Cowpox Research Articles

What a Difference a Gene Makes: Identification of Virulence Factors of Cowpox Virus.

Cowpox virus (CPXV) is a zoonotic orthopoxvirus (OPV) that causes spillover infections from its animal hosts to humans. In 2009, several human CPXV cases occurred through transmission from pet rats. An isolate from a diseased rat, RatPox09, exhibited significantly increased virulence in Wistar rats and caused high mortality compared to that caused by the mildly virulent laboratory strain Brighton Red (BR). The RatPox09 genome encodes four genes which are absent in the BR genome. We hypothesized that their gene products could be major factors influencing the high virulence of RatPox09. To address this hypothesis, we employed several BR-RatPox09 chimeric viruses. Using Red-mediated mutagenesis, we generated BR-based knock-in mutants with single or multiple insertions of the respective RatPox09 genes. High-throughput sequencing was used to verify the genomic integrity of all recombinant viruses, and transcriptomic analyses confirmed that the expression profiles of the genes that were adjacent to the modified ones were unaltered. While the in vitro growth kinetics were comparable to those of BR and RatPox09, we discovered that a knock-in BR mutant containing the four RatPox09-specific genes was as virulent as the RatPox09 isolate, causing death in over 75% of infected Wistar rats. Unexpectedly, the insertion of gCPXV0030 (g7tGP) alone into the BR genome resulted in significantly higher clinical scores and lower survival rates matching the rate for rats infected with RatPox09. The insertion of gCPXV0284, encoding the BTB (broad-complex, tramtrack, and bric-à-brac) domain protein D7L, also increased the virulence of BR, while the other two open reading frames failed to rescue virulence independently. In summary, our results confirmed our hypothesis that a relatively small set of four genes can contribute significantly to CPXV virulence in the natural rat animal model.IMPORTANCE With the cessation of vaccination against smallpox and its assumed cross-protectivity against other OPV infections, waning immunity could open up new niches for related poxviruses. Therefore, the identification of virulence mechanisms in CPXV is of general interest. Here, we aimed to identify virulence markers in an experimental rodent CPXV infection model using bacterial artificial chromosome (BAC)-based virus recombineering. We focused our work on the recent zoonotic CPXV isolate RatPox09, which is highly pathogenic in Wistar rats, unlike the avirulent BR reference strain. In several animal studies, we were able to identify a novel set of CPXV virulence genes. Two of the identified virulence genes, encoding a putative BTB/POZ protein (CPXVD7L) and a B22R-family protein (CPXV7tGP), respectively, have not yet been described to be involved in CPXV virulence. Our results also show that single genes can significantly affect virulence, thus facilitating adaptation to other hosts.

New chemical agents based on adamantane-monoterpene conjugates against orthopoxvirus infections.

Currently, the spectrum of agents against orthopoxviruses, in particular smallpox, is very narrow. Despite the fact that smallpox is well controlled, there is, for many reasons, a real threat of epidemics associated with this or a similar virus. In order to search for new low molecular weight orthopoxvirus inhibitors, a series of amides combining adamantane and monoterpene moieties were synthesized using 1- and 2-adamantanecarboxylic acids as well as myrtenic, citronellic and camphorsulfonic acids as acid components. The produced compounds exhibited high activity against the vaccinia virus (an enveloped virus belonging to the poxvirus family), which was combined with low cytotoxicity. Some compounds had a selectivity index higher than that of the reference drug cidofovir; the highest SI = 1123 was exhibited by 1-adamantanecarboxylic acid amide containing the (-)-10-amino-2-pinene moiety. The produced compounds demonstrated inhibitory activity against other orthopoxviruses: cowpox virus (SI = 30-406) and ectromelia virus (mousepox virus, SI = 39-707).

Investigation of the Antiviral Activity of Experimental Samples Obtained from the Grass and Roots of Alchemilla vulgaris L. Against Vaccinia Virus and Ectromelia Virus

Introduction. The abolition of smallpox vaccination after its elimination in 1980 led to a decrease in the immunocompromised immunity in humans. Zoonotic monkeypox, camelpox, buffalopox and cowpox viruses that are close to the variola virus also pose a danger to humans. In Russia today there are no effective and safe medicines for the prevention and treatment of smallpox and other orthopoxvirus infections in humans and animals. The Lady's mantle (Alchemilla vulgaris) is a promising source for the development of new antiviral drugs. Previous studies have found that Alchemilla vulgaris shows activity against influenza virus and herpes simplex virus.Aim. The aim of this work was to study the chemical composition and antiviral activity of extracts from the roots and the grass of Alchemilla vulgaris against orthopoxviruses.Materials and methods. Qualitative analysis of the samples was performed by high performance liquid chromatography. Quantitative analysis was performed using a complex of spectrophotometric methods. To determine the toxicity and antiviral activity of experimental samples from Alchemilla vulgaris in vitro, a transplantable Vero cell culture was used. Antiviral activity of the obtained preparations was evaluated by reducing the infectivity (titer) of orthopoxviruses in the monolayer of Vero cells infected with orthopoxviruses in the presence of preparations with different concentrations relative to the cell culture infected with orthopoxviruses without the preparations.Results and discussion. It was shown that the experimental sample from Alchemilla vulgaris obtained by the method of ethyl acetate extraction from the roots of the studied plant and purified with chloroform contains mainly catechins and leucoanthocyanins (70 %). In parallel, extract from the raw mass of the grass of the plant purified with chloroform and ethanol contains the amount of flavonoids (71 %). Wherein the content of flavonoids in unpurified ethanol extracts from the roots and the grass of Alchemilla vulgaris composed 5 % and 6 %, respectively. It was revealed that purified preparations obtained from Alchemilla vulgaris roots when using ethyl acetate and ethanol as extractants showed antiviral activity against vaccinia virus and ectromelia virus, as well as preparations obtained from grass by ethanol extraction.Conclusion. Thus, purified ethyl acetate extracts from the roots and ethanol extracts from the wet grass mass of Alchemilla vulgaris exhibit antiviral activity against orthopoxviruses in vitro.

Molecular Detection and Characterization of the First Cowpox Virus Isolate Derived from a Bank Vole.

Cowpox virus (CPXV) is a zoonotic orthopoxvirus (OPV) that infects a wide range of mammals. CPXV-specific DNA and antibodies were detected in different vole species, such as common voles (Microtus arvalis) and bank voles (Myodes glareolus). Therefore, voles are the putative main reservoir host of CPXV. However, CPXV was up to now only isolated from common voles. Here we report the detection and isolation of a bank vole-derived CPXV strain (GerMygEK 938/17) resulting from a large-scale screening of bank voles collected in Thuringia, Germany, during 2017 and 2018. Phylogenetic analysis using the complete viral genome sequence indicated a high similarity of the novel strain to CPXV clade 3 and to OPV “Abatino” but also to Ectromelia virus (ECTV) strains. Phenotypic characterization of CPXV GerMygEK 938/17 using inoculation of embryonated chicken eggs displayed hemorrhagic pock lesions on the chorioallantoic membrane that are typical for CPXV but not for ECTV. CPXV GerMygEK 938/17 replicated in vole-derived kidney cell lines but at lower level than on Vero76 cell line. In conclusion, the first bank vole-derived CPXV isolate provides new insights into the genetic variability of CPXV in the putative reservoir host and is a valuable tool for further studies about CPXV-host interaction and molecular evolution of OPV.

Antibiotics: From the Beginning to the Future: Part 1.

The first written record of intervention against what later came to be known as an infectious disease was in the early seventeenth century by a Buddhist nun. She dried 3 to 4 wk old scabs from patients with mild smallpox and asked well people to inhale the powder. More than a century later in 1796, Edward Jenner described vaccination against smallpox by using cowpox that later was found to be caused by cowpox virus which is non-pathogenic for humans. Another century later in 1890, Robert Koch published the Koch's Postulates allowing the study of pathogenic bacteria and subsequently the study of agents to fight them. The first chemical cure for disease was reported by Paul Erhlich in 1909 in the form of an arsenic compound to treat syphilis. One hundred and ten years since then a lot has happened in the area of preventing and treating infectious diseases with significant contribution to increase in human lifespan. This is the only area of medicine in which treatment (antimicrobial agent) is used to eradicate a replicating biological agent inside the human host. The potential of this second biological agent to mutate under the selection pressure of antibiotics making them resistant was recognized in the 1940s. But the indiscriminate use of antibiotics for over 70 y has led to the present crisis of resistance in major pathogens with increased morbidity and mortality. In this review, we have incorporated all the possible avenues that might be useful in the future. However, none is more important than relearning the judicious use of antibiotics based on microbiology, pharmacology, and genetics.

Cowpox virus encodes a protein that binds B7.1 and B7.2 and subverts T cell costimulation

Costimulation is required for optimal T cell activation, yet it is unclear whether poxviruses dedicatedly subvert costimulation during infection. Here, we report that the secreted M2 protein encoded by cowpox virus (CPXV) specifically interacts with human and murine B7.1 (CD80) and B7.2 (CD86). We also show that M2 competes with CD28 and CTLA4 for binding to cell surface B7 ligands, with stronger efficacy against CD28. Functionally, recombinant M2 and culture supernatants from wild-type (WT) but not M2-deficient (∆M2) CPXV-infected cells can potently suppress B7 ligand-mediated T cell proliferation and interleukin-2 (IL-2) production. Furthermore, we observed increased antiviral CD4 and CD8 T cell responses in C57BL/6 mice challenged by ∆M2 CPXV compared with WT virus. These differences in immune responses to ∆M2 and WT CPXV were not observed in CD28-deficient mice. Taken together, our findings define a mechanism of viral sabotage of T cell activation that highlights the role of CD28 costimulation in host defense against poxvirus infections.

Common vole (Microtus arvalis) and bank vole (Myodes glareolus) derived permanent cell lines differ in their susceptibility and replication kinetics of animal and zoonotic viruses

Pathogenesis and reservoir host adaptation of animal and zoonotic viruses are poorly understood due to missing adequate cell culture and animal models. The bank vole (Myodes glareolus) and common vole (Microtus arvalis) serve as hosts for a variety of zoonotic pathogens. For a better understanding of virus association to a putative animal host, we generated two novel cell lines from bank voles of different evolutionary lineages and two common vole cell lines and assayed their susceptibility, replication and cytopathogenic effect (CPE) formation for rodent-borne, suspected to be rodent-associated or viruses with no obvious rodent association. Already established bank vole cell line BVK168, used as control, was susceptible to almost all viruses tested and efficiently produced infectious virus for almost all of them. The Puumala orthohantavirus strain Vranica/Hällnäs showed efficient replication in a new bank vole kidney cell line, but not in the other four bank and common vole cell lines. Tula orthohantavirus replicated in the kidney cell line of common voles, but was hampered in its replication in the other cell lines. Several zoonotic viruses, such as Cowpox virus, Vaccinia virus, Rift Valley fever virus, and Encephalomyocarditis virus 1 replicated in all cell lines with CPE formation. West Nile virus, Usutu virus, Sindbis virus and Tick-borne encephalitis virus replicated only in a part of the cell lines, perhaps indicating cell line specific factors involved in replication. Rodent specific viruses differed in their replication potential: Murine gammaherpesvirus-68 replicated in the four tested vole cell lines, whereas murine norovirus failed to infect almost all cell lines. Schmallenberg virus and Foot-and-mouth disease virus replicated in some of the cell lines, although these viruses have never been associated to rodents. In conclusion, these newly developed cell lines may represent useful tools to study virus-cell interactions and to identify and characterize host cell factors involved in replication of rodent associated viruses.

Zoonotic Virus Seroprevalence among Bank Voles, Poland, 2002-2010.

Bank voles in Poland are reservoirs of zoonotic viruses. To determine seroprevalence of hantavirus, arenavirus, and cowpox virus and factors affecting seroprevalence, we screened for antibodies against these viruses over 9 years. Cowpox virus was most prevalent and affected by extrinsic and intrinsic factors. Long-term and multisite surveillance is crucial.

Immunogenicity of recombinant fragment of orthopoxvirus p35 protein in mice

Despite the elimination of smallpox, orthopoxviruses continue to be a source of biological danger for humans, as cowpox and monkey pox viruses circulate in nature and the last virus can cause both sporadic cases of human diseases and outbreaks of smallpox-like infection. In addition, periodic vaccination is necessary for representatives of some professions (scientists studying pathogenic orthopoxviruses, medical personnel, etc.). Vaccination against smallpox virus with live vaccinia virus, which was widely used during the elimination of smallpox, induces the formation of long-term immunity in vaccinated people. However, providing a high level of protection, the vaccination is often accompanied by serious post-vaccination complications, the probability of which is particularly great for individuals with compromised immunity. In this regard, the development of preparations for the prevention and treatment of infections caused by orthopoxviruses remains important today. The aim of this study was to assess the immunogenicity in the mouse model of recombinant protein р35Δ12, designed previously on the base of the cowpox virus protein p35. It was previously shown that the protein р35Δ12 was recognized by fully human neutralizing anti-orthopoxviral antibody with high affinity. In this work, recombinant protein р35Δ12 produced in E. coli cells XL1-blue and purified by chromatography was used for two-time immunization of mice. Two weeks after the second immunization, blood samples were taken from mice and serum antibodies were analyzed. It was shown by ELISA and Western-blot analysis that immunized mice sera contained IgG antibodies specific to recombinant protein р35Δ12. Confocal microscopy showed that antibodies induced by the р35Δ12 protein were able to recognize Vero E6 cells infected with the LIVP-GFP vaccinia virus. In addition, the antibodies in the serum of immunized mice were able to neutralize the infectivity of the vaccinia virus LIVP-GFP in the plaque reduction neutralization test in vitro. These experiments have demonstrated promising properties of the р35Δ12 protein if it were used as a component of vaccine for prophylaxis of orthopoxvirus infections.

A Genome-Wide Haploid Genetic Screen Identifies Heparan Sulfate-Associated Genes and the Macropinocytosis Modulator TMED10 as Factors Supporting Vaccinia Virus Infection.

Vaccinia virus is a promising viral vaccine and gene delivery candidate and has historically been used as a model to study poxvirus-host cell interactions. We employed a genome-wide insertional mutagenesis approach in human haploid cells to identify host factors crucial for vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to modified vaccinia virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein trafficking. We validated EXT1, TM9SF2, and TMED10 (TMP21/p23/p24δ) as important host factors for vaccinia virus infection. The critical roles of EXT1 in heparan sulfate synthesis and vaccinia virus infection were confirmed. TM9SF2 was validated as a player mediating heparan sulfate expression, explaining its contribution to vaccinia virus infection. In addition, TMED10 was found to be crucial for virus-induced plasma membrane blebbing and phosphatidylserine-induced macropinocytosis, presumably by regulating the cell surface expression of the TAM receptor Axl.IMPORTANCE Poxviruses are large DNA viruses that can infect a wide range of host species. A number of these viruses are clinically important to humans, including variola virus (smallpox) and vaccinia virus. Since the eradication of smallpox, zoonotic infections with monkeypox virus and cowpox virus are emerging. Additionally, poxviruses can be engineered to specifically target cancer cells and are used as a vaccine vector against tuberculosis, influenza, and coronaviruses. Poxviruses rely on host factors for most stages of their life cycle, including attachment to the cell and entry. These host factors are crucial for virus infectivity and host cell tropism. We used a genome-wide knockout library of host cells to identify host factors necessary for vaccinia virus infection. We confirm a dominant role for heparin sulfate in mediating virus attachment. Additionally, we show that TMED10, previously not implicated in virus infections, facilitates virus uptake by modulating the cellular response to phosphatidylserine.

Identification of CP77 as the Third Orthopoxvirus SAMD9 and SAMD9L Inhibitor with Unique Specificity for a Rodent SAMD9L.

Orthopoxviruses (OPXVs) have a broad host range in mammalian cells, but Chinese hamster ovary (CHO) cells are nonpermissive for vaccinia virus (VACV). Here, we revealed a species-specific difference in host restriction factor SAMD9L as the cause for the restriction and identified orthopoxvirus CP77 as a unique inhibitor capable of antagonizing Chinese hamster SAMD9L (chSAMD9L). Two known VACV inhibitors of SAMD9 and SAMD9L (SAMD9&L), K1 and C7, can bind human and mouse SAMD9&L, but neither can bind chSAMD9L. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 knockout of chSAMD9L from CHO cells removed the restriction for VACV, while ectopic expression of chSAMD9L imposed the restriction for VACV in a human cell line, demonstrating that chSAMD9L is a potent restriction factor for VACV. In contrast to K1 and C7, cowpox virus CP77 can bind chSAMD9L and rescue VACV replication in cells expressing chSAMD9L, indicating that CP77 is yet another SAMD9L inhibitor but has a unique specificity for chSAMD9L. Binding studies showed that the N-terminal 382 amino acids of CP77 were sufficient for binding chSAMD9L and that both K1 and CP77 target a common internal region of SAMD9L. Growth studies with nearly all OPXV species showed that the ability of OPXVs to antagonize chSAMD9L correlates with CP77 gene status and that a functional CP77 ortholog was maintained in many OPXVs, including monkeypox virus. Our data suggest that a species-specific difference in rodent SAMD9L poses a barrier for cross-species OPXV infection and that OPXVs have evolved three SAMD9&L inhibitors with different specificities to overcome this barrier.IMPORTANCE Several OPXV species, including monkeypox virus and cowpox virus, cause zoonotic infection in humans. They are believed to use wild rodents as the reservoir or intermediate hosts, but the host or viral factors that are important for OPXV host range in rodents are unknown. Here, we showed that the abortive replication of several OPXV species in a Chinese hamster cell line was caused by a species-specific difference in the host antiviral factor SAMD9L, suggesting that SAMD9L divergence in different rodent species poses a barrier for cross-species OPXV infection. While the Chinese hamster SAMD9L could not be inhibited by two previously identified OPXV inhibitors of human and mouse SAMD9&L, it can be inhibited by cowpox virus CP77, indicating that OPXVs encode three SAMD9&L inhibitors with different specificities. Our data suggest that OPXV host range in broad rodent species depends on three SAMD9&L inhibitors with different specificities.

CrmA orthologs from diverse poxviruses potently inhibit caspases-1 and -8, yet cleavage site mutagenesis frequently produces caspase-1-specific variants.

Poxviruses encode many proteins that enable them to evade host anti-viral defense mechanisms. Spi-2 proteins, including Cowpox virus CrmA, suppress anti-viral immune responses and contribute to poxviral pathogenesis and lethality. These proteins are 'serpin' protease inhibitors, which function via a pseudosubstrate mechanism involving initial interactions between the protease and a cleavage site within the serpin. A conformational change within the serpin interrupts the cleavage reaction, deforming the protease active site and preventing dissociation. Spi-2 proteins like CrmA potently inhibit caspases-1, -4 and -5, which produce proinflammatory cytokines, and caspase-8, which facilitates cytotoxic lymphocyte-mediated target cell death. It is not clear whether both of these functions are equally perilous for the virus, or whether only one must be suppressed for poxviral infectivity and spread but the other is coincidently inhibited merely because these caspases are biochemically similar. We compared the caspase specificity of CrmA to three orthologs from orthopoxviruses and four from more distant chordopoxviruses. All potently blocked caspases-1, -4, -5 and -8 activity but exhibited negligible inhibition of caspases-2, -3 and -6. The orthologs differed markedly in their propensity to inhibit non-mammalian caspases. We determined the specificity of CrmA mutants bearing various residues in positions P4, P3 and P2 of the cleavage site. Almost all variants retained the ability to inhibit caspase-1, but many lacked caspase-8 inhibitory activity. The retention of Spi-2 proteins' caspase-8 specificity during chordopoxvirus evolution, despite this function being readily lost through cleavage site mutagenesis, suggests that caspase-8 inhibition is crucial for poxviral pathogenesis and spread.

Infections humaines à poxvirus

Les infections à poxvirus (PXV) sont fréquemment responsables de manifestations cutanées. En France, certaines poxviroses sont fréquentes et bénignes (molluscum contagiosum), alors que d’autres sont exceptionnelles mais potentiellement graves (cowpoxvirus [CPXV]). Si seuls le virus de la variole et le molluscum contagiosum virus sont à réservoir humain et à transmission interhumaine, la plupart des infections à poxvirus sont des zoonoses avec un réservoir animal distinct. Seule une partie des poxvirus est responsable d’infections humaines, mais l’augmentation du nombre des nouveaux animaux de compagnie, parfois exotiques, et la rapidité des voyages internationaux exposent au risque de transmission de PXV zoonotiques à de nouveaux vecteurs et leur diffusion, à de nouvelles régions du monde. En France, le molluscum contagiosum, l’orf et le nodule du trayeur sont responsables de multiples consultations et sont bien connus des dermatologues. Toutefois d’autres parapoxvirus de présentations proches de l’orf ne doivent pas être méconnus ; ainsi le CPXV ou le monkeypox virus sont considérés comme des virus potentiellement émergents avec un fort risque d’épidémie et de diffusion du fait de l’exacerbation des transports internationaux et de la perte de la protection vaccinale antivariolique. Enfin, malgré une éradication déclarée, la variole fait l’objet d’une surveillance du fait d’un risque potentiel de réintroduction accidentelle ou volontaire par le bioterrorisme.

RNAi-based small molecule repositioning reveals clinically approved urea-based kinase inhibitors as broadly active antivirals.

Influenza viruses (IVs) tend to rapidly develop resistance to virus-directed vaccines and common antivirals targeting pathogen determinants, but novel host-directed approaches might preclude resistance development. To identify the most promising cellular targets for a host-directed approach against influenza, we performed a comparative small interfering RNA (siRNA) loss-of-function screen of IV replication in A549 cells. Analysis of four different IV strains including a highly pathogenic avian H5N1 strain, an influenza B virus (IBV) and two human influenza A viruses (IAVs) revealed 133 genes required by all four IV strains. According to gene enrichment analyses, these strain-independent host genes were particularly enriched for nucleocytoplasmic trafficking. In addition, 360 strain-specific genes were identified with distinct patterns of usage for IAVs versus IBV and human versus avian IVs. The strain-independent host genes served to define 43 experimental and otherwise clinically approved drugs, targeting reportedly fourteen of the encoded host factors. Amongst the approved drugs, the urea-based kinase inhibitors (UBKIs) regorafenib and sorafenib exhibited a superior therapeutic window of high IV antiviral activity and low cytotoxicity. Both UBKIs appeared to block a cell signaling pathway involved in IV replication after internalization, yet prior to vRNP uncoating. Interestingly, both compounds were active also against unrelated viruses including cowpox virus (CPXV), hantavirus (HTV), herpes simplex virus 1 (HSV1) and vesicular stomatitis virus (VSV) and showed antiviral efficacy in human primary respiratory cells. An in vitro resistance development analysis for regorafenib failed to detect IV resistance development against this drug. Taken together, the otherwise clinically approved UBKIs regorafenib and sorafenib possess high and broad-spectrum antiviral activity along with substantial robustness against resistance development and thus constitute attractive host-directed drug candidates against a range of viral infections including influenza.

Use of Next Generation Sequencing to study two cowpox virus outbreaks.

BackgroundBetween 2008 and 2011 about 40 cases of human cowpox were reported from Germany and France. Infections had been acquired via close contact to infected, young pet rats. An identical and unique sequence of the hemagglutinin gene was found in various cowpox virus (CPXV) isolates pointing to a common source of infection. In a second CPXV outbreak in cats in a small animal clinic in Germany in 2015, four out of five hospitalized cats showed identical hemagglutinin sequences and thus, a hospital-acquired transmission had been assumed. Next-Generation Sequencing was performed in order to re-investigate the outbreaks, as epidemiological data could not confirm all cases.MethodsHomogenates of lesion material from rats, cats and humans were cultivated in cell culture. The genomes of four virus isolates, nine CPXVs from our strain collections and from DNA of three paraffin-embedded lesion materials were determined by Next Generation Sequencing (NGS). For phylogenetic analyses a MAFFT-alignment was generated. A distance matrix based on concatenated SNPs was calculated and plotted as dendrogram using Unweighted Pair Group Method with Arithmetic mean (UPGMA) for visualization.ResultsAligning of about 200.000 nucleotides of 8 virus isolates associated with the pet rat outbreak revealed complete identity of six genomes, the remainder two genomes differed in as little as 3 SNPs. When comparing this dataset with four already published CPXV genomes also associated with the pet rat outbreak, again a maximum difference of 3 SNPs was found. The outbreak which lasted from 2008 till 2011 was indeed caused by a single strain which has maintained an extremely high level of clonality over 4 years. Aligning genomic sequences from four cases of feline cowpox revealed 3 identical sequences and one sequence which differed in 65 nucleotides. Although identical hemagglutinin sequences had been obtained from four hospitalized cats, genomic sequencing proved that a hospital-acquired transmission had occurred in only three cats.ConclusionAnalyzing the rather short sequence of the hemagglutinin gene is not sufficient to conduct molecular trace back analyses. Instead, whole genome sequencing is the method of choice which can even be applied to paraffin-embedded specimens.

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.

Preliminary Screening and In Vitro Confirmation of Orthopoxvirus Antivirals.

The lack of antiviral drugs for the treatment of orthopoxvirus disease represents an unmet medical need, particularly due to the threat of variola virus (the causative agent of smallpox) as an agent of biowarfare or bioterrorism (Henderson, 283:1279-1282, 1999). In addition to variola, monkeypox, cowpox, and vaccinia viruses are orthopoxviruses of concern to human health (Lewis-Jones, 17:81-89, 2004). Smallpox vaccination, using the closely related vaccinia virus, is no longer provided to the general public leading to a worldwide population increasingly susceptible not only to variola but to monkeypox, cowpox, and vaccinia viruses as well. Orthopoxviruses share similar life cycles (Fenner et al., WHO, Geneva, 1988), and significant nucleotide and protein homology, and are immunologically cross-protective against other species within the genus, which was the basis of the highly successful vaccinia virus vaccine. These similarities also serve as the basis for screening for antivirals for dangerous pathogens such as variola and monkeypox virus using generally safer viruses such as cowpox and vaccinia. Methods for preliminary screening and initial characterization of potential orthopoxvirus antivirals in vitro, using vaccinia virus as a relatively safe surrogate for more pathogenic orthopoxviruses, are described herein. They include candidate identification in a viral cytopathic effect (CPE) assay as well as evaluation of the antiviral activity in inhibition assays to determine mean effective (or inhibitory) concentrations (EC50 or IC50). These assays were utilized in the identification and early characterization of tecovirimat (ST-246) (Yang et al., 79:13,139-13,149, 2005). These initial steps in identifying and characterizing the antiviral activity should be followed up with additional in vitro studies including specificity testing (for other orthopoxviruses and against other viruses), single-cycle growth curves, time of addition assays, cytotoxicity testing, and identification of the drug target.

Recombinant Poxviruses: Versatile Tools for Immunological Assays.

The study of antigen processing and presentation is critical to our understanding of the mechanisms that govern immune surveillance. A typical requirement of assays designed to examine antigen processing and presentation is the de novo biosynthesis of a model antigen. Historically, Vaccinia virus, a poxvirus closely related to Cowpox virus, has enjoyed widespread use for this purpose. Recombinant poxvirus-based expression has a number of advantages over other systems. Poxviruses accommodate the insertion of large pieces of recombinant DNA into their genome, and recombination and selection are relatively efficient. Poxviruses readily infect a variety of cell types, and they drive rapid and high levels of antigen expression. Additionally, they can be utilized in a variety of assays to study both MHC class I restricted and MHC class II restricted antigen processing and presentation. Ultimately, the numerous advantages of poxvirus recombinants have made the Vaccinia expression system a mainstay in the study of processing and presentation over the past two decades. In an attempt to address one shortcoming of Vaccinia virus while simultaneously retaining the benefits inherent to poxviruses, our laboratory has begun to engineer recombinant Ectromelia viruses. Ectromelia virus, or mousepox, is a natural pathogen of murine cells and performing experiments in the context of a natural host-pathogen relationship may elucidate unknown factors that influence epitope generation and host response. This chapter will describe several recombinant poxvirus system protocols used to study both MHC class I and class II antigen processing and presentation, as well as provide insight and troubleshooting techniques to improve the reproducibility and fidelity of these experiments.

Mutation and structure guided discovery of an antiviral small molecule that mimics an essential C-Terminal tripeptide of the vaccinia D4 processivity factor

The smallpox virus (variola) remains a bioterrorism threat since a majority of the human population has never been vaccinated. In the event of an outbreak, at least two drugs against different targets of variola are critical to circumvent potential viral mutants that acquire resistance. Vaccinia virus (VACV) is the model virus used in the laboratory for studying smallpox. The VACV processivity factor D4 is an ideal therapeutic target since it is both essential and specific for poxvirus replication. Recently, we identified a tripeptide (Gly-Phe-Ile) motif at the C-terminus of D4 that is conserved among poxviruses and is necessary for maintaining protein function. In the current work, a virtual screening for small molecule mimics of the tripeptide identified a thiophene lead that effectively inhibited VACV, cowpox virus, and rabbitpox virus in cell culture (EC50 = 8.4–19.7 μM) and blocked in vitro processive DNA synthesis (IC50 = 13.4 μM). Compound-binding to D4 was demonstrated through various biophysical methods and a dose-dependent retardation of the proteolysis of D4 proteins. This study highlights an inhibitor design strategy that exploits a susceptible region of the protein and identifies a novel scaffold for a broad-spectrum poxvirus inhibitor.

A System Based-Approach to Examine Cytokine Response in Poxvirus-Infected Macrophages.

The poxviruses are large, linear, double-stranded DNA viruses about 130 to 230 kbp, that have an animal origin and evolved to infect a wide host range. Variola virus (VARV), the causative agent of smallpox, is a poxvirus that infects only humans, but other poxviruses such as monkey poxvirus and cowpox virus (CPXV) have crossed over from animals to infect humans. Therefore understanding the biology of poxviruses can devise antiviral strategies to prevent these human infections. In this study we used a system-based approach to examine the host responses to three orthopoxviruses, CPXV, vaccinia virus (VACV), and ectromelia virus (ECTV) in the murine macrophage RAW 264.7 cell line. Overall, we observed a significant down-regulation of gene expressions for pro-inflammatory cytokines, chemokines, and related receptors. There were also common and virus-specific changes in the immune-regulated gene expressions for each poxvirus-infected RAW cells. Collectively our results showed that the murine macrophage RAW 264.7 cell line is a suitable cell-based model system to study poxvirus host response.