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1.  Real-Time PCR System for Detection of Orthopoxviruses and Simultaneous Identification of Smallpox Virus 
Journal of Clinical Microbiology  2004;42(5):1940-1946.
A screening assay for real-time LightCycler (Roche Applied Science, Mannheim, Germany) PCR identification of smallpox virus DNA was developed and compiled in a kit system under good manufacturing practice conditions with standardized reagents. In search of a sequence region unique to smallpox virus, the nucleotide sequence of the 14-kDa fusion protein gene of each of 14 variola virus isolates of the Russian World Health Organization smallpox virus repository was determined and compared to published sequences. PCR primers were designed to detect all Eurasian-African species of the genus Orthopoxvirus. A single nucleotide mismatch resulting in a unique amino acid substitution in smallpox virus was used to design a hybridization probe pair with a specific sensor probe that allows reliable differentiation of smallpox virus from other orthopoxviruses by melting-curve analysis. The applicability was demonstrated by successful amplification of 120 strains belonging to the orthopoxvirus species variola, vaccinia, camelpox, mousepox, cowpox, and monkeypox virus. The melting temperatures (Tms) determined for 46 strains of variola virus (Tms, 55.9 to 57.8°C) differed significantly (P = 0.005) from those obtained for 11 strains of vaccinia virus (Tms, 61.7 to 62.7°C), 15 strains of monkeypox virus (Tms, 61.9 to 62.2°C), 40 strains of cowpox virus (Tms, 61.3 to 63.7°C), 8 strains of mousepox virus (Tm, 61.9°C), and 8 strains of camelpox virus (Tms, 64.0 to 65.0°C). As most of the smallpox virus samples were derived from infected cell cultures and tissues, smallpox virus DNA could be detected in a background of human DNA. By applying probit regression analysis, the analytical sensitivity was determined to be 4 copies of smallpox virus target DNA per sample. The DNAs of several human herpesviruses as well as poxviruses other than orthopoxviruses were not detected by this method. The assay proved to be a reliable technique for the detection of orthopoxviruses, with the advantage that it can simultaneously identify variola virus.
doi:10.1128/JCM.42.5.1940-1946.2004
PMCID: PMC404623  PMID: 15131152
2.  Comparison of host cell gene expression in cowpox, monkeypox or vaccinia virus-infected cells reveals virus-specific regulation of immune response genes 
Virology Journal  2013;10:61.
Background
Animal-borne orthopoxviruses, like monkeypox, vaccinia and the closely related cowpox virus, are all capable of causing zoonotic infections in humans, representing a potential threat to human health. The disease caused by each virus differs in terms of symptoms and severity, but little is yet know about the reasons for these varying phenotypes. They may be explained by the unique repertoire of immune and host cell modulating factors encoded by each virus. In this study, we analysed the specific modulation of the host cell’s gene expression profile by cowpox, monkeypox and vaccinia virus infection. We aimed to identify mechanisms that are either common to orthopoxvirus infection or specific to certain orthopoxvirus species, allowing a more detailed description of differences in virus-host cell interactions between individual orthopoxviruses. To this end, we analysed changes in host cell gene expression of HeLa cells in response to infection with cowpox, monkeypox and vaccinia virus, using whole-genome gene expression microarrays, and compared these to each other and to non-infected cells.
Results
Despite a dominating non-responsiveness of cellular transcription towards orthopoxvirus infection, we could identify several clusters of infection-modulated genes. These clusters are either commonly regulated by orthopoxvirus infection or are uniquely regulated by infection with a specific orthopoxvirus, with major differences being observed in immune response genes. Most noticeable was an induction of genes involved in leukocyte migration and activation in cowpox and monkeypox virus-infected cells, which was not observed following vaccinia virus infection.
Conclusion
Despite their close genetic relationship, the expression profiles induced by infection with different orthopoxviruses vary significantly. It may be speculated that these differences at the cellular level contribute to the individual characteristics of cowpox, monkeypox and vaccinia virus infections in certain host species.
doi:10.1186/1743-422X-10-61
PMCID: PMC3599072  PMID: 23425254
Orthopoxvirus; Cowpox virus; Vaccinia virus; Monkeypox virus; Microarray; Gene expression; Host cell response
3.  PCR strategy for identification and differentiation of small pox and other orthopoxviruses. 
Journal of Clinical Microbiology  1995;33(8):2069-2076.
Rapid identification and differentiation of orthopoxviruses by PCR were achieved with primers based on genome sequences encoding the hemagglutinin (HA) protein, an infected-cell membrane antigen that distinguishes orthopoxviruses from other poxvirus genera. The initial identification step used a primer pair of consensus sequences for amplifying an HA DNA fragment from the three known North American orthopoxviruses (raccoonpox, skunkpox, and volepox viruses), and a second pair for amplifying virtually the entire HA open reading frame of the Eurasian-African orthopoxviruses (variola, vaccinia, cowpox, monkeypox, camelpox, ectromelia, and gerbilpox viruses). RsaI digest electropherograms of the amplified DNAs of the former subgroup provided species differentiation, and TaqI digests differentiated the Eurasian-African orthopoxviruses, including vaccinia virus from the vaccinia virus subspecies buffalopox virus. Endonuclease HhaI digest patterns distinguished smallpox variola major viruses from alastrim variola minor viruses. For the Eurasian-African orthopoxviruses, a confirmatory step that used a set of higher-sequence-homology primers was developed to provide sensitivity to discern individual virus HA DNAs from cross-contaminated orthopoxvirus DNA samples; TaqI and HhaI digestions of the individual amplified HA DNAs confirmed virus identity. Finally, a set of primers and modified PCR conditions were developed on the basis of base sequence differences within the HA genes of the 10 species, which enabled production of a single DNA fragment of a particular size that indicated the specific species.
PMCID: PMC228337  PMID: 7559950
4.  Critical Role of Perforin-dependent CD8+ T Cell Immunity for Rapid Protective Vaccination in a Murine Model for Human Smallpox 
PLoS Pathogens  2012;8(3):e1002557.
Vaccination is highly effective in preventing various infectious diseases, whereas the constant threat of new emerging pathogens necessitates the development of innovative vaccination principles that also confer rapid protection in a case of emergency. Although increasing evidence points to T cell immunity playing a critical role in vaccination against viral diseases, vaccine efficacy is mostly associated with the induction of antibody responses. Here we analyze the immunological mechanism(s) of rapidly protective vaccinia virus immunization using mousepox as surrogate model for human smallpox. We found that fast protection against lethal systemic poxvirus disease solely depended on CD4 and CD8 T cell responses induced by vaccination with highly attenuated modified vaccinia virus Ankara (MVA) or conventional vaccinia virus. Of note, CD4 T cells were critically required to allow for MVA induced CD8 T cell expansion and perforin-mediated cytotoxicity was a key mechanism of MVA induced protection. In contrast, selected components of the innate immune system and B cell-mediated responses were fully dispensable for prevention of fatal disease by immunization given two days before challenge. In conclusion, our data clearly demonstrate that perforin-dependent CD8 T cell immunity plays a key role in MVA conferred short term protection against lethal mousepox. Rapid induction of T cell immunity might serve as a new paradigm for treatments that need to fit into a scenario of protective emergency vaccination.
Author Summary
Prophylactic use of vaccinia virus allowed eradication of human smallpox, one of the greatest successes in medicine. However there are concerns that variola virus, the infectious agent of smallpox, may be used as bioterroristic weapon and zoonotic monkeypox or cowpox remain threatening infections in humans. Thus, new developments of safe and rapidly protecting orthopoxvirus-specific vaccines have been initiated. The candidate vaccine modified vaccinia virus Ankara (MVA) was recently shown to protect against lethal systemic poxvirus disease even when applied shortly before or after infection of mice with ectromelia virus, the probably best animal model for human smallpox. Surprisingly, little is known about the protective mechanism of early immune responses elicited against orthopoxvirus infections. Here, we used the mousepox model to analyze the immunological basis of rapidly protective MVA vaccination. In contrast to common understanding of orthopoxvirus vaccine efficacy relying mainly on antibody mediated immunity, we observed unimpaired protection also in absence of B cells. Surprisingly, rapid protection by vaccination with MVA or conventional vaccinia virus was solely dependent on T cells, irrespective of the route of injection. Thus, our study suggests a key role for T cell immunity in rapidly protective immunization against orthopoxviruses and potentially other infectious agents.
doi:10.1371/journal.ppat.1002557
PMCID: PMC3291617  PMID: 22396645
5.  Genome-Wide Comparison of Cowpox Viruses Reveals a New Clade Related to Variola Virus 
PLoS ONE  2013;8(12):e79953.
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.
doi:10.1371/journal.pone.0079953
PMCID: PMC3848979  PMID: 24312452
6.  Smallpox: can we still learn from the journey to eradication? 
One of the most celebrated achievements of immunology and modern medicine is the eradication of the dreaded plague smallpox. From the introduction of smallpox vaccination by Edward Jenner, to its popularization by Louis Pasteur, to the eradication effort led by Donald Henderson, this story has many lessons for us today, including the characteristics of the disease and vaccine that permitted its eradication, and the obviousness of the vaccine as a vector for other intractable Infectious diseases. The disease itself, interpreted in the light of modern molecular immunology, is an obvious immunopathological disease, which occurs after a latent interval of 1-2 weeks, and manifests as a systemic cell-mediated delayed type hypersensitivity (DTH) syndrome. The vaccine that slayed this dragon was given the name vaccinia, and was thought to have evolved from cowpox virus, but is now known to be most closely related to a poxvirus isolated from a horse. Of interest is the fact that of the various isolates of orthopox viruses, only variola, vaccinia and monkeypox viruses can infect humans. In contrast to the systemic disease of variola, vaccinia only replicates locally at the site of inoculation, and causes a localized DTH response that usually peaks after 7-10 days. This difference in the pathogenicity of variola vs. vaccinia is thought to be due to the capacity of variola to circumvent innate immunity, which allows it to disseminate widely before the adaptive immune response occurs. Thus, the fact that vaccinia virus is attenuated compared to variola, but is still replication competent, makes for its remarkable efficacy as a vaccine, as the localized infection activates all of the cells and molecules of both innate and adaptive immunity. Accordingly vaccinia itself, and not modified replication incompetent vaccina, is the hope for use as a vector in the eradication of additional pathogenic microbes from the globe.
PMCID: PMC3734679  PMID: 23760373
Eradication; smallpox; smallpox virus
7.  Real-Time PCR Assay To Detect Smallpox Virus 
Journal of Clinical Microbiology  2003;41(8):3835-3839.
We developed a highly sensitive and specific assay for the rapid detection of smallpox virus DNA on both the Smart Cycler and LightCycler platforms. The assay is based on TaqMan chemistry with the orthopoxvirus hemagglutinin gene used as the target sequence. With genomic DNA purified from variola virus Bangladesh 1975, the limit of detection was estimated to be approximately 25 copies on both machines. The assay was evaluated in a blinded study with 322 coded samples that included genomic DNA from 48 different isolates of variola virus; 25 different strains and isolates of camelpox, cowpox, ectromelia, gerbilpox, herpes, monkeypox, myxoma, rabbitpox, raccoonpox, skunkpox, vaccinia, and varicella-zoster viruses; and two rickettsial species at concentrations mostly ranging from 100 fg/μl to 1 ng/μl. Contained within those 322 samples were variola virus DNA, obtained from purified viral preparations, at concentrations of 1 fg/μl to 1 ng/μl. On the Smart Cycler platform, 2 samples with false-positive results were detected among the 116 samples not containing variola virus tested; i.e., the overall specificity of the assay was 98.3%. On the LightCycler platform, five samples with false-positive results were detected (overall specificity, 95.7%). Of the 206 samples that contained variola virus DNA ranging in concentrations from 100 fg/μl to 1 ng/μl, 8 samples were considered negative on the Smart Cycler platform and 1 sample was considered negative on the LightCycler platform. Thus, the clinical sensitivities were 96.1% for the Smart Cycler instrument and 99.5% for the LightCycler instrument. The vast majority of these samples were derived from virus-infected cell cultures and variola virus-infected tissues; thus, the DNA material contained both viral DNA and cellular DNA. Of the 43 samples that contained purified variola virus DNA ranging in concentration from 1 fg/μl to 1 ng/μl, the assay correctly detected the virus in all 43 samples on both the Smart Cycler and the LightCycler platforms. The assay may be useful for the early detection of smallpox virus infections should such infections occur as a result of a deliberate or an accidental recurrence.
doi:10.1128/JCM.41.8.3835-3839.2003
PMCID: PMC179856  PMID: 12904397
8.  In Vitro Efficacy of ST246 against Smallpox and Monkeypox▿  
Since the eradication of smallpox and the cessation of routine childhood vaccination for smallpox, the proportion of the world's population susceptible to infection with orthopoxviruses, such as variola virus (the causative agent of smallpox) and monkeypox virus, has grown substantially. In the United States, the only vaccines for smallpox licensed by the Food and Drug Administration (FDA) have been live virus vaccines. Unfortunately, a substantial number of people cannot receive live virus vaccines due to contraindications. Furthermore, no antiviral drugs have been fully approved by the FDA for the prevention or treatment of orthopoxvirus infection. Here, we show the inhibitory effect of one new antiviral compound, ST-246, on the in vitro growth properties of six variola virus strains and seven monkeypox virus strains. We performed multiple assays to monitor the cytopathic effect and to evaluate the reduction of viral progeny production and release in the presence of the compound. ST-246 had 50% effective concentrations of ≤0.067 μM against variola virus and <0.04 μM against monkeypox virus. In a dose-dependent manner, plaque size and comet tail formation were markedly reduced in the presence of the drug at low, noncytotoxic concentrations between 0.015 and 0.05 μM. Our in vitro phenotype data suggest that ST-246 inhibits variola and monkeypox viruses similarly by reducing the production and release of enveloped orthopoxvirus and support the development of ST-246 as an antiviral therapeutic compound for the treatment of severe systemic orthopoxvirus infections.
doi:10.1128/AAC.01044-08
PMCID: PMC2650551  PMID: 19075062
9.  Identification of the Orthopoxvirus p4c Gene, Which Encodes a Structural Protein That Directs Intracellular Mature Virus Particles into A-Type Inclusions 
Journal of Virology  2002;76(22):11216-11225.
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.
doi:10.1128/JVI.76.22.11216-11225.2002
PMCID: PMC136765  PMID: 12388681
10.  An Orally Bioavailable Antipoxvirus Compound (ST-246) Inhibits Extracellular Virus Formation and Protects Mice from Lethal Orthopoxvirus Challenge 
Journal of Virology  2005;79(20):13139-13149.
ST-246 is a low-molecular-weight compound (molecular weight = 376), that is potent (concentration that inhibited virus replication by 50% = 0.010 μM), selective (concentration of compound that inhibited cell viability by 50% = >40 μM), and active against multiple orthopoxviruses, including vaccinia, monkeypox, camelpox, cowpox, ectromelia (mousepox), and variola viruses. Cowpox virus variants selected in cell culture for resistance to ST-246 were found to have a single amino acid change in the V061 gene. Reengineering this change back into the wild-type cowpox virus genome conferred resistance to ST-246, suggesting that V061 is the target of ST-246 antiviral activity. The cowpox virus V061 gene is homologous to vaccinia virus F13L, which encodes a major envelope protein (p37) required for production of extracellular virus. In cell culture, ST-246 inhibited plaque formation and virus-induced cytopathic effects. In single-cycle growth assays, ST-246 reduced extracellular virus formation by 10 fold relative to untreated controls, while having little effect on the production of intracellular virus. In vivo oral administration of ST-246 protected BALB/c mice from lethal infection, following intranasal inoculation with 10× 50% lethal dose (LD50) of vaccinia virus strain IHD-J. ST-246-treated mice that survived infection acquired protective immunity and were resistant to subsequent challenge with a lethal dose (10× LD50) of vaccinia virus. Orally administered ST-246 also protected A/NCr mice from lethal infection, following intranasal inoculation with 40,000× LD50 of ectromelia virus. Infectious virus titers at day 8 postinfection in liver, spleen, and lung from ST-246-treated animals were below the limits of detection (<10 PFU/ml). In contrast, mean virus titers in liver, spleen, and lung tissues from placebo-treated mice were 6.2 × 107, 5.2 × 107, and 1.8 × 105 PFU/ml, respectively. Finally, oral administration of ST-246 inhibited vaccinia virus-induced tail lesions in Naval Medical Research Institute mice inoculated via the tail vein. Taken together, these results validate F13L as an antiviral target and demonstrate that an inhibitor of extracellular virus formation can protect mice from orthopoxvirus-induced disease.
doi:10.1128/JVI.79.20.13139-13149.2005
PMCID: PMC1235851  PMID: 16189015
11.  Rapid and High-Throughput pan-Orthopoxvirus Detection and Identification using PCR and Mass Spectrometry 
PLoS ONE  2009;4(7):e6342.
The genus Orthopoxvirus contains several species of related viruses, including the causative agent of smallpox (Variola virus). In addition to smallpox, several other members of the genus are capable of causing human infection, including monkeypox, cowpox, and other zoonotic rodent-borne poxviruses. Therefore, a single assay that can accurately identify all orthopoxviruses could provide a valuable tool for rapid broad orthopovirus identification. We have developed a pan-Orthopoxvirus assay for identification of all members of the genus based on four PCR reactions targeting Orthopoxvirus DNA and RNA helicase and polymerase genes. The amplicons are detected using electrospray ionization-mass spectrometry (PCR/ESI-MS) on the Ibis T5000 system. We demonstrate that the assay can detect and identify a diverse collection of orthopoxviruses, provide sub-species information and characterize viruses from the blood of rabbitpox infected rabbits. The assay is sensitive at the stochastic limit of PCR and detected virus in blood containing approximately six plaque-forming units per milliliter from a rabbitpox virus-infected rabbit.
doi:10.1371/journal.pone.0006342
PMCID: PMC2710004  PMID: 19623263
12.  An Increasing Danger of Zoonotic Orthopoxvirus Infections 
PLoS Pathogens  2013;9(12):e1003756.
On May 8, 1980, the World Health Assembly at its 33rd session solemnly declared that the world and all its peoples had won freedom from smallpox and recommended ceasing the vaccination of the population against smallpox. Currently, a larger part of the world population has no immunity not only against smallpox but also against other zoonotic orthopoxvirus infections. Recently, recorded outbreaks of orthopoxvirus diseases not only of domestic animals but also of humans have become more frequent. All this indicates a new situation in the ecology and evolution of zoonotic orthopoxviruses. Analysis of state-of-the-art data on the phylogenetic relationships, ecology, and host range of orthopoxviruses—etiological agents of smallpox (variola virus, VARV), monkeypox (MPXV), cowpox (CPXV), vaccinia (VACV), and camelpox (CMLV)—as well as the patterns of their evolution suggests that a VARV-like virus could emerge in the course of natural evolution of modern zoonotic orthopoxviruses. Thus, there is an insistent need for organization of the international control over the outbreaks of zoonotic orthopoxvirus infections in various countries to provide a rapid response and prevent them from developing into epidemics.
doi:10.1371/journal.ppat.1003756
PMCID: PMC3855571  PMID: 24339772
13.  Naturally Occurring Orthopoxviruses: Potential for Recombination with Vaccine Vectors 
Journal of Clinical Microbiology  1998;36(9):2542-2547.
Orthopoxviruses are being increasingly used as live recombinant vectors for vaccination against numerous infectious diseases in humans, domestic animals, and wildlife. For risk assessments and surveillance, information about the occurrence, distribution and ecology of orthopoxviruses in western Europe is important but has mainly been based on serological investigations. We have examined kidneys, lungs, spleens, and livers of Norwegian small rodents and common shrews (Sorex araneus) for the presence of orthopoxvirus DNA sequences by PCR with primers complementary to the viral thymidine kinase (TK) gene. PCR amplicons were verified as orthopoxvirus specific by hybridization with a vaccinia virus TK-specific probe. A total of 347 animals (1,388 organs) from eight locations in different parts of Norway, collected at different times of the year during 1993 to 1995, were examined. Fifty-two animals (15%) from five locations, up to 1,600 km apart, carried orthopoxvirus DNA in one or more of their organs, most frequently in the lungs. These included 9 of 68 (13%) bank voles (Clethrionomys glareolus), 4 of 13 (31%) gray-sided voles (Clethrionomys rufocanus), 3 of 11 (27%) northern red-backed voles (Clethrionomys rutilus), 16 of 76 (21%) wood mice (Apodemus sylvaticus), and 20 of 157 (13%) common shrews. The previous isolation of cowpox virus from two clinical cases of infection (human and feline) at two of the locations investigated suggests that the viruses detected are cowpox and that some of the virus-carrying small mammalian species should be included among the cowpox virus natural reservoir hosts in Scandinavia and western Europe.
PMCID: PMC105159  PMID: 9705389
14.  Antibody Inhibition of a Viral Type 1 Interferon Decoy Receptor Cures a Viral Disease by Restoring Interferon Signaling in the Liver 
PLoS Pathogens  2012;8(1):e1002475.
Type 1 interferons (T1-IFNs) play a major role in antiviral defense, but when or how they protect during infections that spread through the lympho-hematogenous route is not known. Orthopoxviruses, including those that produce smallpox and mousepox, spread lympho-hematogenously. They also encode a decoy receptor for T1-IFN, the T1-IFN binding protein (T1-IFNbp), which is essential for virulence. We demonstrate that during mousepox, T1-IFNs protect the liver locally rather than systemically, and that the T1-IFNbp attaches to uninfected cells surrounding infected foci in the liver and the spleen to impair their ability to receive T1-IFN signaling, thus facilitating virus spread. Remarkably, this process can be reversed and mousepox cured late in infection by treating with antibodies that block the biological function of the T1-IFNbp. Thus, our findings provide insights on how T1-IFNs function and are evaded during a viral infection in vivo, and unveil a novel mechanism for antibody-mediated antiviral therapy.
Author Summary
Type 1 interferons are molecules important in the defense against viruses. Orthopoxviruses encode a Type 1 interferon binding protein that acts as a decoy for the Type 1 interferon receptor. Here we show that during infection with the Orthopoxvirus ectromelia virus, the agent of mousepox, Type 1 interferons protect the liver locally rather than systemically. We also show that the Type 1 interferon binding protein of ectromelia virus attaches to uninfected cells surrounding infected foci in the liver to impair their ability to receive Type 1 interferon signaling and facilitate virus spread and disease progression. We also show that this process can be reversed and mousepox cured late in infection by treating mice with antibodies that block the biological function of the Type 1 interferon binding protein. Because the Type 1 interferon binding proteins of different Orthopoxviruses are very well conserved, the antibodies also block the biological function of the Type 1 interferon binding proteins from variola virus (the virus of smallpox) and monkeypoxvirus. Thus, our findings provide insights on how Type 1 interferons function and are evaded during a viral infection in vivo, and unveil a novel mechanism for antibody-mediated antiviral therapy.
doi:10.1371/journal.ppat.1002475
PMCID: PMC3252373  PMID: 22241999
15.  The Master Regulator of the Cellular Stress Response (HSF1) Is Critical for Orthopoxvirus Infection 
PLoS Pathogens  2014;10(2):e1003904.
The genus Orthopoxviridae contains a diverse group of human pathogens including monkeypox, smallpox and vaccinia. These viruses are presumed to be less dependent on host functions than other DNA viruses because they have large genomes and replicate in the cytoplasm, but a detailed understanding of the host factors required by orthopoxviruses is lacking. To address this topic, we performed an unbiased, genome-wide pooled RNAi screen targeting over 17,000 human genes to identify the host factors that support orthopoxvirus infection. We used secondary and tertiary assays to validate our screen results. One of the strongest hits was heat shock factor 1 (HSF1), the ancient master regulator of the cytoprotective heat-shock response. In investigating the behavior of HSF1 during vaccinia infection, we found that HSF1 was phosphorylated, translocated to the nucleus, and increased transcription of HSF1 target genes. Activation of HSF1 was supportive for virus replication, as RNAi knockdown and HSF1 small molecule inhibition prevented orthopoxvirus infection. Consistent with its role as a transcriptional activator, inhibition of several HSF1 targets also blocked vaccinia virus replication. These data show that orthopoxviruses co-opt host transcriptional responses for their own benefit, thereby effectively extending their functional genome to include genes residing within the host DNA. The dependence on HSF1 and its chaperone network offers multiple opportunities for antiviral drug development.
Author Summary
Orthopoxviruses bring in many of the factors they need for replication and impair the host cell by preventing the expression of host proteins. Although orthopoxviruses are less reliant on the host than some viruses, host factors are still required for infection. Here, we report results from two genome-scale approaches that identify host proteins used by orthopoxviruses during infection. These approaches showed that the master regulator of the heat shock response, heat shock factor 1 (HSF1), is a critical host factor for orthopoxvirus replication. HSF1-regulated genes are some of the only host genes with expression maintained or increased following virus infection. Our studies show that orthopoxviruses enter the cell and activate a host transcription pathway as part of its own replication process. These proteins are then utilized by the virus during infection and packaged into the virion, essentially extending the viral genome to include genes co-opted from the host nuclear DNA. This is supported by the existence of heat shock proteins in the viral genome of non-orthopoxvirus genera. We further show that small-molecule inhibitors of HSF1 and HSF1-transcribed genes are effective inhibitors of orthopoxvirus replication, suggesting a new avenue for antiviral development.
doi:10.1371/journal.ppat.1003904
PMCID: PMC3916389  PMID: 24516381
16.  Smallpox Virus Resequencing GeneChips Can Also Rapidly Ascertain Species Status for Some Zoonotic Non-Variola Orthopoxviruses▿  
Journal of Clinical Microbiology  2008;46(4):1507-1509.
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.
doi:10.1128/JCM.00158-08
PMCID: PMC2292925  PMID: 18272713
17.  T Cell Inactivation by Poxviral B22 Family Proteins Increases Viral Virulence 
PLoS Pathogens  2014;10(5):e1004123.
Infections with monkeypox, cowpox and weaponized variola virus remain a threat to the increasingly unvaccinated human population, but little is known about their mechanisms of virulence and immune evasion. We now demonstrate that B22 proteins, encoded by the largest genes of these viruses, render human T cells unresponsive to stimulation of the T cell receptor by MHC-dependent antigen presentation or by MHC-independent stimulation. In contrast, stimuli that bypass TCR-signaling are not inhibited. In a non-human primate model of monkeypox, virus lacking the B22R homologue (MPXVΔ197) caused only mild disease with lower viremia and cutaneous pox lesions compared to wild type MPXV which caused high viremia, morbidity and mortality. Since MPXVΔ197-infected animals displayed accelerated T cell responses and less T cell dysregulation than MPXV US2003, we conclude that B22 family proteins cause viral virulence by suppressing T cell control of viral dissemination.
Author Summary
We discovered that the largest gene in the genome of monkeypox viruses and several related viruses, including the virus causing smallpox, but not vaccine strains, encode a protein (B22) that renders the cellular immune system non-responsive. A particularly novel aspect of this work is that B22 proteins directly disable cells of the immune system as compared to previously known molecular strategies that help viruses to hide from the immune system. We further show that monkeypox viruses containing this protein are much more virulent in non-human primates than viruses that lack B22. Our observations suggest that B22 proteins contribute to monkeypox virulence and might have contributed to the severe disease manifestations of variola major virus. However, these data also suggest that B22 proteins could potentially be used to curb undesired immune responses such as autoimmunity or graft versus host disease.
doi:10.1371/journal.ppat.1004123
PMCID: PMC4022744  PMID: 24832205
18.  A protein-based smallpox vaccine protects non-human primates from a lethal monkeypox virus challenge 
Vaccine  2010;28(40):6627-6636.
Concerns about infections caused by orthopoxviruses, such as variola and monkeypox viruses, drive ongoing efforts to develop novel smallpox vaccines that are both effective and safe to use in diverse populations. A subunit smallpox vaccine comprising vaccinia virus membrane proteins A33, B5, L1, A27 and aluminum hydroxide (alum) ± CpG were administered to non-human primates, which were subsequently challenged with a lethal intravenous dose of monkeypox virus. Alum-adjuvanted vaccines provided only partial protection but the addition of CpG provided full protection that was associated with a more homogeneous antibody response and stronger IgG1 responses. These results indicate that it is feasible to develop a highly effective subunit vaccine against orthopoxvirus infections as a safer alternative to live vaccinia virus vaccination.
doi:10.1016/j.vaccine.2010.07.030
PMCID: PMC2939220  PMID: 20659519
smallpox; monkeypox; vaccinia virus; subunit vaccine; CpG; aluminum hydroxide; baculovirus expressed proteins
19.  Use of Internally Controlled Real-Time Genome Amplification for Detection of Variola Virus and Other Orthopoxviruses Infecting Humans▿  
Journal of Clinical Microbiology  2005;44(12):4464-4470.
Smallpox, once a devastating disease caused by Variola virus, a member of the Orthopoxvirus genus, was eradicated in 1980. However, the importance of variola virus infections has been stressed widely in the last few years, particularly following recent social events in the world. Today, variola virus is considered to be one of the most significant agents with potential use as a biological weapon. In this study we developed an internally controlled real-time PCR assay for rapid detection and simultaneous differentiation of variola virus from other orthopoxviruses. The assay is based on TaqMan 3′-minor groove binder (MGB) chemistry and uses generic primers, designed in highly conserved genomic regions of the crmB gene, and three TaqMan MGB probes designed to identify orthopoxviruses, variola virus, and an internal control. The results obtained suggest that the assay is rapid, sensitive, specific, and suitable for the generic detection of orthopoxviruses and the identification of variola virus and avoids false-negative results in a single reaction tube.
doi:10.1128/JCM.00276-06
PMCID: PMC1698395  PMID: 17065259
20.  Side-by-Side Comparison of Gene-Based Smallpox Vaccine with MVA in Nonhuman Primates 
PLoS ONE  2012;7(7):e42353.
Orthopoxviruses remain a threat as biological weapons and zoonoses. The licensed live-virus vaccine is associated with serious health risks, making its general usage unacceptable. Attenuated vaccines are being developed as alternatives, the most advanced of which is modified-vaccinia virus Ankara (MVA). We previously developed a gene-based vaccine, termed 4pox, which targets four orthopoxvirus antigens, A33, B5, A27 and L1. This vaccine protects mice and non-human primates from lethal orthopoxvirus disease. Here, we investigated the capacity of the molecular adjuvants GM-CSF and Escherichia coli heat-labile enterotoxin (LT) to enhance the efficacy of the 4pox gene-based vaccine. Both adjuvants significantly increased protective antibody responses in mice. We directly compared the 4pox plus LT vaccine against MVA in a monkeypox virus (MPXV) nonhuman primate (NHP) challenge model. NHPs were vaccinated twice with MVA by intramuscular injection or the 4pox/LT vaccine delivered using a disposable gene gun device. As a positive control, one NHP was vaccinated with ACAM2000. NHPs vaccinated with each vaccine developed anti-orthopoxvirus antibody responses, including those against the 4pox antigens. After MPXV intravenous challenge, all control NHPs developed severe disease, while the ACAM2000 vaccinated animal was well protected. All NHPs vaccinated with MVA were protected from lethality, but three of five developed severe disease and all animals shed virus. All five NHPs vaccinated with 4pox/LT survived and only one developed severe disease. None of the 4pox/LT-vaccinated animals shed virus. Our findings show, for the first time, that a subunit orthopoxvirus vaccine delivered by the same schedule can provide a degree of protection at least as high as that of MVA.
doi:10.1371/journal.pone.0042353
PMCID: PMC3409187  PMID: 22860117
21.  Vaccinia Virus–Encoded Ribonucleotide Reductase Subunits Are Differentially Required for Replication and Pathogenesis 
PLoS Pathogens  2010;6(7):e1000984.
Ribonucleotide reductases (RRs) are evolutionarily-conserved enzymes that catalyze the rate-limiting step during dNTP synthesis in mammals. RR consists of both large (R1) and small (R2) subunits, which are both required for catalysis by the R12R22 heterotetrameric complex. Poxviruses also encode RR proteins, but while the Orthopoxviruses infecting humans [e.g. vaccinia (VACV), variola, cowpox, and monkeypox viruses] encode both R1 and R2 subunits, the vast majority of Chordopoxviruses encode only R2 subunits. Using plaque morphology, growth curve, and mouse model studies, we investigated the requirement of VACV R1 (I4) and R2 (F4) subunits for replication and pathogenesis using a panel of mutant viruses in which one or more viral RR genes had been inactivated. Surprisingly, VACV F4, but not I4, was required for efficient replication in culture and virulence in mice. The growth defects of VACV strains lacking F4 could be complemented by genes encoding other Chordopoxvirus R2 subunits, suggesting conservation of function between poxvirus R2 proteins. Expression of F4 proteins encoding a point mutation predicted to inactivate RR activity but still allow for interaction with R1 subunits, caused a dominant negative phenotype in growth experiments in the presence or absence of I4. Co-immunoprecipitation studies showed that F4 (as well as other Chordopoxvirus R2 subunits) form hybrid complexes with cellular R1 subunits. Mutant F4 proteins that are unable to interact with host R1 subunits failed to rescue the replication defect of strains lacking F4, suggesting that F4-host R1 complex formation is critical for VACV replication. Our results suggest that poxvirus R2 subunits form functional complexes with host R1 subunits to provide sufficient dNTPs for viral replication. Our results also suggest that R2-deficient poxviruses may be selective oncolytic agents and our bioinformatic analyses provide insights into how poxvirus nucleotide metabolism proteins may have influenced the base composition of these pathogens.
Author Summary
Efficient genome replication is central to the virulence of all DNA viruses, including poxviruses. To ensure replication efficiency, many of the more virulent poxviruses encode their own nucleotide metabolism machinery, including ribonucleotide reductase (RR) enzymes, which act to provide ample DNA precursors for replication. RR enzymes require both large (R1) and small (R2) subunit proteins for activity. Curiously, some poxviruses only encode R2 subunits. Other poxviruses, such as the smallpox vaccine strain, vaccinia virus (VACV), encode both R1 and R2 subunits. We report here that the R2, but not the R1, subunit of VACV RR is required for efficient replication and virulence. We also provide evidence that several poxvirus R2 proteins form novel complexes with host R1 subunits and this interaction is required for efficient VACV replication in primate cells. Our study explains why some poxviruses only encode R2 subunits and identifies a role for these proteins in poxvirus pathogenesis. Furthermore, we provide evidence that mutant poxviruses unable to generate R2 proteins may become entirely dependent upon host RR activity. This may restrict their replication to cells that over-express RR proteins such as cancer cells, making them potential therapeutics for human malignancies.
doi:10.1371/journal.ppat.1000984
PMCID: PMC2900304  PMID: 20628573
22.  Camelpox virus encodes a schlafen-like protein that affects orthopoxvirus virulence 
The Journal of General Virology  2007;88(Pt 6):1667-1676.
Camelpox virus (CMLV) gene 176R encodes a protein with sequence similarity to murine schlafen (m-slfn) proteins. In vivo, short and long members of the m-slfn family inhibited T-cell development, whereas in vitro, only short m-slfns caused arrest of fibroblast growth. CMLV 176 protein (v-slfn) is most closely related to short m-slfns; however, when expressed stably in mammalian cells, v-slfn did not inhibit cell growth. v-slfn is a predominantly cytoplasmic 57 kDa protein that is expressed throughout infection. Several other orthopoxviruses encode v-slfn proteins, but the v-slfn gene is fragmented in all sequenced variola virus and vaccinia virus (VACV) strains. Consistent with this, all 16 VACV strains tested do not express a v-slfn detected by polyclonal serum raised against the CMLV protein. In the absence of a small animal model to study CMLV pathogenesis, the contribution of CMLV v-slfn to orthopoxvirus virulence was studied via its expression in an attenuated strain of VACV. Recombinant viruses expressing wild-type v-slfn or v-slfn tagged at its C terminus with a haemagglutinin (HA) epitope were less virulent than control viruses. However, a virus expressing v-slfn tagged with the HA epitope at its N terminus had similar virulence to controls, implying that the N terminus has an important function. A greater recruitment of lymphocytes into infected lung tissue was observed in the presence of wild-type v-slfn but, interestingly, these cells were less activated. Thus, v-slfn is an orthopoxvirus virulence factor that affects the host immune response to infection.
doi:10.1099/vir.0.82748-0
PMCID: PMC2885618  PMID: 17485525
23.  Detection and Differentiation of Old World Orthopoxviruses: Restriction Fragment Length Polymorphism of the crmB Gene Region 
Journal of Clinical Microbiology  2001;39(1):94-100.
A restriction fragment length polymorphism (RFLP) assay was developed to identify and differentiate Old World, African-Eurasian orthopoxviruses (OPV): variola, vaccinia, cowpox, monkeypox, camelpox, ectromelia, and taterapox viruses. The test uses amplicons produced from virus genome DNA by PCR with a consensus primer pair designed from sequences determined for the cytokine response modifier B (crmB) gene of 43 different OPV strains of known taxonomic origin. The primer pair amplified a single specific product from each of the 115 OPV samples tested. Size-specific amplicons identified and differentiated ectromelia and vaccinia virus strains, which contain a truncated crmB gene, and enabled their differentiation from other OPV species. Restriction digests of amplified products allowed the identification and differentiation of variola, monkeypox, camelpox, vaccinia, and cowpox virus species and strains.
doi:10.1128/JCM.39.1.94-100.2001
PMCID: PMC87686  PMID: 11136755
24.  Antiviral activity of the EB peptide against zoonotic poxviruses 
Virology Journal  2012;9:6.
Background
The EB peptide is a 20-mer that was previously shown to have broad spectrum in vitro activity against several unrelated viruses, including highly pathogenic avian influenza, herpes simplex virus type I, and vaccinia, the prototypic orthopoxvirus. To expand on this work, we evaluated EB for in vitro activity against the zoonotic orthopoxviruses cowpox and monkeypox and for in vivo activity in mice against vaccinia and cowpox.
Findings
In yield reduction assays, EB had an EC50 of 26.7 μM against cowpox and 4.4 μM against monkeypox. The EC50 for plaque reduction was 26.3 μM against cowpox and 48.6 μM against monkeypox. A scrambled peptide had no inhibitory activity against either virus. EB inhibited cowpox in vitro by disrupting virus entry, as evidenced by a reduction of the release of virus cores into the cytoplasm. Monkeypox was also inhibited in vitro by EB, but at the attachment stage of infection. EB showed protective activity in mice infected intranasally with vaccinia when co-administered with the virus, but had no effect when administered prophylactically one day prior to infection or therapeutically one day post-infection. EB had no in vivo activity against cowpox in mice.
Conclusions
While EB did demonstrate some in vivo efficacy against vaccinia in mice, the limited conditions under which it was effective against vaccinia and lack of activity against cowpox suggest EB may be more useful for studying orthopoxvirus entry and attachment in vitro than as a therapeutic against orthopoxviruses in vivo.
doi:10.1186/1743-422X-9-6
PMCID: PMC3275487  PMID: 22225618
EB peptide; vaccinia; cowpox; monkeypox; poxvirus entry; poxvirus attachment
25.  Efficacy of Multiple- or Single-Dose Cidofovir against Vaccinia and Cowpox Virus Infections in Mice 
Antimicrobial Agents and Chemotherapy  2003;47(10):3275-3280.
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.
doi:10.1128/AAC.47.10.3275-3280.2003
PMCID: PMC201130  PMID: 14506041

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