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1.  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
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  Vaccinia viruses: vaccines against smallpox and vectors against infectious diseases and tumors 
Expert review of vaccines  2011;10(8):1221-1240.
Less than 200 years after its introduction, widespread use of vaccinia virus (VACV) as a smallpox vaccine has eradicated variola virus. Along with the remarkable success of the vaccination program, frequent and sometimes severe adverse reactions to VACV were encountered. After eradication, VACV has been reserved for select populations who might be at significant risk for orthopoxvirus infections. Events over the past decade have renewed concerns over the potential use of variola virus as a biological weapon. Accordingly, interest in VACV and attenuated derivatives has increased, both as vaccines against smallpox and as vectors for other vaccines. This article will focus on new developments in the field of orthopoxvirus immunization and will highlight recent advances in the use of vaccinia viruses as vectors for infectious diseases and malignancies.
doi:10.1586/erv.11.79
PMCID: PMC3223417  PMID: 21854314
ACAM2000; clinical trials; Dryvax; IMVAMUNE; MVA; orthopoxviruses; smallpox; vaccine; vaccinia
13.  Smallpox Inhibitor of Complement Enzymes (SPICE): Dissecting Functional Sites and Abrogating Activity1 
Although smallpox was eradicated as a global illness more than 30 years ago, variola virus and other related pathogenic poxviruses, such as monkeypox, remain potential bioterrorist weapons or could re-emerge as natural infections. Poxviruses express virulence factors that down-modulate the host’s immune system. We previously compared functional profiles of the poxviral complement inhibitors of smallpox, vaccinia, and monkeypox known as SPICE, VCP (or VICE), and MOPICE, respectively. SPICE was the most potent regulator of human complement and attached to cells via glycosaminoglycans. The major goals of the present study were to further characterize the complement regulatory and heparin binding sites of SPICE and to evaluate a mAb that abrogates its function. Using substitution mutagenesis, we established that (1) elimination of the three heparin binding sites severely decreases but does not eliminate glycosaminoglycan binding, (2) there is a hierarchy of activity for heparin binding among the three sites, and (3) complement regulatory sites overlap with each of the three heparin binding motifs. By creating chimeras with interchanges of SPICE and VCP residues, a combination of two SPICE amino acids (H77 plus K120) enhances VCP activity ~200-fold. Also, SPICE residue L131 is critical for both complement regulatory function and accounts for the electrophoretic differences between SPICE and VCP. An evolutionary history for these structure-function adaptations of SPICE is proposed. Finally, we identified and characterized a mAb that inhibits the complement regulatory activity of SPICE, MOPICE, and VCP and thus could be used as a therapeutic agent.
doi:10.4049/jimmunol.0901366
PMCID: PMC2899487  PMID: 19667083
14.  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
15.  Ectromelia Virus Infections of Mice as a Model to Support the Licensure of Anti-Orthopoxvirus Therapeutics 
Viruses  2010;2(9):1918-1932.
The absence of herd immunity to orthopoxviruses and the concern that variola or monkeypox viruses could be used for bioterroristic activities has stimulated the development of therapeutics and safer prophylactics. One major limitation in this process is the lack of accessible human orthopoxvirus infections for clinical efficacy trials; however, drug licensure can be based on orthopoxvirus animal challenge models as described in the “Animal Efficacy Rule”. One such challenge model uses ectromelia virus, an orthopoxvirus, whose natural host is the mouse and is the etiological agent of mousepox. The genetic similarity of ectromelia virus to variola and monkeypox viruses, the common features of the resulting disease, and the convenience of the mouse as a laboratory animal underscores its utility in the study of orthopoxvirus pathogenesis and in the development of therapeutics and prophylactics. In this review we outline how mousepox has been used as a model for smallpox. We also discuss mousepox in the context of mouse strain, route of infection, infectious dose, disease progression, and recovery from infection.
doi:10.3390/v2091918
PMCID: PMC3185751  PMID: 21994714
ectromelia; variola; monkeypox; animal model; mousepox; infection route; antiviral; CMX001; ST-246
16.  Comparative Pathology of Smallpox and Monkeypox in Man and Macaques 
Summary
In the three decades since the eradication of smallpox and cessation of routine vaccination, the collective memory of the devastating epidemics caused by this orthopoxvirus has waned, and the human population has become increasingly susceptible to a disease that remains high on the list of possible bioterrorism agents. Research using surrogate orthopoxviruses in their natural hosts, as well as limited variola virus research in animal models, continues worldwide; however, interpretation of findings is often limited by our relative lack of knowledge about the naturally occurring disease. For modern comparative pathologists, many of whom have no first-hand knowledge of naturally occurring smallpox, this work provides a contemporary review of this historical disease, as well as discussion of how it compares with human monkeypox and the corresponding diseases in macaques.
doi:10.1016/j.jcpa.2012.06.007
PMCID: PMC3498598  PMID: 22884034
smallpox; monkeypox; variola; macaque
17.  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
18.  Emergence and Reemergence of Vaccinia-Like Viruses: Global Scenario and Perspectives 
Among the members of the genus Orthopoxvirus (OPXV), vaccinia virus (VACV), the type species of the genus is a double-stranded DNA virus, belongs to the subfamily Chordopoxvirinae of the family Poxviridae. The causative agents of smallpox, VACV and Variola virus are mutually immunogenic and the type species of Orthopoxvirus, cause only mild complications in humans. Therefore, the VACV was used as a smallpox vaccine world over under mass immunization program promoted by World Health Organization, which lead to the variola eradication globally in 1979. Since then, no vaccination of human population has been carried out; however, vaccination has been continued for at-risk laboratory workers, military personnel and others working with recombinant VACV or other non-variola orthopoxviruses (OPXVs). There has now been a surge in the development of safer smallpox vaccines and understanding of the biology of VACV necessitating re-use of this vaccine in most vulnerable population, because of rise in bioterrorist threats globally. Also, globally there has been the emergence and re-emergence of vaccinia-like viruses (VLVs) in Brazil, buffalopox viruses in Egypt, Indonesia, India and its neighbouring countries like Nepal, Pakistan. Bioterrorism as well as emergence and re-emergence of the VLVs constitute a concern as 50 % of the population globally (40 % in USA) <30 years are unvaccinated and most vulnerable for smallpox reemergence. Thus, the search for new generation safer smallpox vaccine entails review of biology of VLVs in the smallpox-free world. In this review, we present occurrence of VLVs in the world with exhaustive discussion particularly on the emergence and re-emergence of these viruses in India and Brazil where VLVs are sufficiently studied.
doi:10.1007/s13337-012-0068-1
PMCID: PMC3550805  PMID: 23729995
Vaccinia virus; Vaccinia-like viruses; Buffalopox; Emergence and reemergence; Zoonosis
19.  A vaccinia virus renaissance 
In 1796, Edward Jenner introduced the concept of vaccination with cowpox virus, an Orthopoxvirus within the family Poxviridae that elicits cross protective immunity against related orthopoxviruses, including smallpox virus (variola virus). Over time, vaccinia virus (VACV) replaced cowpox virus as the smallpox vaccine, and vaccination efforts eventually led to the successful global eradication of smallpox in 1979. VACV has many characteristics that make it an excellent vaccine and that were crucial for the successful eradication of smallpox, including (1) its exceptional thermal stability (a very important but uncommon characteristic in live vaccines), (2) its ability to elicit strong humoral and cell-mediated immune responses, (3) the fact that it is easy to propagate, and (4) that it is not oncogenic, given that VACV replication occurs exclusively within the host cell cytoplasm and there is no evidence that the viral genome integrates into the host genome. Since the eradication of smallpox, VACV has experienced a renaissance of interest as a viral vector for the development of recombinant vaccines, immunotherapies, and oncolytic therapies, as well as the development of next-generation smallpox vaccines. This revival is mainly due to the successful use and extensive characterization of VACV as a vaccine during the smallpox eradication campaign, along with the ability to genetically manipulate its large dsDNA genome while retaining infectivity and immunogenicity, its wide mammalian host range, and its natural tropism for tumor cells that allows its use as an oncolytic vector. This review provides an overview of new uses of VACV that are currently being explored for the development of vaccines, immunotherapeutics, and oncolytic virotherapies.
doi:10.4161/hv.21080
PMCID: PMC3495727  PMID: 22777090
cancer immunotherapy; oncolytic cancer therapy; smallpox; vaccines; vaccinia virus; viral vectors
20.  The Vaccinia Virus Superoxide Dismutase-Like Protein (A45R) Is a Virion Component That Is Nonessential for Virus Replication 
Journal of Virology  2001;75(15):7018-7029.
A characterization of the A45R gene from vaccinia virus (VV) strain Western Reserve is presented. The open reading frame is predicted to encode a 125-amino-acid protein (Mr, of 13,600) with 39% amino acid identity to copper-zinc superoxide dismutase (Cu-Zn SOD). Sequencing of the A45R gene from other orthopoxviruses, here and by others, showed that the protein is highly conserved in all viruses sequenced, including 16 strains of VV, 2 strains of cowpox virus, camelpox virus, and 4 strains of variola virus. In all cases the protein lacks key residues involved in metal ion binding that are important for the catalytic activity. The A45R protein was expressed in Escherichia coli, purified, and tested for SOD activity, but neither enzymatic nor inhibitory SOD activity was detected. Additionally, no virus-encoded SOD activity was detected in infected cells or purified virions. A monoclonal antibody raised against the A45R protein expressed in E. coli identified the A45R gene product as a 13.5-kDa protein that is expressed late during VV infection. Confocal microscopy of VV-infected cells indicated that the A45R protein accumulated predominantly in cytoplasmic viral factories. Electron microscopy and biochemical analyses showed that the A45R protein is incorporated into the virion core. A deletion mutant lacking the majority of the A45R gene and a revertant virus in which the deleted gene was restored were constructed and characterized. The growth properties of the deletion mutant virus were indistinguishable from those of wild-type and revertant viruses in all cell lines tested, including macrophages. Additionally, the virulence and pathogenicity of the three viruses were also comparable in murine and rabbit models of infection. A45R is unusual in being the first VV core protein described that affects neither virus replication nor virulence.
doi:10.1128/JVI.75.15.7018-7029.2001
PMCID: PMC114430  PMID: 11435582
21.  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
22.  Development of Vaccinia Reporter Viruses for Rapid, High Content Analysis of Viral Function at All Stages of Gene Expression 
Antiviral research  2011;91(1):72-80.
Vaccinia virus is the prototypical orthopoxvirus of Poxviridae, a family of viruses that includes the human pathogens Variola (smallpox) and Monkeypox. Core viral functions are conserved among orthopoxviruses, and consequently Vaccinia is routinely used to study poxvirus biology and screen for novel antiviral compounds. Here we describe the development of a series of fluorescent protein-based reporter Vaccinia viruses that provide unprecedented resolution for tracking viral function. The reporter viruses are divided into two sets: 1) single reporter viruses that utilize temporally regulated early, intermediate, or late viral promoters; and 2) multi-reporter viruses that utilize multiple temporally regulated promoters. Promoter and reporter combinations were chosen that yielded high signal-to-background for stage-specific viral outputs. We provide examples for how these viruses can be used in the rapid and accurate monitoring of Vaccinia function and drug action.
doi:10.1016/j.antiviral.2011.04.014
PMCID: PMC3177160  PMID: 21569797
23.  Smallpox DNA Vaccine Protects Nonhuman Primates against Lethal Monkeypox 
Journal of Virology  2004;78(9):4433-4443.
Two decades after a worldwide vaccination campaign was used to successfully eradicate naturally occurring smallpox, the threat of bioterrorism has led to renewed vaccination programs. In addition, sporadic outbreaks of human monkeypox in Africa and a recent outbreak of human monkeypox in the U.S. have made it clear that naturally occurring zoonotic orthopoxvirus diseases remain a public health concern. Much of the threat posed by orthopoxviruses could be eliminated by vaccination; however, because the smallpox vaccine is a live orthopoxvirus vaccine (vaccinia virus) administered to the skin, the vaccine itself can pose a serious health risk. Here, we demonstrate that rhesus macaques vaccinated with a DNA vaccine consisting of four vaccinia virus genes (L1R, A27L, A33R, and B5R) were protected from severe disease after an otherwise lethal challenge with monkeypox virus. Animals vaccinated with a single gene (L1R) which encodes a target of neutralizing antibodies developed severe disease but survived. This is the first demonstration that a subunit vaccine approach to smallpox-monkeypox immunization is feasible.
doi:10.1128/JVI.78.9.4433-4443.2004
PMCID: PMC387704  PMID: 15078924
24.  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
25.  Therapeutic and prophylactic drugs to treat orthopoxvirus infections 
Future virology  2008;3(6):595-612.
With the global eradication of smallpox in 1979, the causative agent, variola, no longer circulates in human populations. Other human poxvirus infections, such as those caused by vaccinia, cowpox virus and molluscum, are usually relatively benign in immunocompetent individuals. Conversely, monkeypox virus infections cause high levels of mortality and morbidity in Africa and the virus appears to be increasing its host range, virulence and demographic environs. Furthermore, there are concerns that clandestine stocks of variola virus exist. The re-introduction of aerosolized variola (or perhaps monkeypox virus) into human populations would result in high levels of morbidity and mortality. The attractiveness of variola as a bioweapon and, to a certain extent, monkeypox virus is its inherent ability to spread from person-to-person. The threat posed by the intentional release of variola or monkeypox virus, or a monkeypox virus epizoonosis, will require the capacity to rapidly diagnose the disease and to intervene with antivirals, as intervention is likely to take place during the initial diagnosis, approximately 10-15 days postinfection. Preimmunization of ‘at-risk populations’ with vaccines will likely not be practical, and the therapeutic use of vaccines has been shown to be ineffective after 4 days of infection with variola. However, a combination of vaccine and antivirals for those infected may be an option. Here we describe historical, current and future therapies to treat orthopoxvirus diseases.
PMCID: PMC2600508  PMID: 19727418
acyclic nucleoside phosphonates; animal model; antiviral; CMX001; monkeypox; orthopoxvirus; smallpox; ST-246; vaccinia

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