Laboratory-confirmed case of human infection with ratpox (cowpox)

The present work describes a case of human disease resulting from the bite of a white rat and caused by a biological variant of cowpox virus. The isolates obtained from the sick man and the white rats which had been the source of this infection proved to be identical and did not differ from the biological variants of cowpox virus, isolated earlier from white rats and carnivorous animals of the family Felidae. Thus, the possibility of ratpox (cowpox) transmission from sick rodents to man was established.     

Genomic expression libraries for the identification of cross-reactive orthopoxvirus antigens

Increasing numbers of human cowpox virus infections that are being observed and that particularly affect young non-vaccinated persons have renewed interest in this zoonotic disease. Usually causing a self-limiting local infection, human cowpox can in fact be fatal for immunocompromised individuals. Conventional smallpox vaccination presumably protects an individual from infections with other Orthopoxviruses, including cowpox virus. However, available live vaccines are causing severe adverse reactions especially in individuals with impaired immunity. Because of a decrease in protective immunity against Orthopoxviruses and a coincident increase in the proportion of immunodeficient individuals in today's population, safer vaccines need to be developed. Recombinant subunit vaccines containing cross-reactive antigens are promising candidates, which avoid the application of infectious virus. However, subunit vaccines should contain carefully selected antigens to confer a solid cross-protection against different Orthopoxvirus species. Little is known about the cross-reactivity of antibodies elicited to cowpox virus proteins. Here, we first identified 21 immunogenic proteins of cowpox and vaccinia virus by serological screenings of genomic Orthopoxvirus expression libraries. Screenings were performed using sera from vaccinated humans and animals as well as clinical sera from patients and animals with a naturally acquired cowpox virus infection. We further analyzed the cross-reactivity of the identified immunogenic proteins. Out of 21 identified proteins 16 were found to be cross-reactive between cowpox and vaccinia virus. The presented findings provide important indications for the design of new-generation recombinant subunit vaccines.     

A cowpox virus gene required for multiplication in Chinese hamster ovary cells.

Cowpox virus, in contrast to vaccinia virus, can multiply in Chinese hamster ovary cells. To study the genetic basis for this difference in host range, recombinants between vaccinia and cowpox viruses were isolated and their DNA restriction patterns were examined. The ability to multiply in Chinese hamster ovary cells could be correlated with the conservation of cowpox virus sequences mapping at the left end of the genome. This was further demonstrated by marker rescue of the host range phenotype with restricted cowpox virus DNA. Marker rescue with cloned restriction fragments of decreasing size enabled the fine localization of the host range function to a 2.3-kilobase-pair fragment. Nucleotide sequencing revealed that the fragment encoded a single major polypeptide of approximately 77,000 daltons. It is suggested that the role of the host range gene from cowpox virus is to prevent the early and extensive shutoff of protein synthesis that normally occurs in Chinese hamster ovary cells infected by vaccinia virus.     

Cowpox: features of spread after cancellation of mandatory pox immunization

Features of spread of cowpox in the contemporary conditions are examined. A decrease of population immunity to pox in the population of Russia caused by cancellation of pox immunization, hidden circulation of cowpox virus in various species of rodents, as well as lack of vigilance to pathogenic orthopoxviurses in healthcare workers were noted to create the real preconditions for the emergence of infection of humans caused by cowpox virus. Thereby presence of means of express laboratory diagnostics of cowpox and means of effective medical protection for the prevention of development of this disease in the population of Russia becomes an actual necessity.     

Cowpox virus infection in natural field vole Microtus agrestis populations: significant negative impacts on survival

1. Cowpox virus is an endemic virus circulating in populations of wild rodents. It has been implicated as a potential cause of population cycles in field voles Microtus agrestis L., in Britain, owing to a delayed density-dependent pattern in prevalence, but its impact on field vole demographic parameters is unknown. This study tests the hypothesis that wild field voles infected with cowpox virus have a lower probability of survival than uninfected individuals. 2. The effect of cowpox virus infection on the probability of an individual surviving to the next month was investigated using longitudinal data collected over 2 years from four grassland sites in Kielder Forest, UK. This effect was also investigated at the population level, by examining whether infection prevalence explained temporal variation in survival rates, once other factors influencing survival had been controlled for. 3. Individuals with a probability of infection, P(I), of 1 at a time when base survival rate was at median levels had a 22.4% lower estimated probability of survival than uninfected individuals, whereas those with a P(I) of 0.5 had a 10.4% lower survival. 4. At the population level, survival rates also decreased with increasing cowpox prevalence, with lower survival rates in months of higher cowpox prevalence. 5. Simple matrix projection models with 28 day time steps and two stages, with 71% of voles experiencing cowpox infection in their second month of life (the average observed seroprevalence at the end of the breeding season) predict a reduction in 28-day population growth rate during the breeding season from lambda = 1.62 to 1.53 for populations with no cowpox infection compared with infected populations. 6. This negative correlation between cowpox virus infection and field vole survival, with its potentially significant effect on population growth rate, is the first for an endemic pathogen in a cyclic population of wild rodents.     

CrmE, a novel soluble tumor necrosis factor receptor encoded by poxviruses

Cytokines and chemokines play a critical role in both the innate and acquired immune responses and constitute prime targets for pathogen sabotage. Molecular mimicry of cytokines and cytokine receptors is a mechanism encoded by large DNA viruses to modulate the host immune response. Three tumor necrosis factor receptors (TNFRs) have been identified in the poxvirus cowpox virus. Here we report the identification and characterization of a fourth distinct soluble TNFR, named cytokine response modifier E (CrmE), encoded by cowpox virus. The crmE gene has been sequenced in strains of the orthopoxviruses cowpox virus, ectromelia virus, and camelpox virus, and was found to be active in cowpox virus. crmE is expressed as a secreted 18-kDa protein with TNF binding activity. CrmE was produced in the baculovirus and vaccinia virus expression systems and was shown to bind human, mouse, and rat TNF, but not human lymphotoxin alpha, conjugates of lymphotoxins alpha and beta, or seven other ligands of the TNF superfamily. However, CrmE protects cells only from the cytolytic activity of human TNF. CrmE is a new member of the TNFR superfamily which is expressed as a soluble molecule that blocks the binding of TNF to high-affinity TNFRs on the cell surface. The remarkable finding of a fourth poxvirus-encoded TNFR suggests that modulation of TNF activity is complex and represents a novel viral immune evasion mechanism.     

Modeling spatial structures of variola and cowpox virus TNF-binding CrmB proteins bound to murine or human TNF

Orthopoxviruses bear in their genomes several genes coding for homologous secreted proteins able to bind tumor necrosis factor. Different species of the genus possess different sets of these tumor necrosis factor-binding proteins. Viriola virus encodes the only one of them named CrmB. Despite sharing high sequence identity, CrmB proteins belonging to distinct orthopoxviral species were shown to significantly differ by their physico-chemical and biological properties. We modeled spatial structures of tumor necrosis factor receptor domains of variola and cowpox virus CrmB proteins bound to either murine, or human or mutated human tumor necrosis factor. In the sequence of last the arginine residue at position 31 is substituted with glutamine that is characteristic for murine tumor necrosis factor. Theoretical analysis of modeled ligand-receptor complexes revealed that the least stable should be the complex of cowpox virus CrmB with human tumor necrosis factor, and that arginine to glutamine substitution at position 31 should significantly stabilize binding of corresponding human tumor necrosis factor mutant to cowpox virus CrmB. Experimental evaluation of recombinant variola and cowpox virus CrmB efficiencies in inhibiting cytotoxic effect of all these tumor necrosis factors have approved our predictions.     

3D structure modeling of complexes formed by CrmB TNF-binding proteins of Variola and cowpox viruses with murine and human TNFs

Orthopoxviral genomes bear genes for a series of homologous secreted proteins binding tumor necrosis factor (TNF). Orthopoxvirus species have different sets of these proteins. Variola virus has only one protein of this series, CrmB. Although CrmB protein sequences are similar to each other, their physicochemical and biological properties show certain species-specific features. We constructed 3D models of complexes formed by TNF-binding domains of variola and cowpox viruses with murine and human TNFs. We also constructed corresponding models with a mutant human TNF. In this mutant TNF, the arginine residue at position 31 involved in receptor binding was replaced by glutamine, characteristic of murine TNF. Analysis of the models showed that the least stable complex should be that formed by cowpox virus CrmB with human TNF, and the Arg31/Gln substitution should significantly stabilize the interaction between cowpox CrmB and mutant human TNF. Experimental comparison of the abilities of recombinant variola and cowpox CrmB proteins to inhibit the cytotoxic action of TNFs confirmed the predictions.     

Vaccinia, cowpox, and camelpox viruses encode soluble gamma interferon receptors with novel broad species specificity.

Soluble receptors for gamma interferon (IFN-gamma) are secreted from cells infected by 17 orthopoxviruses, including vaccinia, cowpox, rabbitpox, buffalopox, elephantpox, and camelpox viruses, representing three species (vaccinia, cowpox, and campelpox viruses). The B8R open reading frame of vaccinia virus strain Western Reserve, which has sequence similarity to the extracellular binding domain of cellular IFN-gamma receptors (IFN-gamma Rs), is shown to encode an IFN-gamma binding activity by expression in recombinant baculovirus. The soluble virus IFN-gamma Rs bind IFN-gamma and, by preventing its interaction with the cellular receptor, interfere with the antiviral effects induced by this cytokine. Interestingly, in contrast to cellular IFN-gamma Rs, which are highly species specific, the vaccinia, cowpox, and camelpox virus IFN-gamma Rs bind and inhibit the biological activity of human, bovine, and rat IFN-gamma but not mouse IFN-gamma. This unique broad species specificity of the IFN-gamma R would aid virus replication in different species and suggests that vaccinia, cowpox, and camelpox viruses may have evolved in several species, possibly including humans but excluding mice. Last, the conservation of an IFN-gamma R in orthopoxviruses emphasizes the importance of IFN-gamma in defense against poxvirus infections.     

Clustered cases of paravaccinia in milkers

Clustered cases of a disease in men and cows firstly diagnosed as cowpox has been described. Clinical manifestation, epidemiology and laboratory diagnostics are presented. Virological and serological investigations of the specimens taken from sick persons and animals proved paravaccinia virus (genus Parapoxvirus) to be etiological agent of the illness. Cowpox virus (genus Orthopoxvirus) greatly differs from the latter by phenotypical markers. The disease in humans is called Milkers' modules, in cows--pseudocowpox. The disease is mostly benign and this is a reason why it does not attract sufficient attention of health personnel. Nevertheless this infection can cause a certain economical loss.     

Chasing Jenner's vaccine: revisiting cowpox virus classification

Cowpox virus (CPXV) is described as the source of the first vaccine used to prevent the onset and spread of an infectious disease. It is one of the earliest described members of the genus Orthopoxvirus, which includes the viruses that cause smallpox and monkeypox in humans. Both the historic and current literature describe "cowpox" as a disease with a single etiologic agent. Genotypic data presented herein indicate that CPXV is not a single species, but a composite of several (up to 5) species that can infect cows, humans, and other animals. The practice of naming agents after the host in which the resultant disease manifests obfuscates the true taxonomic relationships of "cowpox" isolates. These data support the elevation of as many as four new species within the traditional "cowpox" group and suggest that both wild and modern vaccine strains of Vaccinia virus are most closely related to CPXV of continental Europe rather than the United Kingdom, the homeland of the vaccine.     

A Negative Feedback Modulator of Antigen Processing Evolved from a Frameshift in the Cowpox Virus Genome

Coevolution of viruses and their hosts represents a dynamic molecular battle between the immune system and viral factors that mediate immune evasion. After the abandonment of smallpox vaccination, cowpox virus infections are an emerging zoonotic health threat, especially for immunocompromised patients. Here we delineate the mechanistic basis of how cowpox viral CPXV012 interferes with MHC class I antigen processing. This type II membrane protein inhibits the coreTAP complex at the step after peptide binding and peptide-induced conformational change, in blocking ATP binding and hydrolysis. Distinct from other immune evasion mechanisms, TAP inhibition is mediated by a short ER-lumenal fragment of CPXV012, which results from a frameshift in the cowpox virus genome. Tethered to the ER membrane, this fragment mimics a high ER-lumenal peptide concentration, thus provoking a trans-inhibition of antigen translocation as supply for MHC I loading. These findings illuminate the evolution of viral immune modulators and the basis of a fine-balanced regulation of antigen processing.     

Studies on the polypeptides of poxvirus. I. Comparison of structural polypeptides in vaccina, cowpox and Shope fibroma viruses.

Radioactively labeled vaccinia, cowpox and Shope fibroma virions free from any detectable contamination with host cell protein, were dissociated into their constituent polypeptides, and these were then analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. The profiles of constituent polypeptide bands of four strains of vaccinia virus (IHD-W, IHD-J, Lister and DIs) were almost the same, except that a polypeptide of about 41,000 daltons was not detectable in the autoradiogram of strain IHD-W which has no hemagglutinin. The profile of polypeptide bands of cowpox virions was also almost the same as that of vaccinia virions, except for several polypeptides of about 40,000 to 50,000 daltons, but the profile of Shope fibroma virions differed considerably from that of vaccinia or cowpox virions.     

Identification of the orthopoxvirus p4c gene,which encodes a structural protein that directs intracellular mature virus particles into A-type inclusions

The orthopoxvirus gene p4c has been identified in the genome of the vaccinia virus strain Western Reserve. This gene encodes the 58-kDa structural protein P4c present on the surfaces of the intracellular mature virus (IMV) particles. The gene is disrupted in the genome of cowpox virus Brighton Red (BR), demonstrating that although the P4c protein may be advantageous for virus replication in vivo, it is not essential for virus replication in vitro. Complementation and recombination analyses with the p4c gene have shown that the P4c protein is required to direct the IMV into the A-type inclusions (ATIs) produced by cowpox virus BR. The p4c gene is highly conserved among most members of the orthopoxvirus genus, including viruses that produce ATIs, such as cowpox, ectromelia, and raccoonpox viruses, as well as those such as variola, monkeypox, vaccinia, and camelpox viruses, which do not. The conservation of the p4c gene among the orthopoxviruses, irrespective of their capacities to produce ATIs, suggests that the P4c protein provides functions in addition to that of directing IMV into ATIs. These findings, and the presence of the P4c protein in IMV but not extracellular enveloped virus (D. Ulaeto, D. Grosenbach, and D. E. Hruby, J. Virol. 70:3372-3377, 1996), suggest a model in which the P4c protein may play a role in the retrograde movement of IMV particles, thereby contributing to the retention of IMV particles within the cytoplasm and within ATIs when they are present. In this way, the P4c protein may affect both viral morphogenesis and processes of virus dissemination.     

The ps/hr gene (B5R open reading frame homolog) of rabbitpox virus controls pock color, is a component of extracellular enveloped virus, and is secreted into the medium.

Wild-type rabbitpox virus (RPV) produces red hemorrhagic pocks on the chorioallantoic membranes (CAMs) of embryonated chicken eggs. Like the crmA (SPI-2) gene of cowpox virus, disruption of the RPV ps/hr gene results in a mutant which produces white pocks on the CAMs. An examination of the properties of the RPV(ps/hr) mutant in cell culture also reveals a significantly reduced host range, defined as the inability to form plaques, compared with wild-type virus. One of several cell types on which RPV(ps/hr) mutants fail to produce plaques is chicken embryo fibroblasts, cells which have been traditionally used to propagate spontaneously arising white pock mutants isolated from CAMs. The inability of the RPV(ps/hr) mutant to form plaques in chicken embryo fibroblasts correlates with a failure of a low multiplicity of infection to spread to neighboring cells and to form extracellular enveloped virus (EEV), although the formation and yields of infectious intracellular naked virus appear relatively normal. The gene product of the ps/hr gene, initially synthesized as a 45-kDa glycoprotein, is found as a component of EEV, but not intracellular naked virus, and as a smaller, secreted soluble protein of 35 kDa. Production of the secreted 35-kDa protein was found to be independent of any viral morphogenesis, suggesting two distinct pathways for release of the ps/hr gene product from the cell, i.e., as a component of the EEV particle and as a separately secreted glycoprotein.     

Cowpox Virus Outbreak in Banded Mongooses (Mungos mungo) and Jaguarundis (Herpailurus yagouaroundi) with a Time-Delayed Infection to Humans

Background

Often described as an extremely rare zoonosis, cowpox virus (CPXV) infections are on the increase in Germany. CPXV is rodent-borne with a broad host range and contains the largest and most complete genome of all poxviruses, including parts with high homology to variola virus (smallpox). So far, most CPXV cases have occurred individually in unvaccinated animals and humans and were caused by genetically distinguishable virus strains.

Methodology/Principal Findings

Generalized CPXV infections in banded mongooses (Mungos mungo) and jaguarundis (Herpailurus yagouaroundi) at a Zoological Garden were observed with a prevalence of the affected animal group of 100% and a mortality of 30%. A subsequent serological investigation of other exotic animal species provided evidence of subclinical cases before the onset of the outbreak. Moreover, a time-delayed human cowpox virus infection caused by the identical virus strain occurred in a different geographical area indicating that handling/feeding food rats might be the common source of infection.

Conclusions/Significance

Reports on the increased zoonotic transmission of orthopoxviruses have renewed interest in understanding interactions between these viruses and their hosts. The list of animals known to be susceptible to CPXV is still growing. Thus, the likely existence of unknown CPXV hosts and their distribution may present a risk for other exotic animals but also for the general public, as was shown in this outbreak. Animal breeders and suppliers of food rats represent potential multipliers and distributors of CPXV, in the context of increasingly pan-European trading. Taking the cessation of vaccination against smallpox into account, this situation contributes to the increased incidence of CPXV infections in man, particularly in younger age groups, with more complicated courses of clinical infections.     

Genome-Wide Comparison of Cowpox Viruses Reveals a New Clade Related to Variola Virus

Zoonotic infections caused by several orthopoxviruses (OPV) like monkeypox virus or vaccinia virus have a significant impact on human health. In Europe, the number of diagnosed infections with cowpox viruses (CPXV) is increasing in animals as well as in humans. CPXV used to be enzootic in cattle; however, such infections were not being diagnosed over the last decades. Instead, individual cases of cowpox are being found in cats or exotic zoo animals that transmit the infection to humans. Both animals and humans reveal local exanthema on arms and legs or on the face. Although cowpox is generally regarded as a self-limiting disease, immunosuppressed patients can develop a lethal systemic disease resembling smallpox. To date, only limited information on the complex and, compared to other OPV, sparsely conserved CPXV genomes is available. Since CPXV displays the widest host range of all OPV known, it seems important to comprehend the genetic repertoire of CPXV which in turn may help elucidate specific mechanisms of CPXV pathogenesis and origin. Therefore, 22 genomes of independent CPXV strains from clinical cases, involving ten humans, four rats, two cats, two jaguarundis, one beaver, one elephant, one marah and one mongoose, were sequenced by using massive parallel pyrosequencing. The extensive phylogenetic analysis showed that the CPXV strains sequenced clearly cluster into several distinct clades, some of which are closely related to Vaccinia viruses while others represent different clades in a CPXV cluster. Particularly one CPXV clade is more closely related to Camelpox virus, Taterapox virus and Variola virus than to any other known OPV. These results support and extend recent data from other groups who postulate that CPXV does not form a monophyletic clade and should be divided into multiple lineages.     

The Ugi reaction in the generation of new nucleosides as potential antiviral and antileishmanial agents

5-Formyl-2'-deoxyuridine-3',5'-diacetate was converted to a small library of 5-substituted pyrimidine nucleoside N-acylamino acid amides by means of a Ugi multicomponent reaction. The reaction allowed introduction of various substituents at the acyl moiety, at the amino acid alpha-amide group, and at the amino acid carboxyl function. Evaluation of these novel 5-substituted nucleosides against vaccinia virus and cowpox virus provided one compound with discernable activity against cowpox virus but five- to eightfold less active than the Cidofovir standard. More promising activity was seen for the inhibition of Leishmania donovani promastigotes. Several synthetic products showed antileishmanial activity in the 10(-5)M range. When compared to earlier studies demonstrating anti-orthopoxviral and antileishmanial activity of 5-substituted pyrimidine nucleosides, these results imply that the 5-(N-acylamino acid amide)-derivatized pyrimidine nucleosides may possess more steric bulk, greater hydrophobicity, and more flexibility than is compatible with these particular biological activities.     

Inference of cowpox virus transmission rates between wild rodent host classes using space-time interaction

There have been virtually no studies of 'who acquires infection from whom' in wildlife populations, but patterns of transmission within and between different classes of host are likely to be reflected in the spatiotemporal distribution of infection among those host classes. Here, we use a modified form of K-function analysis to test for space-time interaction among bank voles and wood mice infectious with cowpox virus. There was no evidence for transmission between the two host species, supporting previous evidence that they act as separate reservoirs for cowpox. Among wood mice, results suggested that transmission took place primarily between individuals of the opposite sex, raising the possibility that cowpox is sexually transmitted in this species. Results for bank voles indicated that infected females might be a more important source of infection to either sex than are males. The suggestion of different modes of transmission in the two species is itself consistent with the apparent absence of transmission between species.