[Human poxvirus infections]. / Infections humaines à poxvirus.

Poxvirus (PXV) infections are a common cause of cutaneous signs. In France, certain forms of poxvirus are frequent and benign (molluscum contagiosum), while others are rare but potentially serious (cowpox virus [CPXV]). Whereas only smallpox and molluscum contagiosum viruses have a human reservoir and are transmitted between humans, most poxvirus infections are zoonoses having only animal reservoirs. Only a small number of poxviruses are responsible for infection in humans, but the increasing number of new pets, some of which are exotic, coupled with the rapid rise in international travel are creating a greater risk of transmission of zoonotic PXV to new vectors and of spread of these diseases to new regions throughout the world. In France, molluscum contagiosum, orf and milkers' nodule give rise to numerous consultations and are well known to dermatologists. However, dermatologists must also be able to identify other parapoxviruses of similar presentation to orf; thus, CPXV and monkeypox are considered potentially emergent viruses with a high risk of epidemic and spread due to increasing international transport and the loss of the maximum protection against smallpox. Finally, despite its declared eradication, smallpox is currently being monitored because of the potential risk of reintroduction, whether accidentally or deliberately through bioterrorism.

Cowpox after a cat scratch - case report from Poland.

Cowpox in humans is a rare zoonotic disease; its recognition is therefore problematic due to the lack of clinical experience. The differential diagnosis includes other poxvirus infections and also infections with herpesviruses or selected bacteria. The clinical course can be complicated and the improvement may take weeks. Late diagnosis is one of the causes of unnecessary combined antibiotic therapy or surgical intervention. A case of cowpox after a cat scratch in a 15-year-old girl is presented, with a summary of the available clinical data on cowpox infections.

Emergence of cowpox: study of the virulence of clinical strains and evaluation of antivirals.

The last years, cowpox infections are being increasingly reported through Eurasia. Cowpox viruses (CPXVs) have been reported to have different genotypes and may be subdivided in at least five genetically distinct monophyletic clusters. However, little is known about their in vitro and in vivo features. In this report, five genetically diverse CPXVs, including one reference strain (CPXV strain Brighton) and four clinical isolates from human and animal cases, were compared with regard to growth in cells, pathogenicity in mice and inhibition by antivirals. While all CPXVs replicated similarly in vitro and showed comparable antiviral susceptibility, marked discrepancies were seen in vivo, including differences in virulence with recorded mortality rates of 0%, 20% and 100%. The four CPXV clinical isolates appeared less pathogenic than two reference strains, CPXV Brighton and vaccinia virus Western-Reserve. Disease severity seemed to correlate with high viral DNA loads in several organs, virus titers in lung tissues and levels of IL-6 cytokine in the sera. Our study highlighted that the species CPXV consists of viruses that not only differ considerably in their genotypes but also in their in vivo phenotypes, indicating that CPXVs should not be longer classified as a single species. Lung virus titers and IL-6 cytokine level in mice may be used as biomarkers for predicting disease severity. We further demonstrated the potential benefit of cidofovir, CMX001 and ST-246 use as antiviral therapy.

Feline cowpoxvirus infections in Germany: clinical and epidemiological aspects.

Clinical and epidemiological aspects of cats with cowpox in Germany from the years 2004 to 2010 are described and discussed. Questionnaires were sent to veterinarians and owners of affected cats identified with the help of a number of pathology laboratories. Of 69 mailed questionnaires, 45 veterinary and 26 owner questionnaires were returned and a total of 46 feline poxcases were evaluated. The cases were distributed all over Germany although there was an accumulation of cases in specific geographic areas. The clinical and epidemiological observations match those of other studies. The majority of cats were outdoor cats, came from a rural environment and developed clinical signs in late summer or autumn. All cats showed skin lesions which were predominantly localized on the anterior part of the body, 61% of the cats showed other clinical signs in addition to the skin lesions. Approximately half of the cats lived in a multi-pet household, but in only one case clinical signs typical for cowpox were observed in another cat of the household. In two cases a cat-to-human transmission was assumed. In addition, to evaluate the prevalence of pox virus infections in outdoor cats in areas with previous reports of such infections, 92 apparently unaffected outdoor cats were tested for orthopoxvirus antibodies using an indirect immunofluorescence assay. Sixteen (17%) of the tested serum samples were seropositive against orthopoxvirus (titre between 1:20 and 1:40).This is a higher serum prevalence than in previously published studies from Germany. A possible explanation is selection of a population of outdoor cats from regions with previous known clinical cases.

Antiviral activity of the EB peptide against zoonotic poxviruses.

BACKGROUND: The EB peptide is a 20-mer that was previously shown to have broad spectrum in vitro activity against several unrelated viruses, including highly pathogenic avian influenza, herpes simplex virus type I, and vaccinia, the prototypic orthopoxvirus. To expand on this work, we evaluated EB for in vitro activity against the zoonotic orthopoxviruses cowpox and monkeypox and for in vivo activity in mice against vaccinia and cowpox. FINDINGS: In yield reduction assays, EB had an EC50 of 26.7 µM against cowpox and 4.4 µM against monkeypox. The EC50 for plaque reduction was 26.3 µM against cowpox and 48.6 µM against monkeypox. A scrambled peptide had no inhibitory activity against either virus. EB inhibited cowpox in vitro by disrupting virus entry, as evidenced by a reduction of the release of virus cores into the cytoplasm. Monkeypox was also inhibited in vitro by EB, but at the attachment stage of infection. EB showed protective activity in mice infected intranasally with vaccinia when co-administered with the virus, but had no effect when administered prophylactically one day prior to infection or therapeutically one day post-infection. EB had no in vivo activity against cowpox in mice. CONCLUSIONS: While EB did demonstrate some in vivo efficacy against vaccinia in mice, the limited conditions under which it was effective against vaccinia and lack of activity against cowpox suggest EB may be more useful for studying orthopoxvirus entry and attachment in vitro than as a therapeutic against orthopoxviruses in vivo.

Inhibition of cowpox virus and monkeypox virus infection by mitoxantrone.

Mitoxantrone, an FDA-approved therapeutic for the treatment of cancer and multiple sclerosis, was previously reported to exhibit antiviral activity against vaccinia virus. To determine whether this activity extends to other orthopoxviruses, mitoxantrone was tested against cowpox and monkeypox. Mitoxantrone demonstrated an EC(50) of 0.25 µM against cowpox and 0.8 µM against monkeypox. Intraperitoneal treatment of cowpox virus-challenged C57Bl/6 mice with 0.5 mg/kg mitoxantrone resulted in 25% survival and a significant increase in survival time. In an effort to improve its efficacy, mitoxantrone was tested for synergistic activity with cidofovir. In vitro tests demonstrated significant synergy between the two drugs against cowpox; however, no synergistic effect on animal survival or median time-to-death was seen in intranasally-infected BALB/c mice. Significantly fewer animals survived when treated with a combination of 0.5 mg/kg mitoxantrone and 100 mg/kg cidofovir than with 100 mg/kg cidofovir alone. This is, to our knowledge, the first report of limited anti-orthopoxvirus activity by mitoxantrone in an animal model.

Survival of a cat with pneumonia due to cowpox virus and feline herpesvirus infection.

Cowpox virus infection in cats is rare and usually leads to cutaneous lesions alone. Pulmonary infection and pneumonia have been documented occasionally but all such cases described to date have been fatal. Although usually affecting the upper respiratory tract, feline herpesvirus can also induce pneumonia. The present report describes the case of a cat that recovered from a pneumonia in which both poxvirus and feline herpesvirus were demonstrated.

Activities of certain 5-substituted 4'-thiopyrimidine nucleosides against orthopoxvirus infections.

As part of a program to identify new compounds that have activity against orthopoxviruses, a number of 4'-thionucleosides were synthesized and evaluated for their efficacies against vaccinia and cowpox viruses. Seven compounds that were active at about 1 microM against both viruses in human cells but that did not have significant toxicity were identified. The 5-iodo analog, 1-(2-deoxy-4-thio-beta-d-ribofuranosyl)-5-iodouracil (4'-thioIDU), was selected as a representative molecule; and this compound also inhibited viral DNA synthesis at less than 1 microM but only partially inhibited the replication of a recombinant vaccinia virus that lacked a thymidine kinase. This compound retained complete activity against cidofovir- and ST-246-resistant mutants. To determine if this analog had activity in an animal model, mice were infected intranasally with vaccinia or cowpox virus and treatment with 4'-thioIDU was given intraperitoneally or orally twice daily at 50, 15, 5, or 1.5 mg/kg of body weight beginning at 24 to 120 h postinfection and was continued for 5 days. Almost complete protection (87%) was observed when treatment with 1.5 mg/kg was begun at 72 h postinfection, and significant protection (73%) was still obtained when treatment with 5 mg/kg was initiated at 96 h. Virus titers in the liver, spleen, and kidney were reduced by about 4 log(10) units and about 2 log(10) units in mice infected with vaccinia virus and cowpox virus, respectively. These results indicate that 4'-thioIDU is a potent, nontoxic inhibitor of orthopoxvirus replication in cell culture and experimental animal infections and suggest that it may have potential for use in the treatment of orthopoxvirus infections in animals and humans.

Cowpox virus infection: an emerging health threat.

PURPOSE OF REVIEW: Human cowpox, a rare zoonotic infection, evokes a self-limited disease, except for immunocompromised and eczematous patients, particularly children, where it can become severe. The causative agent, cowpox virus, is distributed in Europe, west former USSR, and adjacent areas of Northern and Central Asia, with an increasing number of reports in Europe. The purpose of this paper is to review cowpox with an emphasis on its epidemiology and management. RECENT FINDINGS: Numerous reports of human cowpox affecting young people in Europe indicate that lack of smallpox vaccination, which has been abandoned since 1977, may render the population more vulnerable to cowpox virus. The ownership of wild and exotic animal pets is becoming more popular, and the range of recognized wild and domestic animal hosts is expanding, SUMMARY: Cowpox as a human emerging zoonotic hazard raises public health concerns as well as a question about the production of effective vaccine and antiviral agents.

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.

Effect of oral treatment with hexadecyloxypropyl-[(S)-9-(3-hydroxy-2- phosphonylmethoxypropyl)adenine] [(S)-HPMPA] or octadecyloxyethyl-(S)-HPMPA on cowpox or vaccinia virus infections in mice.

We have previously reported that (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine, or (S)-HPMPA, is active in vitro against cowpox virus (CV) and vaccinia virus (VV) but is not active orally in animals. However, the ether lipid esters of (S)-HPMPA, hexadecyloxypropyl-[(S)-HPMPA] [HDP-(S)-HPMPA] and octadecyloxyethyl-[(S)-HPMPA] [ODE-(S)-HPMPA], had significantly enhanced activity in vitro and are orally bioavailable in mice. In the current study, HDP-(S)-HPMPA and ODE-(S)-HPMPA were prepared in water and administered once daily by oral gavage to mice at doses of 30, 10, and 3 mg/kg of body weight for 5 days beginning 24, 48, or 72 h after inoculation with CV or VV. Oral HDP-(S)-HPMPA and ODE-(S)-HPMPA were both highly effective (P < 0.001) at preventing mortality due to CV at 30 mg/kg, even when treatments were delayed until up to 72 h postinfection. ODE-(S)-HPMPA or HDP-(S)-HPMPA were also highly effective (P < 0.001) at preventing mortality in mice infected with VV at 30 mg/kg when treatments were delayed until to 48 or 72 h postinfection, respectively. Protection against both viruses was associated with a significant reduction of virus replication in the liver, spleen, and kidney but not in the lung. These data indicate that HDP-(S)-HPMPA and ODE-(S)-HPMPA are active when given orally against lethal CV and VV infections in mice, and further evaluation is warranted to provide additional information on the potential of these orally active compounds for treatment of human orthopoxvirus infection.

In vivo imaging of cidofovir treatment of cowpox virus infection.

Variola virus and other members of the genus Orthopoxviruses constitute a prominent bioterrorism and public health threat. Treatment with the anti-viral drug cidofovir inhibits replication of orthopoxviruses in vitro and in vivo. In this study, we visualized the effect of cidofovir on viral kinetics in orthopoxvirus infected mice by using whole-body fluorescence imaging (FI). We engineered a cowpox virus (CPV) expressing the enhanced green fluorescent protein (GFP). Single-step growth curves and calculated 50% lethal doses (LD(50)) of wild-type CPX (Wt-CPV) and GFP-expressing CPX (GFP-CPV) were comparable. Whole-body FI first detected GFP fluorescence in the mesenteric tissue of untreated animals on post-infection day (PID) 1. On PID 3 GFP signal was detected throughout the mesentery, in all abdominal organs by PID 5 and in most major organs, except for the heart and brain by PID 6. Infected animals treated with 25mg/kg of cidofovir also began showing signs of viral replication on PID 1, however, the fluorescent signal was limited only to discrete foci throughout the course of the infection. This work describes the first use of an established Orthopox model of infection to evaluate drug efficacy and track virus progression on a macroscopic level.

Synthesis and antiviral evaluation of alkoxyalkyl-phosphate conjugates of cidofovir and adefovir.

Esterification of cidofovir (CDV), an antiviral nucleoside phosphonate, with alkyl or alkoxyalkyl groups increases antiviral activity by enhancing cell uptake and conversion to CDV diphosphate. Hexadecyloxypropyl-CDV (HDP-CDV) has been shown to be 40-100 times more active than CDV in vitro in cells infected with herpes group viruses, variola, cowpox, vaccinia or ectromelia viruses. Since the first phosphorylation of CDV may be rate limiting, we synthesized the hexadecyloxypropyl-phosphate (HDP-P-) and octadecyloxyethyl-phosphate (ODE-P-) conjugates of CDV and phosphonomethoxy-ethyl-adenine (PMEA, adefovir). We tested the CDV analogs in cells infected with human cytomegalovirus, herpes simplex virus, cowpox virus and vaccinia virus; the analogs of PMEA were tested in cells infected with the human immunodeficiency virus, type 1. In general, the alkoxyalkyl-phosphate conjugates of CDV were substantially more active than CDV. HDP-P-CDV and ODE-P-CDV were 4.6-40 times more active against HCMV and 7-30 times more active against cowpox and vaccinia in vitro. Although the compounds of this type were more cytotoxic than the unmodified bases, their selectivity for virally infected cells was generally greater than the parent nucleotides except that HDP-P-PMEA showed little or no selectivity in HIV-1 infected MT-2 cells. Although the new compounds with an interposed phosphate were generally less active than the corresponding alkoxyalkyl esters of CDV and PMEA, the present approach provides a possible alternative method for enhancing the antiviral activity of drugs of this class.

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.

Effects of cidofovir treatment on cytokine induction in murine models of cowpox and vaccinia virus infection.

Cytokine profiles during cowpox and vaccinia (WR strain) virus infections were characterized in intranasal (i.n.) and intraperitoneal (i.p.) models in BALB/c mice. The time-course of induction and effects of cidofovir treatment on interferon (IFN)-gamma, IFN-gamma inducible protein (IP)-10, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1 were determined. The four mouse infection models have distinct patterns of cytokine induction. Cowpox virus i.p. and vaccinia virus i.n. infections showed increased induction throughout the time studied. Cowpox virus i.n. infection resulted in delayed induction of IFN-gamma and IP-10. Cytokine levels were fairly constant during vaccinia virus i.p. infections. Cidofovir treatment (100mg/kg/day i.p. for 2 days) significantly suppressed certain cytokine (IFN- gamma, IL-6, IL-10, IL-11, IP-10, LIF, MCP-1, MCP-3, MCP-5, MIP-1 gamma, and TIMP-1) levels to near normal relative to uninfected animals, as well as prevented mortality and reduced virus titers significantly. Characterization of cytokine responses has implications for understanding the immune responses and pathogeneses of viral infections in these mouse models.

Cutaneous infections of mice with vaccinia or cowpox viruses and efficacy of cidofovir.

Orthopoxviruses, including smallpox, monkeypox and molluscipox, pose risks to human health through bioterrorist acts or natural transmission. There is no approved therapy for orthopoxvirus infections; however, cidofovir (CDV) has been approved as an investigational new drug for emergency treatment of adverse effects following smallpox vaccination. For evaluation of new therapies directed against orthopoxvirus infections, we have utilized immunocompetent, hairless mice (SKH-1) inoculated by a cutaneous route with cowpox virus (CV) or vaccinia virus (VV). Mice subsequently developed skin lesions and virus was recovered from the site of inoculation and quantified. Skin biopsies were evaluated microscopically, revealing brick-like eosinophilic, intracytoplasmic inclusion bodies characteristic of orthopoxvirus infection. SKH-1 mice fully recovered from either CV or VV infection. Immunodeficient Athymic or Rhino mice inoculated with CV or VV had more lesions and severe disease than SKH-1 mice. CV-infected SKH-1 mice were treated either with systemic or topical CDV. Although some protection was achieved with systemic treatment, 5% topical CDV was most effective at reducing virus titers in skin, lung, kidney, and spleen. These models may provide a means for evaluating efficacy of new therapies directed against orthopoxvirus diseases and further confirm the topical activity of CDV against cutaneous infections.