Cowpox Virus Infection Research Articles

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)-γ, IFN-γ 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-γ and IP-10. Cytokine levels were fairly constant during vaccinia virus i.p. infections. Cidofovir treatment (100 mg/kg/day i.p. for 2 days) significantly suppressed certain cytokine (IFN- γ, IL-6, IL-10, IL-11, IP-10, LIF, MCP-1, MCP-3, MCP-5, MIP-1γ, 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.

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=0.6–2.2μM, rabbit=52–90μM, monkey=87 to >1000μM, and human=39–220μM. 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 1000μM. 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 30mg/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.

In vitro and in vivo evaluation of isatin-β-thiosemicarbazone and marboran against vaccinia and cowpox virus infections

It has been reported previously that some thiosemicarbazone compounds have prophylactic activity against smallpox disease and therapeutic activity against vaccinia virus (VV) infections. In these studies, isatin-β-thiosemicarbazone (IBT) and marboran were administered once daily by intraperitoneal (ip) injection to mice using 30, 10 or 3 mg/kg for 5 days beginning 24, 48 or 72 h after inoculation with VV or cowpox virus (CV). Both compounds were highly effective ( p < 0.01) at preventing mortality due to VV even when treatment was delayed up to 72 h postinfection. In CV-infected mice, neither IBT nor Marboran were effective in preventing mortality at any dosage tested when administered at 24 h postinoculation. Viral replication in liver, spleen and kidney was delayed or reduced by 100–to 10,000-fold by 10 mg/kg of marboran, but not IBT, in VV infections. Neither compound was effective against CV infection. Neither IBT nor marboran treatment of mice cutaneously infected with VV or CV reduced viral replication or clinical disease. These results suggest that this class of compound has little therapeutic potential for orthopoxvirus infections since the in vivo activity against CV, a surrogate virus for variola, is lacking.

Space–time clustering of cowpox virus infection in wild rodent populations

SummaryThe spatial and temporal scale at which infectious animals pose a transmission risk to others may be inferred by a study of space–time interaction in the clustering of cases of infection.We analyse the distribution of bank voles and wood mice infectious with cowpox virus, using 7 years of monthly live‐trapping data from a woodland in north‐west England.A published method,K‐function analysis, is adapted to measure space–time interaction among infectious animals trapped on a grid system, where the populations at risk may vary in density.We hypothesized that the risk of cowpox transmission from an infectious animal would be contained within a temporal scale of one infectious period and a spatial scale of one home‐range diameter.Cowpox cases among wood mice showed significantly greater spatial clustering than the population at risk at the smallest spatial scale, corresponding to animals caught in the same location. At larger scales, and for bank voles at all scales, cowpox cases showed no spatial clustering beyond that expected from an association with host distribution.Cowpox cases among wood mice showed strong evidence of temporal clustering beyond that of the host population, indicating short‐lived outbreaks. No such effect was apparent among bank voles.Significant space–time interaction among cowpox cases was detected at the hypothesized spatial and temporal scales in both host species. This suggests that the risk of transmission is concentrated locally.Results are discussed in terms of transmission, including consideration of the appropriateness of distance and nearest‐neighbour based clustering tests to infections transmitted by local density and frequency dependent mechanisms.

Identification of poxvirus CD8+ T cell determinants to enable rational design and characterization of smallpox vaccines

The large size of poxvirus genomes has stymied attempts to identify determinants recognized by CD8+ T cells and greatly impeded development of mouse smallpox vaccination models. Here, we use a vaccinia virus (VACV) expression library containing each of the predicted 258 open reading frames to identify five peptide determinants that account for approximately half of the VACV-specific CD8+ T cell response in C57BL/6 mice. We show that the primary immunodominance hierarchy is greatly affected by the route of VACV infection and the poxvirus strain used. Modified vaccinia virus ankara (MVA), a candidate replacement smallpox vaccine, failed to induce responses to two of the defined determinants. This could not be predicted by genomic comparison of viruses and is not due strictly to limited MVA replication in mice. Several determinants are immunogenic in cowpox and ectromelia (mousepox) virus infections, and immunization with the immunodominant determinant provided significant protection against lethal mousepox. These findings have important implications for understanding poxvirus immunity in animal models and bench-marking immune responses to poxvirus vaccines in humans.

Cowpox Virus Infection Associated with a Streptococcal Septicaemia in a Foal

Cowpox virus infection associated with a streptococcal septicaemia was diagnosed in a weak German Warmblood filly, born 29 days prematurely, and humanely destroyed on the sixth day of life. At necropsy, ulcerative lesions in the alimentary tract, colitis, polyarthritis and nephritis were observed. Transmission electron microscopical examination of specimens from ulcerative lesions revealed typical orthopox virions. Cowpox virus was unequivocally identified by virological and molecular-biological methods.

Oral Treatment of Cowpox and Vaccinia Virus Infections in Mice with Ether Lipid Esters of Cidofovir

Oral Treatment of Cowpox and Vaccinia Virus Infections in Mice with Ether Lipid Esters of Cidofovir

Infection à cowpox virus chez l’enfant

Although human cowpox virus infection is rare nowadays, an animal reservoir of this virus still exists. The general course of cowpox virus infections is usually benign but the diagnosis is difficult and often late. Case report. – An 11-year-old boy, owner of two cats, presented with an infected sacral wound lesion associated with fever and lymph nodes. The wound became necrotic and other cutaneous and mucous membrane lesions developed secondarily. Blood tests did not show hyperleukocytosis or a systemic inflammatory response. Concurrently one of the cats was examined by a veterinary because of multifocal cutaneous lesions. Evocative skin biopsy specimens from the animal and, secondarily from the patient, allowed the identification of orthopoxvirus. Evolution was slowly favourable under symptomatic treatment. Conclusion. – Poxviruses are responsible for many animal and human diseases, the most famous of them being smallpox which today is considered eradicated. Vaccination against smallpox is no longer performed since 1977. Whether the arrest of vaccinations against smallpox may induce the apparition of other poxviruses infections or alter their clinical expression is an open question.

Efficacy of multiple- or single-dose cidofovir against vaccinia and cowpox virus infections in mice.

Orthopoxviruses, including variola and monkeypox, pose risks to human health through natural transmission or potential bioterrorist activities. Since vaccination has not recently been utilized for control of these infections, there is renewed effort in the development of antiviral agents not only for postexposure smallpox therapy but also for treatment of adverse reactions following vaccination. The objectives of this study were to expand on the results of others that cidofovir (CDV) is effective in mice inoculated with cowpox virus (CV) or vaccinia virus (VV) and to document the efficacy of single and interval dosing beginning prior to or after infection, particularly including evaluations using suboptimal doses of CDV. We utilized BALB/c or SCID mice inoculated with CV or VV as models for systemic poxvirus infections. BALB/c mice were inoculated intranasally with CV or VV and treated with CDV prior to or after virus inoculation. CDV, at concentrations as low as 0.7 to 6.7 mg/kg of body weight/day for 5 days, conferred significant protection when treatment was initiated as late as 72 to 96 h postinfection. A single-dose pretreatment or posttreatment with CDV at 3 to 100 mg/kg was effective when given as early as 5 days prior to infection or as late as 3 days after infection with either VV or CV. Interval treatments given every third day beginning 72 h postinfection using 6.7 or 2 mg of CDV/kg also proved effective against CV infections. When SCID mice were inoculated intraperitoneally with CV or VV and treated for 7 to 30 days with CDV, all the mice eventually died during or after cessation of treatment; however, significant delays in time to death and reduction of virus replication in organs occurred in most treated groups, and no resistance to CDV was detected.

Feline orthopoxvirus infection transmitted from cat to human

We report the case of a 56-year-old female patient who presented with an inflamed, ulcerated lesion on the left side of her neck after contact (scratch) with a cat living in the patient's house. Satellite lesions developed despite local treatment and parenteral clindamycin. Histopatholgic examination and the Tzanck test showed evidence of a viral infection. Subsequent transmission electron microscopy of scrap tissue and material from a fresh pustule exhibited multiple typical poxvirus particles, predominantly in remnants of scaled-off layers of degenerated keratinocytes, and virus particles in intermingled phagocytes, leading to the diagnosis of feline Orthopoxvirus (cowpox virus) infection. These results were verified by polymerase chain reaction and sequencing. Concern has been raised as to whether discontinuation of smallpox vaccine would cause an increase in Orthopoxvirus infection, but this has not yet shown to be the case.

Rodents, cowpox virus and islands: densities, numbers and thresholds

SummaryThe population dynamics of bank voles and wood mice, and of cowpox virus infection in these two species, was studied over a 2‐year period in a mainland population and in 14 nearby island populations of varying sizes.For both species, there was no intrinsic variation in the pattern of host dynamics with island size: small island populations behaved as though they were small subsamples of a larger population, displaying no more than the expected random variation from the general pattern.None the less, the relative numbers of bank voles to wood mice increased markedly with decreasing island size; and bank vole densities tended to be higher on smaller islands.Only 22 animals were discovered to have moved either between islands or between the mainland and the islands, out of 1883 captured in all. None the less, it was apparent that males were more likely to move than females.Overall patterns of cowpox virus dynamics were similar in all cases. However, on all islands there were extended periods when cowpox virus infection was apparently absent, and on the small islands the numbers of infected individuals were mostly very low and in many cases infection was never found.For both host species, there was no evidence for a threshold population size for cowpox virus (critical community size) in terms of density, but clear evidence for one in terms of the numerical size of populations. This suggests little support for density‐dependent transmission, despite this having been the usual default assumption for non‐sexually transmitted infections.There was also evidence for a separate invasion threshold (between ecological and epidemiological invasion) and persistence threshold (between epidemiological invasion and persistence). This is contrary to the output of the most‐quoted (deterministic) models – persistence and invasion threshold one and the same – highlighting the fact that little attention has been paid in the past to the practical meaning of the theoretical concept of a threshold.In the case of the wood mice, a superficial similarity to the bank vole thresholds was potentially misleading. Wood mouse thresholds were influenced at least as much by the bank vole thresholds as they were by the dynamics within the wood mouse populations themselves.

Increased antiviral activity of 1-O-hexadecyloxypropyl-[2-(14)C]cidofovir in MRC-5 human lung fibroblasts is explained by unique cellular uptake and metabolism.

Recently, there has been renewed interest in finding orally active drugs against smallpox. Cidofovir (CDV) given by parenteral injection has been shown to protect against lethal poxvirus infection. We have been interested in the synthesis and evaluation of orally active derivatives of CDV. Previous studies showed that the CDV and cyclic cidofovir (cCDV) analogs 1-O-hexa-decyloxypropyl-CDV (HDP-CDV) and 1-O-hexadecyloxypropyl-cCDV (HDP-cCDV), show >100-fold increases in antiviral activity versus the unmodified nucleosides against cells infected with orthopoxviruses, cowpox, and vaccinia virus. In contrast to CDV, HDP-CDV is orally bioavailable and has been reported to be orally active in lethal cowpox virus infection in mice. To assess the metabolic basis for the increased antiviral activity of HDP-CDV in vitro, we studied the cellular uptake and anabolic metabolism of (14)C-labeled CDV, cCDV, and their alkoxyalkanol esters HDP-CDV and HDP-cCDV. HDP-CDV and HDP-cCDV were taken up rapidly by MRC-5 human lung fibroblasts in vitro, but uptake of CDV and cCDV was much slower. Analysis of cellular metabolites showed that levels of cidofovir diphosphate (CDV-DP), the active antiviral compound, were >100 times greater with HDP-CDV than levels observed with CDV. When cells were exposed to HDP-CDV, the intracellular half-life of CDV-DP was 10 days versus 2.7 days reported when cells are exposed to CDV. HDP-CDV seems to circumvent poor cellular uptake by rapid association with cellular membrane phospholipids, whereas CDV uptake proceeds via the slow process of fluid endocytosis.

Plasma membrane localization and fusion inhibitory activity of the cowpox virus serpin SPI-3 require a functional signal sequence and the virus encoded hemagglutinin

The cowpox virus (CPV) glycoprotein serpin SPI-3, a functional protease inhibitor, and the viral hemagglutinin (HA) are required to prevent fusion of wt CPV infected cells. SPI-3 and HA from CPV infected cells co-localize to the plasma membrane and are found in extracellular enveloped virus (EEV). We also show that an N-terminal SPI-3 signal sequence, but not glycosylation, is required for membrane localization and fusion inhibition. In the absence of HA (CPVΔHA), no SPI-3 is found on the membrane and infected cells fuse. Conversely, HA from both wt CPV and CPVΔSPI-3 infections is on the membrane, indicating a requirement of HA for SPI-3 plasma membrane localization. In the absence of HA, secretion of SPI-3 or SPI-3 N-glyc(−) was markedly enhanced, suggesting HA serves to retain SPI-3 on the plasma membrane,thereby preventing cell fusion.

Therapy and short-term prophylaxis of poxvirus infections: historical background and perspectives

The era of antiviral chemotherapy started more than 50 years with the findings by Domagk and his colleagues that thiosemicarbazones showed activity against vaccinia virus. One of the derivatives, methisazone, was even investigated in the prophylaxis of smallpox. With the successful implementation of the smallpox vaccine, the use of methisazone was not further pursued. Should there be a threat of smallpox or other poxvirus infections, that could not be immediately controlled by vaccination, a therapeutic intervention could be envisaged based on several therapeutic strategies targeted at such cellular enzymes as IMP dehydrogenase, SAH hydrolase, OMP decarboxylase and CTP synthetase, as well as viral enzymes such as the DNA polymerase. Most advanced as a therapeutic or early prophylactic modality to tackle poxvirus infection is cidofovir, which was found active (i) in vitro against all poxviruses studied so far; (ii) in vivo, against vaccinia and cowpox virus infections in experimental animal models; as well as (iii) some human poxvirus infections, such as molluscum contagiosum. In case of an inadvertent poxvirus epidemic, antiviral therapy (i.e. with cidofovir) will offer the possibility to provide short-term prophylaxis, or therapy. Cidofovir should also allow to treat severe complications of vaccination as may happen in for example immunosuppressed patients.

Characterization of wild-type and cidofovir-resistant strains of camelpox, cowpox, monkeypox, and vaccinia viruses.

Cidofovir ([(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine] [HPMPC])-resistant forms of camelpox, cowpox, monkeypox, and vaccinia viruses were developed by prolonged passage in Vero 76 cells in the presence of drug. Eight- to 27-fold-higher concentrations of cidofovir were required to inhibit the resistant viruses than were needed to inhibit the wild-type (WT) viruses. Resistant viruses were characterized by determining their cross-resistance to other antiviral compounds, examining their different replication abilities in two cell lines, studying the biochemical basis of their drug resistance, and assessing the degrees of their virulence in mice. These viruses were cross resistant to cyclic HPMPC and, with the exception of vaccinia virus, to (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)adenine. Three of the four resistant cowpox and monkeypox viruses exhibited reduced abilities to infect and replicate in 3T3 cells compared to their abilities in Vero 76 cells. Compared to the WT virus polymers the resistant cowpox virus DNA polymerase was 8.5-fold less sensitive to inhibition by cidofovir diphosphate, the active form of the drug. Intracellular phosphorylation of [3H]cidofovir was not stimulated or inhibited by infection with resistant cowpox virus. In intranasally infected BALB/c mice, WT cowpox virus was 80-fold more virulent than the resistant virus. Cidofovir treatment (100 mg/kg of body weight, given one time only as early as 5 min after virus challenge) of a resistant cowpox virus infection could not protect mice from mortality. However, the drug prevented mortality in 80 to 100% of the mice treated with a single 100-mg/kg dose at 1, 2, 3, or 4 days after WT virus challenge. By application of these results to human orthopoxvirus infections, it is anticipated that resistant viruses may be untreatable with cidofovir but their virulence may be attenuated. Studies will need to be conducted with cidofovir-resistant monkeypox virus in monkeys to further support these hypotheses.

Treatment of aerosolized cowpox virus infection in mice with aerosolized cidofovir

The Brighton strain of cowpox virus causes lethal bronchopneumonia when delivered as a small-particle (1 μm) aerosol to weanling BALB/c mice. We showed previously that this disease can be prevented or cured with one subcutaneous injection of cidofovir (HPMPC, Vistide®). To determine whether even better results could be obtained by delivering the drug directly to the respiratory tract, we administered cidofovir by small-particle aqueous aerosol before or after aerosolized cowpox infection. In a series of five experiments, aerosol doses of 0.5–5 mg/kg were always more effective than 25 mg/kg and sometimes more effective than 100 mg/kg injected subcutaneously, as measured by changes in body and lung weight, lung viral titers, pulmonary pathology and survival. A cyclic analog ((1-[( S)-2-hydroxy-2-oxo-1,4,2-dioxaphosphorinan-5-yl)methyl] cytosine) (cHPMPC) was less protective. The results suggest that aerosolized cidofovir would be effective for prophylaxis or early post-exposure therapy of human smallpox or monkeypox virus infection.

Treatment of lethal cowpox virus respiratory infections in mice with 2-amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine and its orally active diacetate ester prodrug

The acyclic purine nucleoside analog, 2-amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine (S2242) and its orally active diacetate ester prodrug (HOE961) were reported to be potent inhibitors of vaccinia virus replication in cell culture and in infected mice. These compounds were evaluated further, using infections with the related cowpox virus. Against a wild-type (WT) cowpox virus strain in mouse C127I cell culture, 50% effective concentrations (EC 50, determined by plaque reduction assays) of S2242 and cidofovir (a positive control) were 3.5 and 1.0 μM, respectively. EC 50 values obtained against a cidofovir-resistant strain of the virus were 33 and 230 μM, respectively. Compounds were at least ten-fold less potent against WT virus in Vero cells than C127I cells. S2242 and cidofovir were 50% inhibitory to the proliferation of uninfected C127I cells at 340 and 180 μM, respectively, but neither compound inhibited Vero cell growth at 1000 μM. Mice were lethally infected with cowpox virus by intranasal inoculation, followed 24 h later by antiviral treatment for 5 consecutive days. Once or twice daily intraperitoneal (i.p.) treatments with either S2242 or HOE961 at 100 mg/kg per day resulted in ≥70 survival compared with no survivors in the placebo group. Lower doses of these compounds (10 and 30 mg/kg per day) were not protective, however. Cidofovir was 100% protective at 30 mg/kg per day. A 10-day course of treatment gave comparable survival results and demonstrated the oral efficacy of HOE961. Treatments with S2242 (100 mg/kg per day) and cidofovir (30 mg/kg per day) each reduced lung and nasal virus titers by approximately ten-fold, whereas, HOE961 (100 mg/kg per day) was less active. Overall, S2242 and HOE961 were found to be effective against cowpox virus infections in mice but were less potent than cidofovir. Since, HOE961 was orally active, it may have advantages over the other parenterally administered compounds for treating orthopoxvirus infections.

Intranasal treatment of cowpox virus respiratory infections in mice with cidofovir

Orthopoxvirus infections in mice have been effectively treated with cidofovir, a clinically approved drug given by intravenous infusion to treat cytomegalovirus infections. In a bioterrorist scenario it would be technically difficult to give this drug to a large number of exposed individuals. New treatment approaches are being sought, which include giving cidofovir by alternative routes or designing oral prodrugs of cidofovir. In this report, intranasal cidofovir was investigated as a treatment of pulmonary cowpox virus infections in BALB/c mice. Ninety to 100% of animals given a single intranasal drug treatment (10, 20 or 40 mg/kg) 24 h after virus challenge survived the infection, whereas all placebo-treated mice died. Doses of 2.5 and 5 mg/kg resulted in 60 and 80% survival, respectively. Single treatments of 20 and 40 mg/kg could be given up to 3 days after virus inoculation and still be 80–90% protective. A single 40 mg/kg treatment of infected mice given 1 or 2 days after infection also resulted in statistically significant decreases in virus titer in lungs and nose/sinus compared to the placebo group. Drug efficacy was found to be contingent upon treatment volume. A 10 mg/kg intranasal dose given 24 h after virus challenge was 100 and 50% effective in volumes of 40 and 20 μl, respectively. The same dose in 5 and 10 μl volumes caused no decrease in mortality. The results of these studies establish the utility of cidofovir treatment of poxvirus infections in mice by intranasal route. The data suggest the possibility that aerosol delivery of cidofovir to human lungs may be a viable alternative to intravenous dosing.

Cowpox virus infection in an 11-year-old girl

We describe an 11-year-old girl with a cowpox virus infection, who presented with a 14-day-old crusted, ulcerated nodule on the chin/neck and a 6-day-old eroded blister on the left leg. The girl lived in a rural environment, had close contact to several cats from the neighborhood, and had an atopic predisposition. The presence of orthopox virus in the lesion on the left leg was demonstrated by electron microscopy (negative staining, transmission electron microscopy) and virus isolation. Classification as a cowpox virus was determined by polymerase chain reaction (PCR), followed by restriction enzyme digestion of the PCR product. (J Am Acad Dermatol 2000;42:892-4.)

Cidofovir protects mice against lethal aerosol or intranasal cowpox virus challenge.

The efficacy of cidofovir for treatment of cowpox virus infection in BALB/c mice was investigated in an effort to evaluate new therapies for virulent orthopoxvirus infections of the respiratory tract in a small animal model. Exposure to 2(-5)x10(6) pfu of cowpox virus by aerosol or intranasally (inl) was lethal in 3- to 7-week-old animals. One inoculation of 100 mg/kg cidofovir on day 0, 2, or 4, with respect to aerosol infection, resulted in 90%-100% survival. Treatment on day 0 reduced peak pulmonary virus titers 10- to 100-fold, reduced the severity of viral pneumonitis, and prevented pulmonary hemorrhage. The same dose on day -6 to 2 protected 80%-100% of inl infected mice, whereas 1 inoculation on day -16 to -8 or day 3 to 6 was partially protective. Cidofovir delayed but did not prevent the death of inl infected mice with severe combined immunodeficiency. Treatment at the time of tail scarification with vaccinia virus did not block vaccination efficacy.