatments were also significantly protected against weight loss (p < 0.001). UDA also effectively reduced lung pathology scores. At day 6 after virus exposure, all groups of mice receiving UDA had much lower lung weights than did the placebo-treated mice. Thus, our data suggest that UDA treatment of SARS infection in mice leads to a substantial therapeutic effect that protects mice against death and weight loss. Furthermore, the mode of action of UDA in vitro was further investigated using live SARS-CoV Urbani strain virus and retroviral particles pseudotyped with SARS-CoV spike (S). UDA specifically inhibited the replication of live SARS-CoV or SARS-CoV pseudotyped virus when added just before, but not after, adsorption. These data suggested that UDA likely inhibits SARS-CoV infection by targeting early stages of the replication cycle, namely, adsorption or penetration. In addition, we demonstrated that UDA neutralizes the virus infectivity, presumably by binding to the SARS-CoV spike (S) glycoprotein. Finally, the target molecule for the inhibition of virus replication was partially characterized. When UDA was exposed to N-acetylglucosamine and then UDA was added to cells just prior to adsorption, UDA did not inhibit the virus infection. These data support the conclusion that UDA might bind to N-acetylglucosamine-like residues present on the glycosylated envelope glycoproteins, thereby preventing virus attachment to cells.

Single-dose intranasal administration with mDEF201 (adenovirus vectored mouse interferon-alpha) confers protection from mortality in a lethal SARS-CoV BALB/c mouse model.

Interferons (IFNs) are a first line of defense against viral infection. Herein we describe the use of an adenovirus vectored mouse IFN alpha gene (mDEF201) as a prophylactic and treatment countermeasure in a SARS-CoV-infected BALB/c mouse model. Complete survival protection was observed in mice given a single dose of mDEF201 administered intranasally 1, 3, 5, 7, or 14 days prior to lethal SARS-CoV challenge (p<0.001), and body weights of these treated mice were unaffected by the challenge. In addition, low doses of mDEF201 protected lungs in a dose dependent manner as measured by a reduction in gross pathology. Intranasal treatment with mDEF201 ranging from 10(6) to 10(8)PFU significantly protected mice against a lethal SARS-CoV infection in a dose dependent manner up to 12h post infection (p<0.001). The data suggest that mDEF201 is a new class of antiviral agent further development as treatment for SARS-CoV infections.

Induction of interferon-gamma-inducible protein 10 by SARS-CoV infection, interferon alfacon 1 and interferon inducer in human bronchial epithelial Calu-3 cells and BALB/c mice.

BACKGROUND: The pathogenesis of severe acute respiratory syndrome coronavirus (SARS-CoV) is poorly understood. Several mechanisms involving both direct effects on target cells and indirect effects via the immune system might exist. SARS-CoV has been shown in vitro to induce changes of cytokines and chemokines in various human and animal cells. We previously reported that interferon (IFN) alfacon-1 was more active against SARS-CoV infection in human bronchial epithelial Calu-3 cells than in African green monkey kidney epithelial cells on day 3 post-infection. METHODS: In the current study, we first evaluated the efficacy of IFN-alfacon 1 in Calu-3 cells during the first 7 days of virus infection. We then used the two-antibody sandwich ELISA method to detect IFN-gamma-inducible protein 10 (IP-10). We further evaluated the efficacy of antivirals directed against SARS-CoV infection in BALB/c mice. RESULTS: A potent, prolonged inhibition of SARS-CoV replication in Calu-3 cells with IFN-alfacon 1 was observed. Furthermore, IP-10, an IFN-inducible leukocyte chemoattractant, was detected in Calu-3 cells after SARS-CoV infection. Interestingly, IP-10 expression was shown to be significantly increased when SARS-CoV-infected Calu-3 cells were treated with IFN alfacon-1. IP-10 expression was detected in the lungs of SARS-CoV-infected BALB/c mice. Significantly high levels of mouse IP-10 in BALB/c mice was also detected when SARS-CoV-infected mice were treated with the interferon inducer, polyriboinosinic-polyribocytidylic acid stabilized with poly-L-lysine and carboxymethyl cellulose (poly IC:LC). Treatment with poly IC:LC by intranasal route were effective in protecting mice against a lethal infection with mouse-adapted SARS-CoV and reduced the viral lung titres. CONCLUSIONS: Our data might provide an important insight into the mechanism of pathogenesis of SARS-CoV and these properties might be therapeutically advantageous.

Interferon alfacon 1 inhibits SARS-CoV infection in human bronchial epithelial Calu-3 cells.

The primary targets for SARS-CoV infection are the epithelial cells in the respiratory and intestinal tract. The angiotensin-converting enzyme 2 (ACE-2) has been identified as a functional receptor for SARS-CoV. ACE-2 has been shown to be expressed at the apical domain of polarized Calu-3 cells. In this report, interferon alfacon 1 was examined for inhibitory activities against SARS-CoV on human lung carcinoma epithelial Calu-3 cell line and the other three African green monkey kidney epithelial cell lines. Interferon alfacon 1 demonstrated significant antiviral activity in neutral red uptake assay and virus yield reduction assay. The data might provide an important insight into the mechanism of pathogenesis of SARS-CoV allowing further development of antiviral therapies for treating SARS infections.

Differential pathogenesis of cowpox virus intranasal infections in mice induced by low and high inoculum volumes and effects of cidofovir treatment.

The causes of death from intranasal cowpox virus infections in mice remain unclear. Hypotheses include severe pneumonitis, hepatitis and/or hyperproduction of cytokines and chemokines. This work explores these hypotheses by studying the influence of low- and high-volume virus inocula on viral pathogenesis. BALB/c mice were infected intranasally with a syncytium-forming variant of cowpox virus in 5 microL or 50 microL volumes containing the same infectious virus challenge dose. The 50 microL infection produced a more rapidly lethal disease associated with severe pneumonitis, high lung and nasal virus titres and increased cytokine and chemokine levels in the lungs and nasal tissue, whilst liver infection was minimal. The 5 microL inoculum infection was also lethal, but the infection was primarily confined to the upper respiratory tract and included elevated nasal cytokine and chemokine levels. Levels of the pro-inflammatory cytokine interleukin-6 were particularly high in both infections. Treatment of the infections with cidofovir (100mg/kg/day for 2 days starting 24h after virus exposure) led to survival and suppression of tissue virus titres. Treatment reduced pneumonitis in the 50 microL infection and lessened cytokine hyperproduction in both infections. We conclude that a 5 microL volume inoculum of cowpox virus causes a lethal upper respiratory tract infection, whilst the 50 microL inoculum targets both upper and lower respiratory tracts, with excessive release of systemic pro-inflammatory factors. Cidofovir effectively treated both infections and slowed viral replication sufficiently to subdue the exaggerated release of pro-inflammatory mediators.

Cell line dependency for antiviral activity and in vivo efficacy of N-methanocarbathymidine against orthopoxvirus infections in mice.

A novel carbocyclic thymidine analog, N-methanocarbathymidine [(N)-MCT], was evaluated for inhibition of orthopoxvirus infections. Efficacy in vitro was assessed by plaque reduction assays against wild-type and cidofovir-resistant strains of cowpox and vaccinia viruses in nine different cell lines. Minimal differences were seen in antiviral activity against wild-type and cidofovir-resistant viruses. (N)-MCT's efficacy was affected by the cell line used for assay, with 50% poxvirus-inhibitory concentrations in cells as follows: mouse=0.6-2.2 microM, rabbit=52-90 microM, monkey=87 to >1000 microM, and human=39-220 microM. Limited studies performed with carbocyclic thymidine indicated a similar cell line dependency for antiviral activity. (N)-MCT did not inhibit actively dividing uninfected cells at 1000 microM. The potency of (N)-MCT against an S-variant thymidine kinase-deficient vaccinia virus was similar to that seen against S-variant and wild-type viruses in mouse, monkey, and human cells, implicating a cellular enzyme in the phosphorylation of the compound. Mice were intranasally infected with cowpox and vaccinia viruses followed 24h later by intraperitoneal treatment with (N)-MCT (twice a day for 7 days) or cidofovir (once a day for 2 days). (N)-MCT treatment at 100 and 30 mg/kg/day resulted in 90 and 20% survival from cowpox virus infection, respectively, compared to 0% survival in the placebo group. Statistically significant reductions in lung virus titers on day 5 occurred in 10, 30, and 100mg/kg/day treated mice. These same doses were also active against a lethal vaccinia virus (WR strain) challenge, and protection was seen down to 10mg/kg/day against a lethal vaccinia virus (IHD strain) infection. Cidofovir (100mg/kg/day) protected animals from death in all three infections.

Influenza A (H1N1) virus resistance to cyanovirin-N arises naturally during adaptation to mice and by passage in cell culture in the presence of the inhibitor.

Influenza A/New Caledonia/20/99 (H1N1) virus was studied for development of resistance to cyanovirin-N (CVN). CVN neutralizes virus infectivity by binding to specific high-mannose oligosaccharides on the viral haemagglutinin 1 (HA1) subunit. During virus adaptation to mice in the absence of CVN treatment the virus became resistant to CVN (CVN-MR virus), as did virus passaged in cell culture in the presence of CVN (CVN-R virus). The CVN-R virus possessed a single amino acid change at position 94a (Asn94aAsp) of HA1 that eliminated this glycosylation site. The CVN-MR virus at mouse passage 7 was a mixture of clones, consisting of a single mutation (Asp225Gly) and double mutations (Asn63Ser+Asp225Gly or Asn94a+Asp225Gly), eliminating glycosylation sites. CVN did not bind well to the CVN-R and CVN-MR viruses. Propagating these viruses in cells treated with 1 mM deoxymannojirimycin (dMJ, mannosidase inhibitor) increased sensitivity to CVN, suggesting that glycans attached at other sites on HA1 that typically are not high-mannosidic became so due to dMJ treatment. Further evaluation showed that the Asp225Gly mutant virus was sensitive to the inhibitor and did not kill mice or induce weight loss. The CVN-R virus was also avirulent to mice. The double-mutant CVN-MR viruses were resistant to CVN and caused deaths and severe weight loss in mice. CVN-R virus subjected to mouse adaptation acquired the 225 mutation and a lethal phenotype. Thus, the 225 mutation in the HA receptor-binding site in combination with a loss of glycan at Asn (63 or 94a) are important for mouse adaptation in this virus. The mutations reported here causing resistance to CVN are consistent with its known mode of action.

Anti-influenza virus activities of 4-[(1,2-dihydro-2-oxo-3H-indol-3-ylidene)amino]-N-(4,6-dimethyl-2-pyrimidin-2-yl)benzenesulphonamide and its derivatives.

4-[(1,2-Dihydro-2-oxo-3H-indol-3-ylidene)amino]-N-(4,6-dimethyl-2-pyrimidinyl)-benzenesulphonamide (SPIII-5H) and related compounds were tested for antiviral activity against influenza A (H1N1, H3N2, and H5N1) and B viruses in Madin Darby canine kidney (MDCK) cell culture. Among the compounds tested, SPIII-5H and four derivatives (5-chloro [SPill-5Cl], 5-bromo [SPIII-5Br], 5-methyl [SPIII-5Me] and N-acetyl [SPIII-NA]) showed similar antiviral potencies, with only the 5-fluoro (SPIII-5F) derivative being ineffective. Fifty percent effective concentration (EC50) values were determined in cytopathic effect (CPE) inhibition assays quantified by neutral red dye uptake. By this method, the active compounds were inhibitory to the H1N1 strain of influenza A at 2.7-5.2 microg/ml, to the H3N2 strain of influenza A at 13.8-26.0 microg/ml, to the H5N1 strain of influenza A at 3.1-6.3 microg/ml and to influenza B at 7.7-11.5 microg/ml. Confirmatory virus yield reduction studies against influenza A (H1N1) virus demonstrated antiviral activity (90% inhibition) at concentrations of 2-10 microg/ml. No cytotoxic effects were evident in actively growing uninfected cells or stationary monolayers at 100 microg/ml. Potencies of the compounds were similar to those of ribavirin, but much less than those of oseltamivir carboxylate against the various viruses. Time-of-addition studies indicated the compounds inhibited an early step in the virus replication cycle, probably virus adsorption/penetration, and no virucidal activity was evident. The basic molecule is amenable to diverse chemical modifications, which may improve water solubility and antiviral potency.

Sialidase fusion protein as a novel broad-spectrum inhibitor of influenza virus infection.

Influenza is a highly infectious disease characterized by recurrent annual epidemics and unpredictable major worldwide pandemics. Rapid spread of the highly pathogenic avian H5N1 strain and escalating human infections by the virus have set off the alarm for a global pandemic. To provide an urgently needed alternative treatment modality for influenza, we have generated a recombinant fusion protein composed of a sialidase catalytic domain derived from Actinomyces viscosus fused with a cell surface-anchoring sequence. The sialidase fusion protein is to be applied topically as an inhalant to remove the influenza viral receptors, sialic acids, from the airway epithelium. We demonstrate that a sialidase fusion construct, DAS181, effectively cleaves sialic acid receptors used by both human and avian influenza viruses. The treatment provides long-lasting effect and is nontoxic to the cells. DAS181 demonstrated potent antiviral and cell protective efficacies against a panel of laboratory strains and clinical isolates of IFV A and IFV B, with virus replication inhibition 50% effective concentrations in the range of 0.04 to 0.9 nM. Mouse and ferret studies confirmed significant in vivo efficacy of the sialidase fusion in both prophylactic and treatment modes.

Characterization and treatment of cidofovir-resistant vaccinia (WR strain) virus infections in cell culture and in mice.

The wild-type (WT) vaccinia (WR strain) virus is highly virulent to mice by intranasal inoculation, yet death can be prevented by cidofovir treatment. A cidofovir-resistant (CDV-R) mutant of the virus was developed by 15 Vero cell culture passages in order to determine cross-resistance to other inhibitors, growth characteristics, virulence in infected mice, and suitability of the animal model for studying antiviral therapies. Comparisons were made to the original WT virus and to a WT virus passaged 15 times in culture (WTp15 virus). Cidofovir inhibited WT, WTp15, and CDV-R viruses by 50% at 61, 56 and 790 microM, respectively, in plaque reduction assays, with similar inhibition seen in virus yield studies. Cross-resistance occurred with compounds related to cidofovir, but not with unrelated nucleosides. The resistant virus produced 300-fold fewer infectious particles (PFU) than WT and WTp15 viruses in mouse C1271 cells, yet replicated similarly in Vero (monkey) cells. The CDV-R virus was completely attenuated for virulence at 10(7) PFU per mouse in normal BALB/c mice and in severe combined immunodeficient (SCID) mice. The WTp15 virus was 100-fold less virulent than WT virus in BALB/c mice. Thus, the lack of virulence of the resistant virus in the animal model is explained partly by its reduced ability to replicate in mouse cells and by attenuation occurring as a result of extensive cell culturing (inferred from what occurred with the WTp15 virus). Lung and snout virus titre reduction parameters were used to assess antiviral activity of compounds in BALB/c mice infected intranasally with the CDV-R virus. Cidofovir, HDP-cidofovir and arabinofuranosyladenine treatments reduced lung virus titres or = eight-fold. The animal model appears to have limited utility in drug efficacy testing.

Treatment of mannan-enhanced influenza B virus infections in mice with oseltamivir, ribavirin and viramidine.

Mannan, a polysaccharide preparation from Saccharomyces cerevisiae, has previously been shown to enhance influenza virus replication in mice by inhibiting host defense collectins. The use of mannan in infections may serve to broaden the types of influenza viruses that can be studied in rodent infection models. When mannan was co-administered with influenza B/Sichuan/379/99 virus to mice, the animals died from the infection, whereas mice infected with only virus survived. Three types of influenza A (H1N1) and another influenza B (Hong Kong/330/01) virus infection were also enhanced by mannan, but not four types of influenza A (H3N2) viruses. Mannan was used at 0.16 or 0.5 mg/mouse for optimal disease-enhancing activity using influenza B/Sichuan/379/99 virus. Using this model, influenza B/Sichuan/379/99 infections were treated with oseltamivir, ribavirin or viramidine (the carboxamidine derivative of ribavirin). When oral gavage treatments started 4 h before virus and mannan challenge, oseltamivir was effective at 2.5, 5 and 10 mg/kg/day. Ribavirin was active at 20, 40 and 80 mg/kg/day. Viramidine was effective at 80 and 160 mg/kg/day but not at 40 mg/kg/day. Active drug doses improved lung consolidation scores and lung weights, with decreases in lung virus titres also noted. Arterial oxygen saturation values in treated groups were significantly better than those of the placebo group on days 7-11 of the infection. Oseltamivir (5 mg/kg/day) and ribavirin (40 mg/kg/day) were used alone and in combination to determine how late after infection they could be beneficially administered. Ribavirin alone was very effective (90-100% survival of mice) when treatments started as late as 3 days after infection. Forty percent survival was evident even when treatments started 4 days post-infection. Oseltamivir was active starting treatments 1 day after virus exposure, but lost considerable efficacy when treatments began after that time. The combination of ribavirin and oseltamivir appeared to be no better than ribavirin alone, due to the stronger beneficial effect of ribavirin in this model. The overall results demonstrate that mannan can be used to enhance certain non-lethal influenza virus infections sufficiently to allow antiviral studies.

Topical cidofovir is more effective than is parenteral therapy for treatment of progressive vaccinia in immunocompromised mice.

BACKGROUND: Severe complications may arise as a result of virus dissemination after smallpox (live vaccinia virus) vaccination, particularly in immunocompromised individuals. We developed a new mouse model for studying the effects of antiviral agents on progressive vaccinia virus infections. METHODS: Hairless mice were treated with cyclophosphamide (100 mg/kg/day) every 4 days starting 1 day before vaccinia virus exposure to wounded skin. Primary lesions progressed in severity, satellite lesions developed, and the infection eventually killed the mice. RESULTS: Topical treatment with 1%-cidofovir cream (twice daily for 7 days) was much more effective in reducing the severity of primary lesions and the number of satellite lesions than was parenteral cidofovir treatment (100 mg/kg/day, given every 3 days). Both forms of treatment delayed death. Topical drug treatment markedly reduced virus titers in the skin and snout, whereas parenteral treatment did not, suggesting that the latter treatment resulted in lower drug exposure to skin. Topical treatment starting 9 days after infection delayed death by 10 days, compared with treatment with placebo. Combining topical and parenteral cidofovir treatments provided the greatest reduction in lesion severity and prolongation of life. CONCLUSIONS: Topical cidofovir treatment was superior to parenteral treatment. This new animal model may be useful in evaluation of the efficacy of treatment regimens against complications from smallpox vaccination.