In Vivo Characterization of a Bank Vole-Derived Cowpox Virus Isolate in Natural Hosts and the Rat Model.

Cowpox virus (CPXV) belongs to the genus Orthopoxvirus in the Poxviridae family and is endemic in western Eurasia. Based on seroprevalence studies in different voles from continental Europe and UK, voles are suspected to be the major reservoir host. Recently, a CPXV was isolated from a bank vole (Myodes glareolus) in Germany that showed a high genetic similarity to another isolate originating from a Cotton-top tamarin (Saguinus oedipus). Here we characterize this first bank vole-derived CPXV isolate in comparison to the related tamarin-derived isolate. Both isolates grouped genetically within the provisionally called CPXV-like 3 clade. Previous phylogenetic analysis indicated that CPXV is polyphyletic and CPXV-like 3 clade represents probably a different species if categorized by the rules used for other orthopoxviruses. Experimental infection studies with bank voles, common voles (Microtusarvalis) and Wistar rats showed very clear differences. The bank vole isolate was avirulent in both common voles and Wistar rats with seroconversion seen only in the rats. In contrast, inoculated bank voles exhibited viral shedding and seroconversion for both tested CPXV isolates. In addition, bank voles infected with the tamarin-derived isolate experienced a marked weight loss. Our findings allow for the conclusion that CPXV isolates might differ in their replication capacity in different vole species and rats depending on their original host. Moreover, the results indicate host-specific differences concerning CPXV-specific virulence. Further experiments are needed to identify individual virulence and host factors involved in the susceptibility and outcome of CPXV-infections in the different reservoir hosts.

Atypical Cowpox Virus Infection in Smallpox-Vaccinated Patient, France.

We report a case of atypical cowpox virus infection in France in 2016. The patient sought care for thoracic lesions after injury from the sharp end of a metallic guardrail previously stored in the ground. We isolated a cowpox virus from the lesions and sequenced its whole genome. The patient reported that he had been previously vaccinated against smallpox. We describe an alternative route of cowpox virus infection and raise questions about the immunological status of smallpox-vaccinated patients for circulating orthopoxviruses.

Revisiting Jenner's mysteries, the role of the Beaugency lymph in the evolutionary path of ancient smallpox vaccines.

In 1796, Edward Jenner developed the smallpox vaccine consisting of pustular material obtained from lesions on cows affected by so-called cow-pox. The disease, caused by cowpox virus, confers crossprotection against smallpox. However, historical evidence suggests that Jenner might have used vaccinia virus or even horsepox virus instead of cowpox virus. Mysteries surrounding the origin and nature of the smallpox vaccine persisted during the 19th century, a period of intense exchange of vaccine strains, including the Beaugency lymph. This lymph was obtained from spontaneous cases of cow-pox in France in 1866 and then distributed worldwide. A detailed Historical Review of the distribution of the Beaugency lymph supports recent genetic analyses of extant vaccine strains, suggesting the lymph was probably a vaccinia strain or a horsepox-like virus. This Review is a historical investigation that revisits the mysteries of the smallpox vaccine and reveals an intricate evolutionary relationship of extant vaccinia strains.

Classification of Cowpox Viruses into Several Distinct Clades and Identification of a Novel Lineage.

Cowpox virus (CPXV) was considered as uniform species within the genus Orthopoxvirus (OPV). Previous phylogenetic analysis indicated that CPXV is polyphyletic and isolates may cluster into different clades with two of these clades showing genetic similarities to either variola (VARV) or vaccinia viruses (VACV). Further analyses were initiated to assess both the genetic diversity and the evolutionary background of circulating CPXVs. Here we report the full-length sequences of 20 CPXV strains isolated from different animal species and humans in Germany. A phylogenetic analysis of altogether 83 full-length OPV genomes confirmed the polyphyletic character of the species CPXV and suggested at least four different clades. The German isolates from this study mainly clustered into two CPXV-like clades, and VARV- and VACV-like strains were not observed. A single strain, isolated from a cotton-top tamarin, clustered distantly from all other CPXVs and might represent a novel and unique evolutionary lineage. The classification of CPXV strains into clades roughly followed their geographic origin, with the highest clade diversity so far observed for Germany. Furthermore, we found evidence for recombination between OPV clades without significant disruption of the observed clustering. In conclusion, this analysis markedly expands the number of available CPXV full-length sequences and confirms the co-circulation of several CPXV clades in Germany, and provides the first data about a new evolutionary CPXV lineage.

Cowpox virus: What's in a Name?

Traditionally, virus taxonomy relied on phenotypic properties; however, a sequence-based virus taxonomy has become essential since the recent requirement of a species to exhibit monophyly. The species Cowpox virus has failed to meet this requirement, necessitating a reexamination of this species. Here, we report the genomic sequences of nine Cowpox viruses and, by combining them with the available data of 37 additional genomes, confirm polyphyly of Cowpox viruses and find statistical support based on genetic data for more than a dozen species. These results are discussed in light of the current International Committee on Taxonomy of Viruses species definition, as well as immediate and future implications for poxvirus taxonomic classification schemes. Data support the recognition of five monophyletic clades of Cowpox viruses as valid species.

Cowpox in a human, Russia, 2015.

We investigated the first laboratory-confirmed human case of cowpox virus infection in Russia since 1991. Phylogenetic studies of haemagglutinin, TNF-α receptor-like protein and thymidine kinase regions showed significant differences with known orthopoxviruses, including unique amino-acid substitutions and deletions. The described cowpox virus strain, taking into account differences, is genetically closely related to strains isolated years ago in the same geographical region (European part of Russia and Finland), which suggests circulation of viral strains with common origin in wild rodents without spread over long distances and appearance in other parts of the world.

Buffalopox.

Buffalopox is a contagious viral disease affecting milch buffaloes (Bubalus Bubalis) and, rarely, cows. The disease has zoonotic implications, as outbreaks are frequently associated with human infections, particularly in the milkers. Buffalopox is associated with high morbidity (80%). The clinical symptoms of the disease are characterized by wartline lesions on the udder, teats, inguinal region, base of the ears, and over the parotid. In the severe form, generalized rash is observed. Although the disease does not lead to high mortality, it has an adverse effect on the productivity and working capacity of the animals resulting in large economic losses. The outbreaks of buffalopox occurred frequently in India, Pakistan, Bangladesh, Nepal, Iran, Egypt, and Indonesia, where buffaloes are reared as milch animals. The buffalopox is closely related with other Orthopoxviruses. In particular, it is close to the vaccinia virus. There is a view that the buffalopox virus might be derived from the vaccinia virus. It is possible that it became pathogenic to humans and animals through adaptive evolution of the genome by obtaining the virulence genes. PCR is performed for the C18L gene for the purpose of specific detection and differentiation of the buffalopox virus from other orthopoxviruses. The C18L gene encodes the ankyrin repeat protein, which determines the virus host range. The open reading frame of this gene is only 150-nucleotide long as against 453 nucleotide in the vaccinia virus, 756 - in the camelpox virus, and 759 - in the cowpox virus. It can be concluded that a systematic study based on the epidemiology of the virus, existence of reservoirs, biological transmission, and the molecular organization of the buffalopox virus from buffalo, cow, and humans may pave the way to a better understanding of the circulating virus and contribute to the control of the disease using the suitable diagnostic and prophylactic measures.

Pipasic: similarity and expression correction for strain-level identification and quantification in metaproteomics.

MOTIVATION: Metaproteomic analysis allows studying the interplay of organisms or functional groups and has become increasingly popular also for diagnostic purposes. However, difficulties arise owing to the high sequence similarity between related organisms. Further, the state of conservation of proteins between species can be correlated with their expression level, which can lead to significant bias in results and interpretation. These challenges are similar but not identical to the challenges arising in the analysis of metagenomic samples and require specific solutions. RESULTS: We introduce Pipasic (peptide intensity-weighted proteome abundance similarity correction) as a tool that corrects identification and spectral counting-based quantification results using peptide similarity estimation and expression level weighting within a non-negative lasso framework. Pipasic has distinct advantages over approaches only regarding unique peptides or aggregating results to the lowest common ancestor, as demonstrated on examples of viral diagnostics and an acid mine drainage dataset. AVAILABILITY AND IMPLEMENTATION: Pipasic source code is freely available from https://sourceforge.net/projects/pipasic/. CONTACT: RenardB@rki.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Molecular characterization and phylogenetics of Fennoscandian cowpox virus isolates based on the p4c and atip genes.

BACKGROUND: Cowpox virus (CPXV), a rodent-borne Orthopoxvirus (OPV) that is indigenous to Eurasia can infect humans, cattle, felidae and other animals. Molecular characterization of CPXVs isolated from different geographic locations is important for the understanding of their biology, geographic distribution, classification and evolution. Our aim was to characterize CPXVs isolated from Fennoscandia on the basis of A-type inclusion (ATI) phenotype, restriction fragment length polymorphism (RFLP) profiles of atip gene fragment amplicon, and phylogenetic tree topology in conjunction with the patristic and genetic distances based on full length DNA sequence of the atip and p4c genes. METHODS: ATI phenotypes were determined by transmission electron microcopy and RFLP profiles were obtained by restriction enzyme digestion of the atip gene fragment PCR product. A 6.2 kbp region spanning the entire atip and p4c genes of Fennoscandian CPXV isolates was amplified and sequenced. The phylogenetic affinity of Fennoscandian CPXV isolates to OPVs isolated from other geographic regions was determined on the basis of the atip and p4c genes. RESULTS: Fennoscandian CPXV isolates encoded full length atip and p4c genes. They produce wild type V+ ATI except for CPXV-No-H2. CPXVs were resolved into six and seven species clusters based on the phylogeny of the atip and p4c genes respectively. The CPXVs isolated from Fennoscandia were grouped into three distinct clusters that corresponded to isolates from Norway, Sweden and Finland. CONCLUSION: CPXV is a polyphyletic assemblage of six or seven distinct clusters and the current classification in which CPXVs are united as one single species should be re-considered. Our results are of significance to the classification and evolution of OPVs.

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.

Concomitant human infections with 2 cowpox virus strains in related cases, France, 2011.

We investigated 4 related human cases of cowpox virus infection reported in France during 2011. Three patients were infected by the same strain, probably transmitted by imported pet rats, and the fourth patient was infected by another strain. The 2 strains were genetically related to viruses previously isolated from humans with cowpox infection in Europe.

Cowpox virus infection of cynomolgus macaques as a model of hemorrhagic smallpox.

Hemorrhagic smallpox was a rare but severe manifestation of variola virus infection that resulted in nearly 100% mortality. Here we describe intravenous (IV) inoculation of cowpox virus Brighton Red strain in cynomolgus macaques (Macaca fascicularis) which resulted in disease similar in presentation to hemorrhagic smallpox in humans. IV inoculation of macaques resulted in a uniformly lethal disease within 12 days post-inoculation in two independent experiments. Clinical observations and hematological and histopathological findings support hemorrhagic disease. Cowpox virus replicated to high levels in blood (8.0-9.0 log(10) gene copies/mL) and tissues including lymph nodes, thymus, spleen, bone marrow, and lungs. This unique model of hemorrhagic orthopoxvirus infection provides an accessible means to further study orthopoxvirus pathogenesis and to identify virus-specific and nonspecific therapies. Such studies will serve to complement the existing nonhuman primate models of more classical poxviral disease.

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.

Development of real-time PCR assay for specific detection of cowpox virus.

BACKGROUND: The number of recorded human cowpox cases are recently increasing. The symptoms caused by cowpox virus (CPXV) in a number of human cases are close to the symptoms characteristic of the orthopoxviral human infections caused by monkeypox or smallpox (variola) viruses. Any rapid and reliable real-time PCR method for distinguishing cowpox from smallpox and monkeypox is yet absent. OBJECTIVES: The aim of this study was to develop a quick and reliable real-time TaqMan PCR assay for specific detection of cowpox virus and to determine the sensitivity and specificity of this method. STUDY DESIGN: Based on aligned nucleotide sequences of orthopoxviruses, we found a virus-specific region in the CPXV genome and selected the oligonucleotide primers and hybridization probe within this region. The specificity of the developed method was tested using a panel of various orthopoxvirus (OPV) DNAs. The sensitivity was determined using the recombinant plasmid carrying a fragment of CPXV DNA and genomic DNA of the CPXV strain GRI-90. RESULTS: The analytical specificity of this method was determined using DNAs of 17 strains of four OPV species pathogenic for humans and amounted to 100%. The method allows 6 copies of plasmid DNA and 20 copies of CPXV DNA in the reaction mixture to be detected. CONCLUSION: A quick and reliable TaqMan PCR assay providing for a highly sensitive and specific detection of CPXV DNA was developed.

Comparison and phylogenetic analysis of cowpox viruses isolated from cats and humans in Fennoscandia.

Cowpox virus (CPXV), a member of the genus Orthopoxvirus (OPV), has reservoirs in small mammals and may cause disease in humans, felidae and other animals. In this study we compared CPXVs isolated from humans and cats in Fennoscandia by restriction enzyme and DNA sequence analysis. The HindIII restriction profiles clearly distinguished geographically distinct CPXV isolates, whereas only minor differences were found between the profiles of geographically linked isolates. The complete gene sequences encoding the cytokine response modifier B, the hemagglutinin and the Chinese hamster ovary host range protein were determined for the same isolates and included in phylogenetic analysis. By including representative OPV sequences from GenBank, detailed comparative analyses were performed showing pronounced heterogeneity among CPXVs compared to members of other OPV species. However, a close relationship between the Norwegian (3 of 4 isolates) and Swedish isolates was detected, whereas the isolate from Finland was more closely related to a Russian isolate for all three genes compared. We infer that the investigated CPXVs have distinct evolutionary histories in different rodent lineages.

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.

Smallpox virus resequencing GeneChips can also rapidly ascertain species status for some zoonotic non-variola orthopoxviruses.

We recently developed a set of seven resequencing GeneChips for the rapid sequencing of Variola virus strains in the WHO Repository of the Centers for Disease Control and Prevention. In this study, we attempted to hybridize these GeneChips with some known non-Variola orthopoxvirus isolates, including monkeypox, cowpox, and vaccinia viruses, for rapid detection.