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A. Taxonomy Information
B. Lifecycle Information (Henderson et al., 1999):
- Species:
- Variola virus (Website 4):
- GenBank Taxonomy No.: 10255
- Description: The poxviruses (of the family Poxviridae) are a family of large, enveloped deoxyribonucleic acid (DNA) viruses. The most notorious poxvirus is variola, the causative agent of smallpox. Smallpox was an important cause of morbidity and mortality in the developing world until recent times. Since the host range of the variola virus is confined to humans, aggressive case identification and contact vaccination were ultimately successful in controlling the disease. The last occurrence of endemic smallpox was in Somalia in 1977, and the last human cases were laboratory-acquired infections in 1978. By 1980, the World Health Organization (WHO) General Assembly ratified the declaration of success made by the Global Commission for the Certification of Smallpox Eradication (Website 1).
- Variant(s):
- Variola major virus (Website 5):
- GenBank Taxonomy No.: 12870
- Parent: Variola major virus
- Description: Variola major is the severe and most common form of smallpox, with a more extensive rash and high fever. There are four types of variola major smallpox: ordinary (the most frequent type, accounting for 90% or more of cases); modified (mild and occurring in previously vaccinated persons); flat; and hemorrhagic (both rare and very severe). Historically, variola major has an overall fatality rate of about 30%; however, flat and hemorrhagic smallpox usually are fatal (Website 13).
- Variola minor (Website 6, Website 2):
- GenBank Taxonomy No.: 52358
- Parent: Variola major virus
- Description: In 1904 Korte described a very mild smallpox-like disease with a case-fatality rate of 1% or less in unvaccinated persons that had occurred in South Africa for several years and was known locally as kaffir-pox, or "amaas" (Fenner et al., 1988). Subsequently, Chapin recognized that a similar mild disease had been occurring in North America since about 1896, and had subsequently been exported from there to South America, Europe, and Australia (Fenner et al., 1988). Virological studies showed that there was no doubt that 'amass' and 'alastrim' as it is called in South America were indeed mild varieties of smallpox. Although many other names were used, this clinico-epidemiological variety of smallpox has come to be called variola minor, a designation that led to the use of the term variola major for classical smallpox (Fenner et al., 1988).
C. Genome Summary:
- Virion (Henderson et al., 1999):
- Size: The virus particles are brick-shaped to ovoid and measure approximately 300 by 200 by 100 um (Klietmann and Ruoff, 2001).
- Shape: The virus particles are brick-shaped to ovoid and measure approximately 300 by 200 by 100 um (Klietmann and Ruoff, 2001).
- Picture(s):
- Smallpox virus by negative stain electron microscopy (Website 15):
Description: Smallpox virus, single virion, as seen by negative stain electron microscopy. The brick-shaped virion is covered with what looks like filaments (although in reality this outer layer is not really like a ball of string). This virion is from a human skin lesion, from a diagnostic specimen that came to the Centers for Disease Control in 1966 as part of the WHO Global Smallpox Eradication Program. Magnification about x150,000. Micrograph from F. A. Murphy, School of Veterinary Medicine, University of California, Davis.
- Other:
- Kaffir-pox Korte (1904) described a very mild pox-like disease that had occurred in South Africa for several years and was known locally as kaffir-pox, or "amaas" (Fenner et al., 1988B).
- Amaas Korte (1904) described a very mild pox-like disease that had occurred in South Africa for several years and was known locally as kaffir-pox, or "amaas" (Fenner et al., 1988B).
- Genome of Variola virus
- Description: VAR strain India-1967 was isolated from a patient in India in 1967. The strain had undergone 5 passages on chorioallantoic membranes of chick embryos and is maintained in the collection of WHO Collaborating Center on Smallpox and Related Infections, Institute for Viral Preparations, Moscow, Russian Federation (Shchelkunov et al., 1993).
- Variola virus Genome Information (Website 7, Website 11):
- GenBank Accession Number: NC_001611, X69198
- Size: 185578 bp (Website 7, Website 11)
- Gene Count: There are 187 putative proteins identified from the sequencing of variola virus (Website 1).
- Description: Possessing one of the largest genomes of any virus, an Orthopoxvirus consists of one piece of double-stranded DNA, which is cross-linked at each end. With their brick-shaped morphology, poxviruses have a biconcave core containing the DNA genome. The virus-encoded enzymes in the core are critical to transcription of the viral DNA. Genes encoding the nonessential functions important for virus virulence are arrayed near the ends of the genome; as would be expected, the greatest heterogeneity between poxviruses is at these genomic ends (Website 1).
- Genome of Variola major virus (Massung et al., 1994):
- Description: This virus strain was isolated from scab material from the last naturally occurring case of variola major in the world, which occurred in a 3-year-old girl who survived ordinary smallpox diagnosed on October 16, 1975, in Kuralia, South Dingaldi, Bhola Island, Bangladesh (Massung et al., 1994).
- Variola major virus Bangladesh-1975 Genome Information (Website 8):
- GenBank Accession Number: L22579
- Size: 186103 bp (Website 8)
- Gene Count: 187 (Massung et al., 1994)
- Description: Massung et al. (1994) analyzed the 186,102 base pairs (bp) that constitute the entire DNA genome of a highly virulent variola virus isolated from Bangladesh in 1975. They found that the linear, double-stranded molecule has relatively small (725 bp) inverted terminal repeat (ITR) sequences containing three 69-bp direct repeat elements, a 54-bp partial repeat element, and a 105-base telomeric end-loop that can be maximally base-paired to contain 17 mismatches. Proximal to the right-end ITR sequences are another seven 69-bp elements and a 53- and a 27-bp partial element. Sequence analysis showed 187 closely spaced open reading frames specifying putative major proteins containing greater than or equal to 65 amino acids. Most of the virus proteins correspond to proteins in current databases, including 150 proteins that have greater than 90% identity to major gene products encoded by vaccinia virus, the smallpox vaccine. Variola virus has a group of proteins that are truncated compared with vaccinia virus counterparts and a smaller group of proteins that are elongated. The terminal regions encode several novel proteins and variants of other poxvirus proteins that potentially augment variola virus transmissibility and virulence for its only natural host, humans (Massung et al., 1994).
- Genome of Variola minor (Shchelkunov et al., 2000):
- Description: This virus strain (Variola minor Garcia-1966) was isolated from skin lesions on a patient in Sao Paulo during an alastrim outbreak in Brazil in 1966 that was associated with a 0.8% case fatality rate. The isolate has been used as a diagnostic reference strain at the Adolpho Lutz Institute, Sao Paulo, and was provided to CDC in the late 1960's. It was also used as a reference strain at CDC (Shchelkunov et al., 2000).
- Variola minor Garcia-1966 Genome Information (Website 9, Website 10):
- GenBank Accession Number: X72086, Y16780
- Size: 186986 bp (Shchelkunov et al., 2000)
- Gene Count: By computer analysis, Shchelkunov et al. (2000) identified 206 non-overlapping potential open reading frames containing greater than or equal to 60 amino acids (Shchelkunov et al., 2000).
- Description: Shchelkunov et al. (2000) analyzed the alastrim variola minor virus, which causes mild smallpox, and was first recognized in Florida and South America in the late 19th century. Genome linear double-stranded DNA sequences (186,986 bp) of the alastrim virus Garcia-1966, a laboratory reference strain from an outbreak associated with 0.8% case fatalities in Brazil in 1966, were determined except for a 530-bp fragment of hairpin-loop sequences at each terminus. The DNA sequences showed 206 potential open reading frames for proteins containing greater than or equal to 60 amino acids. The amino acid sequences of the putative proteins were compared with those reported for vaccinia virus strain Copenhagen and the Asian variola major strains India-1967 and Bangladesh-1975. About one-third of the alastrim viral proteins were 100% identical to correlates in the variola major strains and the remainder were greater than or equal to 95% identical. Compared with variola major virus DNA, alastrim virus DNA has additional segments of 898 and 627 bp, respectively, within the left and right terminal regions. The former segment aligns well with sequences in other orthopoxviruses, particularly cowpox and vaccinia viruses, and the latter is apparently alastrim-specific (Shchelkunov et al., 2000).
A. Outbreak Locations:
B. Transmission Information:
- By the time vaccination was introduced at the end of the 18th century, the distribution of smallpox was world-wide. It was endemic everywhere, except in remote areas with sparse populations, such as Australia, New Zealand and the islands of the Pacific, Atlantic, and Indian Oceans (Fenner et al., 1988C). In 1959, the Twelfth World Health Assembly adopted a resolution introduced by the USSR calling for the world-wide eradication of smallpox (Fenner et al., 1988C). By 1976 the only remaining endemic country was Ethiopia, in which variola minor persisted and spread to Somalia (Fenner et al., 1988C). On October 26, 1977, the last case of naturally acquired smallpox occurred in the Merca District of Somalia. In May 1980, the World Health Assembly certified the world free of naturally occurring smallpox (MMWR, 2002).
C. Environment:
- From: Homo sapiens To: Homo sapiens , With Destination: Homo sapiens (Fenner et al., 1988C):
Mechanism: Smallpox is a viral disease unique to humans. To sustain itself, the virus must pass from person to person in a continuing chain of infection and is spread by inhalation of air droplets or aerosols (Henderson, 1999). There are three principal routes of viral infection corresponding to the three principal surfaces of the body: the respiratory tract, the alimentary tract, and the skin. Minor routes of infection include the urinary and genital tracts and the conjunctiva. Although congenital infection occasionally occurred in smallpox, it was of no epidemiological importance (Fenner et al., 1988C).
No environment information is currently available here.
D. Intentional Releases:
- Intentional Release information :
- Description: Smallpox probably was first used as a biological weapon during the French and Indian Wars (1754-1767) by British forces in North America. Soldiers distributed blankets that had been used by smallpox patients with the intent of initiating outbreaks among American Indians. Epidemics occurred, killing more than 50% of many affected tribes (Henderson et al., 1999).
- Emergency contact: If you believe that you have been exposed to a biological or chemical agent, or if you believe an intentional biological threat will occur or is occurring, please contact your local health department and/or your local police or other law enforcement agency (Website 12). A possible case of smallpox is a public health emergency and of utmost international concern. State health officials should be contacted immediately, and the diagnosis confirmed in a Biological Safety Level 4 laboratory where staff members have been vaccinated. The state officials should contact the CDC (Centers for Disease Control and Prevention) at any time of the day or night, telephone number, 770-488-7100 (Breman and Henderson, 2002).
- Delivery mechanism: Recent allegations from Ken Alibek, a former deputy director of the Soviet Union's civilian bioweapons program, have heightened concern that smallpox might be used as a bioweapon. Alibek reported that beginning in 1980, the Soviet government embarked on a successful program to produce the smallpox virus in large quantities and adapt it for use in bombs and intercontinental ballistic missiles; the program had an industrial capacity capable of producing many tons of smallpox virus annually. Furthermore, Alibek reports that Russia even now has a research program that seeks to produce more virulent and contagious recombinant strains. Because financial support for laboratories in Russia has sharply declined in recent years, there are increasing concerns that existing expertise and equipment might fall into non-Russian hands (Henderson et al., 1999).
- Containment: Medical personnel must be prepared to recognize a vesicular exanthem in possible biowarfare theaters as potentially smallpox, and to initiate appropriate countermeasures. Any confirmed case should be considered an international emergency, with an immediate report made not only to the chain of command but also to public health authorities. Strict quarantine with respiratory isolation should be applied for 17 days to all persons in direct contact with the index case or cases, especially the unvaccinated. Immediate vaccination should also be undertaken for all personnel exposed to either weaponized variola or monkeypox virus or a clinical case of smallpox. Medical personnel should have a history of vaccination and should undergo immediate revaccination to ensure solid immunity (Website 1).
A. Biosafety Information:
B. Culturing Information:
- Biosafety information for : Variola virus (Henderson et al., 1999):
- Biosafety Level: Laboratory examination requires high-containment (BL-4) facilities and should be undertaken only in designated laboratories with the appropriate training and equipment.
- Precautions:
- Laboratory confirmation of the diagnosis in a smallpox outbreak is important. Specimens should be collected by someone who has recently been vaccinated (or is vaccinated that day) and who wears gloves and a mask. To obtain vesicular or pustular fluid, it is often necessary to open lesions with the blunt edge of a scalpel. The fluid can then be harvested on a cotton swab. Scabs can be picked off with forceps. Specimens should be deposited in a vacutainer tube that should be sealed with adhesive tape at the juncture of stopper and tube. This tube, in turn, should be enclosed in a second durable, watertight container. State or local health department laboratories should immediately be contacted regarding the shipping of specimens (Henderson et al., 1999).
C. Diagnostic Tests :
- Variola virus Culturing Methods :
- Description: Most orthopox viruses can be grown in one or another kind of cultured cell and assayed by plaque counts in suitable susceptible cells (Fenner et al., 1988B). Species with a restricted host range, such as variola virus, replicate in a narrower range of cells and often produce hyperplastic foci. However, on serial passage, adaptation occurs readily and may involve change to a more lytic plaque. Monolayers infected with viruses that produce hyperplastic foci usually yield much less virus than those infected with viruses that produce lytic plaques, since most cells in the monolayer remain uninfected. Differential growth capacity in particular cell lines (e.g., the rabbit cell line RK 13 and pig embryo kidney cells) may be useful in distinguishing between variola and monkeypox viruses when these viruses are first inoculated into such cells; however, adaptation occurs readily (Fenner et al., 1988B).
- Medium:
- Most human and non-human primate cells, and some cells derived from other species (rabbit kidney and pig embryo kidney cells) are susceptible to infection with variola virus (Fenner et al., 1988B). Growth in pig embryo kidney cells was sometimes used to differentiate between variola and monkeypox viruses (Fenner et al., 1988B).
- Note: Out of 186 specimens that were tested by all four methods, 182 were positive by electron microscopy, 135 by tissue culture inoculation and 117 by chorioallantoic membrane inoculation. Gel precipitation was the least sensitive technique (Fenner et al., 1988B).
- Organism Detection Tests:
- Variola virus Particles Detection:
- Time to Perform: minutes-to-1-hour
- Description: Under light microscopy, aggregations of variola virus particles, called Guarnieri bodies, correspond to B-type poxvirus inclusions. These cytoplasmic inclusions are hematoxylinophilic, stain reddish purple with Giemsa stain, and contain Feulgen-positive material (Website 1). The appearance of characteristic virions on electron microscopy or Guarnieri bodies under light microscopy is useful but does not discriminate variola from vaccinia, monkeypox, or cowpox (Franz et al., 1997).
- Variola virus Electron Microscopy:
- Time to Perform: 1-hour-to-1-day
- Description: Widespread use of electron microscopy as a diagnostic method was not feasible until the negative staining technique was introduced (Fenner et al., 1988B). Others have shown the value of this method for recognizing poxvirus or herpes virus particles in vesicle fluid and scabs taken directly from patients (Fenner et al., 1988B). Electron microscopy had the advantage of being much the most rapid method of making a presumptive diagnosis, which was a very important requirement, especially in nonendemic countries. In scabs or material that had been some time in transit, it was also the most sensitive, although fields might have to be searched for as long as 30 minutes before a specimen was declared negative (Fenner et al., 1988B). The appearance of characteristic virions on electron microscopy or Guarnieri bodies under light microscopy is useful but does not discriminate variola from vaccinia, monkeypox, or cowpox (Franz et al., 1997).
- False Positive: Out of 186 specimens that were tested by all four methods, 182 were positive by electron microscopy, 135 by tissue culture inoculation and 117 by chorioallantoic membrane inoculation. Gel precipitation was the least sensitive technique (Fenner et al., 1988B).
- Gel diffusion or gel precipitation:
- Description: The gel diffusion test, in which vesicular fluid from a pox lesion was incubated with vaccinia hyperimmune serum, constitutes a rapid and inexpensive method when microscopy is not available (Website 1).
- False Positive: Out of 186 specimens that were tested by all four methods, 182 were positive by electron microscopy, 135 by tissue culture inoculation and 117 by chorioallantoic membrane inoculation. Gel precipitation was the least sensitive technique (Fenner et al., 1988B).
- False Negative: The gel precipitation was the least sensitive technique and was often negative in lesion material that had been exposed to ambient temperatures for several days (Fenner et al., 1988B).
- Inoculation of the chorioallantoic membrane:
- Description: Although cell culture was sometimes more sensitive, inoculation on the chorioallantoic membrane was generally a more useful test with field material, since positive results could be obtained even with scabs that were contaminated with bacteria, which typically destroyed cell cultures. Also, if the content of viable virus was low it often took several days, and perhaps serial passage, before characteristic lesions occurred in cultured cells, whereas a result could always be obtained within 3 days by chorioallantoic membrane inoculation (Fenner et al., 1988B). Inoculation on the chorioallantoic membrane had the great advantage of allowing differentiation between the 4 orthopoxviruses that can infect man (variola, monkeypox, cowpox, and vaccinia viruses). It was also the most sensitive with fresh specimens of vesicular fluid, since one infectious particle was potentially capable of producing a pox (Fenner et al., 1988B).
- False Positive: Out of 186 specimens that were tested by all four methods, 182 were positive by electron microscopy, 135 by tissue culture inoculation and 117 by chorioallantoic membrane inoculation. Gel precipitation was the least sensitive technique (Fenner et al., 1988B).
- False Negative: In 1979 Nakano found that the susceptibility of the chorioallantoic membrane, although usually quite satisfactory, was sometimes unacceptably low, as judged by control inoculation in cultured cells. For this reason he found it useful to make inoculations on cultured cells, especially with critical specimens in which recovery of the responsible virus was very desirable (Fenner et al., 1988B).
- Immunoassay Tests:
No immuno-assay tests available here.
- Nucleic Acid Detection Tests: :
- Ibrahim's PCR Detection:
- Description: 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/microl. to 1 ng/microl. Contained within those 322 samples were variola virus DNA, obtained from purified viral preparations, at concentrations of 1 fg/ microl to 1 ng/ microl. 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/ microl to 1 ng/ microl, 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/ microl to 1 ng/ microl, 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 (Ibrahim et al., 2003).
- Primers:
- OPXJ7R3U, OPXJ7R3L and probe VARJ7R3P
- Forward: TCATACTGGAGAATCCACAACA (Ibrahim et al., 2003)
- Reverse: CATCATTGGCGGTTGATTTA (Ibrahim et al., 2003)
- Real-time-probe: CAAGACGTCGGGACCAATTACTAATA (Ibrahim et al., 2003)
- Product
- Size: Over 942 nucleotides (Ibrahim et al., 2003).
- Product source: Website 4
- False Positive: 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%) (Ibrahim et al., 2003).
- False Negative: Of the 206 samples that contained variola virus DNA ranging in concentrations from 100 fg/ microl to 1 ng/ microl, 8 samples were considered negative on the Smart Cycler platform and 1 sample was considered negative on the LightCycler platform (Ibrahim et al., 2003).
- Ropp's PCR Detection:
- Description: 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 (NACP1 and NACP2) 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 EACP1 and EACP2) 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 (Ropp et al., 1995).
- Primers:
- Espy's PCR Detection (Espy et al., 2002):
- Description: A 300-bp plasmid fragment of the hemagglutinin gene was used as target DNA to develop a rapid real-time LightCycler (Roche Applied Science, Indianapolis, Ind.) PCR assay for laboratory detection of smallpox virus. PCR primers and probes were designed specifically for detection of smallpox virus DNA, but all viruses of the genus Orthopoxvirus tested could be detected by use of the hemagglutinin gene target sequence. Base pair mismatches in the 204-bp amplicon allowed discrimination of cowpox virus (melting temperature [Tm], 56.40 C), monkeypox virus (Tm, 56.24 C), and vaccinia virus (Tm, 56.72 C), including the Dryvax vaccine strain, from smallpox virus (Tm, 62.45 C) by melting curve analysis. The analytical sensitivity was 5 to 10 copies of target DNA per sample. The assay was specific for members of the genus Orthopoxvirus; the DNAs of herpes simplex virus and varicella-zoster virus were not detected by the smallpox virus LightCycler PCR (Espy et al., 2002).
- Primers:
- Pair of primers
- Forward: CTA ATA TCA TTA GTA TAC GCT ACA C
- Reverse: GAG TCG TAA GAT ATT TTA TCC
- Product
- Size: 204 bp
- Restriction fragment length polymorphism (RFLP) assay (Loparev et al., 2001):
- Description: 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 (Loparev et al., 2001).
- Microarray method to detect Orthopox viruses:
- Time to Perform: 1-hour-to-1-day
- Description: We have developed a new microarray-based method that detects simultaneously and discriminates four orthopoxvirus (OPV) species pathogenic for humans (variola, monkeypox, cowpox, and vaccinia viruses) and distinguishes them from chickenpox virus (varicella-zoster virus or VZV). The OPV gene C23L/B29R, encoding the CC-chemokine binding protein, was sequenced for 41 strains of seven species of orthopox viruses obtained from different geographical regions. Those C23L/B29R sequences and the ORF 62 sequences from 13 strains of VZV (selected from GenBank) were used to design oligonucleotide probes that were immobilized on an aldehyde-coated glass surface (a total of 57 probes). The microchip contained several unique 13-21 bases long oligonucleotide probes specific to each virus species to ensure redundancy and robustness of the assay. A region approximately 1100 bases long was amplified from samples of viral DNA and fluorescently labeled with Cy5-modified dNTPs, and single-stranded DNA was prepared by strand separation. Hybridization was carried out under plastic coverslips, resulting in a fluorescent pattern that was quantified using a confocal laser scanner. 49 known and blinded samples of OPV DNA, representing different OPV species, and two VZV strains were tested. The oligonucleotide microarray hybridization technique identified reliably and correctly all samples. This new procedure takes only 3 h, and it can be used for parallel testing of multiple samples (Laassri et al., 2003).
- Other Types of Diagnostic Tests:
No other tests available here.
A. Journal References:Breman and Henderson, 2002: Breman Joel G. , Henderson D.A. Diagnosis and Management of smallpox. The New England Journal of Medicine. 2002 ; 346 ( 17 ): 1300 - 1308 . [PubMed: 11923491].Espy et al., 2002: Espy M.J. , Cockerill III F.R. , Meyer R.F. , Bowen MD, Poland GA, Hadfield TL, Smith TF. Detection of smallpox virus DNA by LightCycler PCR. J Clin Microbiol . 2002 ; 40 ( 6 ): 1985 - 1988 . [PubMed: 12037052].Franz et al., 1997: Franz David R. , Jahrling Peter B. , Friedlander Arthur M. , McClain DJ, Hoover DL, Bryne WR, Pavlin JA, Christopher GW, Eitzen EM Jr. Clinical recognition and management of patients exposed to biological warfare agents. JAMA . 1997 ; 278 ( 5 ): 399 - 411 . [PubMed: 9244332].Goldstein et al., 1975: Goldstein J.A. , Neff J.M. , Lane J.M. , Koplan. Smallpox vaccination reactions, prophylaxis, and therapy of complications. Pediatrics . 1975 ; 55 ( 3 ): 342 - 347 . [PubMed: 238178].Hammarlund et al., 2003: Hammarlund E, Lewis MW, Hansen SG, Strelow LI, Nelson JA, Sexton GJ, Hanifin JM, Slifka MK. Duration of antiviral immunity after smallpox vaccination. Nat Med.. 2003; 9(9): 1131 - 1137. [PubMed: 12925846].Hassett, 2003: Hassett DE Smallpox infections during pregnancy, lessons on pathogenesis from nonpregnant animal models of infection. J Reprod Immunol. 2003; 60(1): 13 - 24. [PubMed: 14568674].Henderson, 1999: Henderson D.A. Smallpox: clinical and epidemiologic features. Emerg Infect Dis . 1999 ; 5 ( 4 ): 537 - 539 . [PubMed: 10458961].Henderson et al., 1999: Henderson D.A. , Inglesby T.V. , Bartlett J.G. , Ascher MS, Eitzen E, Jahrling PB, Hauer J, Layton M, McDade J, Osterholm MT, O'Toole T, Parker G, Perl T, Russell PK, Tonat K. Smallpox as a biological weapon: medical and public health management. Working Group on Civilian Biodefense. JAMA . 1999 ; 281 ( 22 ): 2127 - 2137 . [PubMed: 10367824].Ibrahim et al., 2003: Ibrahim MS, Kulesh DA, Saleh SS, Damon IK, Esposito JJ, Schmaljohn AL, Jahrling PB. Real-time PCR assay to detect smallpox virus. J Clin Microbiol.. 2003; 41(8): 3835 - 3839. [PubMed: 12904397].Klietmann and Ruoff, 2001: Klietmann W.F. , Ruoff K.L. Bioterrorism: implications for the clinical microbiologist. Clin Microbiol Rev. 2001 ; 14 ( 2 ): 364 - 381 . [PubMed: 11292643].Laassri et al., 2003: Laassri M, Chizhikov V, Mikheev M, Shchelkunov S, Chumakov K. Detection and discrimination of orthopoxviruses using microarrays of immobilized oligonucleotides. J Virol Methods. 2003; 112(1-2): 67 - 78. [PubMed: 12951214].Loparev et al., 2001: Loparev V.N. , Massung R.F. , Esposito J.J. , Meyer H. Detection and differentiation of old world orthopoxviruses: restriction fragment length polymorphism of the crmB gene region. J Clin Microbiol. 2001 ; 39 ( 1 ): 94 - 100 . [PubMed: 11136755].Mahoney et al., 2003: Mahoney MC, Symons AB, Kimmel SR. Smallpox: clinical highlights and considerations for vaccination. J Postgrad Med.. 2003; 49(2): 141 - 147. [PubMed: 12867690].Massoudi et al., 2003: Massoudi MS, Barker L, Schwartz B. Effectiveness of postexposure vaccination for the prevention of smallpox: results of a delphi analysis. J Infect Dis. 2003; 188(7): 973 - 976. [PubMed: 14513416].Massung et al., 1994: Massung R.F. , Liu L.I. , Knight J.C. , Knight JC, Yuran TE, Kerlavage AR, Parsons JM, Venter JC, Esposito JJ. Analysis of the complete genome of smallpox variola major virus strain Bangladesh-1975. Virology . 1994 ; 201 ( 2 ): 215 - 240 . [PubMed: 8184534].Maurer et al., 2003: Maurer DM, Harrington B, Lane LM. Smallpox vaccine: contraindications, administration, and adverse reactions. Am Fam Physician. 2003; 68(5): 889 - 896. [PubMed: 13678138].MMWR, 2002: 25th Anniversary of the last case of naturally acquired smallpox. MMWR Morb Mortal Wkly Rep . 2002 ; 51 ( 42 ): 952 - 952 . [PubMed: 12442741].Quenelle et al., 2003: Quenelle DC, Collins DJ, Kern ER Efficacy of multiple- or single-dose cidofovir against vaccinia and cowpox virus infections in mice. Antimicrob Agents Chemother.. 2003; 47(10): 3275 - 3280. [PubMed: 14506041].Ropp et al., 1995: Ropp S.L. , Jin Q. , Knight J.C. , Massung RF, Esposito JJ. PCR strategy for identification and differentiation of smallpox and other orthopoxviruses. J Clin Microbiol. 1995 ; 33 ( 8 ): 2069 - 2076 . [PubMed: 7559950].Shchelkunov et al., 1993: Shchelkunov Sergei N. , Blinov Vladimir M. , Sandakhchiev Lev S. Genes of variola and vaccinia viruses necessary to overcome the host protective mechanisms. FEBS Lett . 1993 ; 319 ( 1-2 ): 80 - 83 . [PubMed: 8384129].Shchelkunov et al., 2000: Shchelkunov S.N. , Totmenin A.V. , Loparev V.N. , Safronov PF, Gutorov VV, Chizhikov VE, Knight JC, Parsons JM, Massung RF, Esposito JJ. Alastrim smallpox variola minor virus genome DNA sequences. Virology. 2000 ; 266 ( 2 ): 361 - 386 . [PubMed: 10639322].
B. Book References:Fenner et al., 1988: Fenner F. , Henderson D.A. , Arita I. , Jezek Z. , Ladnyi I.D. Chapter 1: The clinical features of smallpox. 1 - 68 . In: Smallpox and its eradication 1988 . World Health Organization (WHO) , .Fenner et al., 1988B: Fenner F. , Henderson D.A. , Arita I. , Jezek Z. , Ladnyi I.D. Chapter 2: Variola virus and other orthopoxviruses>. 69 - 119 . In: Smallpox and its eradication 1988 . World Health Organization (WHO) , .Fenner et al., 1988C: Fenner F. , Henderson D.A. , Arita I. , Jezek Z. , Ladnyi I.D. Chapter 4: The epidemiology of smallpox . 169 - 208 . In: Smallpox and its eradication 1988 . World Health Organization (WHO) , .
C. Website References:Website 1: Smallpox [ http://www.vnh.org/MedAspChemBioWar/chapters/chapter_27.htm ].Website 2: Smallpox [ http://www.emedicine.com/aaem/topic550.htm ].Website 3: Homo sapiens [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Tree&id=9606&lvl=3&keep=1&srchmode=1&unlock ].Website 4: Variola virus [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10255&lvl=3&lin=f&keep=1&srchmode=1&unlock ].Website 5: Variola major virus [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=12870&lvl=3&keep=1&srchmode=1&unlock ].Website 6: Variola minor virus [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=53258&lvl=3&keep=1&srchmode=1&unlock ].Website 7: Variola virus, complete genome [ http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=nucleotide&list_uids=9627521&dopt=GenBank ].Website 8: Variola major virus (strain Bangladesh-1975) complete genome [ http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=nucleotide&list_uids=623595&dopt=GenBank ].Website 9: Variola minor virus complete genome [ http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=nucleotide&list_uids=1150658&dopt=GenBank ].Website 10: variola minor virus complete genome [ http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=nucleotide&list_uids=5830555&dopt=GenBank ].Website 11: Variola virus DNA complete genome [ http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=nucleotide&list_uids=456758&dopt=GenBank ].Website 12: CDC: Public Health Emergency Preparedness and Response [ http://www.bt.cdc.gov/emcontact/index.asp ].Website 13: Smallpox Fact Sheet. Smallpox Overview [ http://www.bt.cdc.gov/agent/smallpox/overview/disease-facts.asp ].Website 14: SMALLPOX FACT SHEET People Who Should NOT Get the Smallpox Vaccine (Unless they are exposed to smallpox) [ http://www.bt.cdc.gov/agent/smallpox/vaccination/contraindications-public.asp ].Website 15: UCDavis School Of Veterinary Medicine Virus Images [ http://www.vetmed.ucdavis.edu/viruses/download.html ].Website 16: Smallpox Disease Images at CDC [ http://www.bt.cdc.gov/agent/smallpox/smallpoximages.asp ].
D. Thesis References:
No thesis or dissertation references used.

subset: commoncrawl


text:
Massive global tsunami competition down to final five
An astonishing 379 entries from 43 countries have been whittled down to a shortlist of five in the international competition to design a tsunami memorial in Thailand.The final five ( pictured above and below), who are anonymous, will now battle it out to win the contest to build one of the most significant memorials since the Second World War. The competition received an extraordinary 679 expressions of interest from all around the world. The five will now pick up a US$25,000 (£14,000) honorarium to work up a more detailed design that meets the brief's demand that the final scheme should be both 'appropriate' and 'interactive'. The final designs of the five entries are due for submission on 2 May 2006. The selection process by the Stage II Jury is expected to be completed by 14 May and the winning design will be submitted to the Thai government on 22 May. For further information, visit www.tsunamimemorial.or.thby Ed Dorrell
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text: << Back Header Content Recruit LA: Alumni Only Career ExpoDate: 3/20/2014Time: 10:00 AM to 2:30 PM Oregon State University alumni are invited to attend the Alumni Only Career Expo Thursday, March 20, 2014 10:00 AM to 2:30 PM University of Southern California Troudale Parkway Los Angeles, CA Hiring or looking for a new career? Whether you're hiring or looking to network or for a new career in finance, investment banking, marketing, technology, media, nonprofit, consulting and more, you will find all the best talent, employers and career opportunities at Recruit LA. We are expecting 150 top employers and 2,000 elite alumni attendees. ALUMNI WELCOME FROM The University of Southern California (host), Pepperdine University, California Institute of Technology, University of California at Berkeley, California State Polytechnic University at Pomona, University of California at Irvine, Stanford University, Loyola Marymount University, Harvey Mudd College, California State University at Fullerton, Chapman University, California State University at Northridge, Oregon State University, Carnegie Mellon University, Dartmouth University, Emory University, New York University, Arizona State University, Notre Dame, George Washington University, University of Maryland, University of Oregon, University of Iowa, Pomona College, LaSalle University, The University of La Verne, University of California San Diego, Harvey Mudd, Case Western Reserve University, Occidental College, University of Arizona, University of Indiana, Penn State, Ohio State University. Early bird registration: Employers Job Seekers Register for Early VIP Registration by March 7th – The VIP ticket highlights your resume in the resume book that is presented to all the attending employers. $10.00 plus fee Final Registration – March 20th - $15.00 plus fee Question? Contact Kelly Grunther: 917-841-2657 or email@example.com. Contact InformationOSU Alumni Association 204 CH2M HILL Alumni Center Corvallis, OR 97331 (541) 737-2351 Toll free: (877) OSTATER (877-678-2837) email@example.com Date & LocationDate: 3/20/2014Time: 10:00 AM to 2:30 PMLocation: University of Southern California Troudale Parkway Los Angeles, CA 90089


text:
The Phillies rode the bats of Pat Burrell and Chase Utley through April, but May has proven to be the kryptonite for these men of steel.
Following a 6-3 loss, there's really no need to get into the same, tired Adam Eaton rant because it's the same issue over and over. He caught the fat part of the plate more than you should with a fastball he had trouble controlling early. He made dumb mistakes and didn't put away batters when he had the chance. Rod Barajas' grand slam pitch wasn't a bad one, but for the most part, the Blue Jays waited for Eaton to get behind or make mistakes and hit him harder than the box score suggests.
Ryan Howard had his fifth three-strikeout 0-fer of the season, as the home fans continue their unrest. But it wasn't Howard who blew the biggest opportunity. That honor went to Chase Utley, who grounded out on one pitch with the bases juiced in the third inning. Utley has seen his average fall from .369 to .310 this month and has one extra-base hit since May 6.
For the first time since April 3, Pat Burrell's OPS fell below 1.000 after an 0-for-4 night. After a scorching start, voted as one of the runner-ups to Utley in National League Player of the Month voting, his line now reads like that of a human being - .286/.427/.564 - and he hasn't gone deep since May 2. Comparatively speaking, Howard has been the better producer than these two lately, although you'd never know it by the tone being fostered at Citizens Bank Park.


text:
The Hamilton County school board decided Thursday not to take away medical benefits from contract bus drivers.
Superintendent Rick Smith said he would submit another version of the budget next week.
Smith also told the board that the cuts proposed are too severe and he will go to the County Commission to ask for more funding.
Joan Garrett McClane has been a staff writer for the Times Free Press since August 2007. Before becoming a general assignment writer for the paper, she wrote about business, higher education and the court systems. She grew up the oldest of five sisters near Birmingham, Ala., and graduated with a master's and bachelor's degrees in journalism from the University of Alabama. Before landing her first full-time job as a reporter at the Times Free Press, ...
The donation to Rhonda Thurman from the contract bus drivers was just a smidgen under the $1,000 maximum allowed. Turned out to be a pretty good investment I guess. http://elect.hamiltontn.gov/candidate/current/part2/Thurman,%20Rhonda/2004-07-28%20Fin.%20Disc.pdf
"Board members were told there is still no contract with the contract bus drivers with school fast approaching. There are 49 spots alloted for the private operators. The rest of the school bus program is by the Durham Transportation firm.
Board member Rhonda Thurman said the contract bus drivers "take a hit" every year, while Durham is spared."
A little campaign donation makes a good friend of Rhonda Thurman. Some people think that Thurman looks out for the regular folks. They are right that she will "speak her mind" but the the mind she speaks looks out for her cronies and donors, like any typical politician.
"Board members said they did not want to cut medical benefits for contract school bus drivers, which means drawing down another $490,000 from the fund balance."
Yep, always worried about cutting costs, that Rhonda Thurman.
So we're quoting Chattanoogan.com on the TFP website? Interesting.


text:
Molotov cocktail thrown at door of missionary building in Detroit
Someone threw a Molotov cocktail at the door of a missionary building in Detroit Wednesday morning.
Fire officials said the incident happened just after 7:30 a.m. at PIME Missionaries on Quincy Street.
No one was hurt and damage was minimal.
Read more Local News headlines:
- Rash of fires break out in SW Detroit Wednesday morning
- Campbell Ewald moving to Detroit, bringing jobs
- Detroit City Council mulls next move under financial crisis
- Shred old documents to protect privacy
- New search launched to find missing Canton man
- Memorial services set for slain Detroit mother
- Detroit police search for missing 13-year-old
- Man to stand trial for 72-year-old Detroit woman's shooting
- Detroit schools save big by not printing pay statements
The views expressed below are not those of Click On Detroit, WDIV, or its affiliated companies. By clicking on "Post," you acknowledge that you have read the Terms of Service and your comment is in compliance with such terms. Readers, please help keep this discussion respectful and on topic by flagging comments that are offensive or inappropriate (hover over the commenter's name and you'll see the flag option appear on right side of that line). And remember, respect goes both ways: Tolerance of others' opinions is important in a free discourse. If you're easily offended by strong opinions, you might skip reading comments entirely.


text: Flickr / Basecampbaker 1154 [ Gearing Up for Holiday Shopping By Amy Wright November 2, 2013 It's already November 2nd and not too early to plan for your holiday shopping. Here's some information to get "your ducks in order." Read More|Comment Category: Christmas, Shopping, Weekends Tags: black friday, Cyber Monday, holiday shopping, Small Business Saturday Send to a friend! Share on Facebook Share on Twitter Pin it! Reddit This! Share on Tumblr!


text:
A new day, a new month. I have a good feeling about October – what do you all hope to do/see/accomplish this month? (…) Read more »
Tagged: calendarFree People Blog
Download Our September Calendar!Sep 01, 2014
I hope everyone is enjoying the holiday weekend! September may be upon us but summer is not over yet :). Visit our flickr page or click on the image below to download the large version of our September calendar! (…) Read more »
Download Our June Calendar!Jun 03, 2014
Sorry for the delay in posting this month's calendar! June seemed to creep up out of nowhere, but I sure am happy it's here :). Summer is oh-so-close! Visit our flickr page to download the large version of the calendar or click on the image below. (…) Read more »
Download Our April Calendar!Apr 01, 2014
April! At long last, you're here… the month that leaves winter in its dust, offering us hope and renewal with each rain shower that helps coax the flowers out of hibernation. (…) Read more »
Download Our February Calendar!Feb 01, 2014
Happy first day of February! Head over to our flickr page to download our calendar, or click on the image below to be taken directly to the large version. (…) Read more »
Download Our October Calendar!Oct 01, 2013
October always strikes me as a month where everything gets a little bit darker… the days are shorter, and with Halloween waiting at the end of the month, a mysterious mood sinks in. It's a month to embrace our darker side a bit, to take long walks in the moonlight and wrap ourselves in cozy (…) Read more »
November Calendar & Wallpaper Download!Nov 01, 2012
This month we have two downloads for you! Our November calendar, which you can download from our flickr page in the horizontal or vertical version, and some pretty desktop wallpaper! (…) Read more »
April CalendarApr 01, 2012
Happy first of April! Head over to our flickr page to download the large size of the vertical or horizontal version of the calendar! (…) Read more »
Saturday DOMar 17, 2012
DO: plan a vacation (…) Read more »
March CalendarMar 01, 2012
I love March – you know why? Because it's the month when Spring starts :) Visit our flickr page to download the vertical or horizontal large version of the calendar! (…) Read more »
February CalendarFeb 01, 2012
It definitely doesn't feel like the first day of February here in Philly… it's supposed to be almost 65 degrees out today! But hey, I'll take it. And, there's a February 29th this year! Click here to download our February calendar in the horizontal or vertical version. (…) Read more »
The 2012 Calendar is Here – UPDATE!Jan 03, 2012
*UPDATE – here is the horizontal version!* Our lovely graphic design team did a great job putting this calendar together! You can download the January calendar from our flickr page, and we'll post them each month here on the blog! Enjoy :) *UPDATE: a horizontal version is now available! Download it on our flickr page. (…) Read more »


text:
The biweekly Senate Meeting resumes and tomorrow! Join us as we pass legislation and discuss important… http://t.co/RFZXErIfjN(about 1 day ago)
RT @gsusignal: SGA votes to send basketball legislation to Athletics http://t.co/a1CUStojig(about 17 days ago)
Come out to plaza today and Meet the people who represent your voice at #GSU! Also there will be a… http://t.co/veR4t44PjM(about 23 days ago)
Come to the @gsupanthers Football Game This Saturday at 2 p.m. And have the chance to win $50, $100,… http://t.co/sjQEl7IDHZ(about 42 days ago)
Let's Go State #PantherFamily #AllBlueAllIn #WeWearBlue(about 49 days ago)


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Snorkeling, Whale Watching, Sunset Cocktail Cruises, Captain Cook Historical Cruises and Private Charters


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Man charged with baseball bat assaultJuly 24,2013Charge is denied
in assault with bat
A man who allegedly struck another man multiple times with an aluminum baseball bat during a fight Monday in Poultney denied a felony charge in Rutland criminal court Tuesday.
Timothy S. Petty, 46, of Poultney pleaded innocent to aggravated assault, which carries a potential 15-year jail sentence.
Judge Theresa DiMauro released Petty on a $5,000 unsecured bond and on court-ordered conditions that he stay away from Eugene Webster, the man he allegedly hit with the bat.
Vermont State Police said they arrested Petty after a fight in front of his home on Maple Street in Poultney at around 6:15 p.m. Monday. Police said Petty used the bat to hit Webster in the leg, arm and head.MORE IN This Just InSki area operators in Vermont and across northern New England were giddy Wednesday at the... Full StoryCounty-wide storm Full StoryA New York woman is facing an arson charge for setting a scarecrow on fire. Full Story
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text:
Love Letter 3
ALTHOUGH, my Mistress, it
has not pleased you to remem-
ber the promise you made me when I
was last with you — that is, to hear
good news from you, and to have an
answer to my last letter; yet it seems
to me that it belongs to a true servant
(seeing that otherwise he can know
nothing) to inquire the health of his
mistress, and to acquit myself of the
duty of a true servant, I send you this
letter, beseeching you to apprise me
of your welfare, which I pray to God
may continue as long as I desire mine
own. And to cause you yet oftener
to remember me, I send you, by the
bearer of this, a buck killed late last
night by my own hand, hoping that
when you eat of it you may think of
the hunter; and thus, for want of
room, I must end my letter, written
by the hand of your servant, who very
often wishes for you instead of your
brother. H. R.
6 Responses to "Love Letter 3"
Claire, what does Henry mean by "who very often wishes for you instead of your brother???
Claire Reply:
February 8th, 2012 at 8:49 am
Hi Liz,
George was one of Henry VIII's favoured courtiers and so spent a lot of time with the King socially. He was also the one who delivered the love letters to Anne. Hope that helps!
When I read this letter and other love letters that he sent her, I find it so hard to understand how Henry who waited so long,,(seven years at least) to finally be with Anne,he could of then treated her the way that he did and her brother as well, who was a favourite courtier…The lovely and brave Anne Boleyn was and always will be my favourite Queen of all time.. I have followed her life story for over 45 years and it still never fails to this day to give me a lump in my throat when I read about her…
Lexi Reply:
July 14th, 2014 at 12:04 am
Henry was also a spoiled brat. Not getting what he wanted for so long made it even more important to him, but then when he finally got it, it wasn't what he expected. He wanted Anne, after they married, to no longer be the bold woman she was during their courting days. He wanted her to suddenly be meek and obedient, to ignore his affairs and to give him a son. Or two. Or seven. She couldn't do any of that – it wasn't who se was (except for the son thing – idk why she couldn't have a son), and so when he realized that, he discarded her like a piece of trash. God, I love Anne so much and her end was so tragic and extremely saddening. I wish she could have married Henry Percy and Katherine had been able to give Henry a boy. Wouldn't the world have been a much better place? I believe so.
Ok sorry for my rant. Lol. I love the Tudors. XD
I have always suspected that as a man and a hunter, Henry enjoyed the chase more than he enjoyed the catch. I think they spent so long building up In their heads what their relationship would be like, and when it finally happened it just wasn't what he thought it would be.
PHS Reply:
April 12th, 2014 at 10:43 am
Very well said, I think you're right.


text:
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Thread: Fitness DVD
03-02-12, 13:57 #1Member
- Join Date
- Feb 2007
- Location
- Bed? mmmm maybe not!
- Posts
- 193
Fitness DVD
Hi Ladies
Could anyone recommend a good at home fitness DVD?
I have lost 2 stone and now need to do some regular exercise! I need something for beginners and also not too high impact as I suffer from back problems, also I have not done any exercise for years so need something really simple!!! (so that I can follow the exercises, terrible co-ordination lol)
Thanks for any recommendations!
Joanne x
03-02-12, 14:42 #2Member
- Join Date
- Oct 2008
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- 162
jillian michaels 30 day shred


text:
this is a beautiful piece and i hope you don't mind but i actually got it tattooed on me
Wow, am I ever out of it. I just went in to reformat this design for the new iphone4 and happened upon your comment. You probably won't read this because it was so long ago, but of course I don't mind and am in fact very honoured. Awesome-looking tattoo, they did a really great job with it.
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1/3 Photos of Scottish Shortbread Cookies
Prep Time:
Cook Time:
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25 mins
This is a great recipe for light and flaky cookies! My family loves them. I like to have one with a cup of coffee....
Serves: 48
Units: US | Metric
Serving Size: 1 (25 g)
Servings Per Recipe: 48
Make this season merry, bright and manageable with our easy ideas.


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Dancer, Performer, Aspiring Drag Queen, Interested in Fashion, Music Lover, TV Watcher, Love of the Unique and Strange, Looking to give support and find support


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GreatSchools Rating
Teacher quality
Principal leadership
Parent involvement
Wonderful school! Teachers are caring and involved, and so is the principal. My only complaint is that they seem to focus on the Common Core curriculum, which I disagree with. I realize that most schools are adopting this curriculum but I wish LPS would reconsider.
Can't say enough about how happy we have been with Lenski. The teachers are exceptional and really have their eyes on pushing each child to their fullest potential (which I think/hope is also driven and supported by our great principal). The parents involvement is off the charts. We were nervous about "real school" and have been very very happy.
As a parent of a 5 year old, this is the first time my wife and I have dealt with "real" school since we were kids. The horror stories and our own bad experiences made us yearn for private school. It was too cost prohibitive. LENSKI IS Incredible. It has turned out to be everything we had hoped for in private school. It should be a model for other schools. My wife and I are allowed to volunteer at school and we are so glad we could because it allows us to see how school can be run with good direction. We could not be happier with the staff at all levels. My son was considerably behind (through no fault of his own) and as a kindegardner they are successfully bringing him up to speed. They have exceptional teachers and programs and I am amazed at how many of the names of each child they know from all over the school.
Lenski is a wonderful school with fantastic academic achievement. There is a large percentage of parent involvement and a very active PTO organization. While class size has increased over the past couple of years due to budget cuts statewide, the level of education has not suffered. The school has many resources for reading, computers , library sciences, etc. and pulls from these resources when needed for various learning needs and opportunities. The children in grades 3rd, 4th and 5th touch a computer daily and the younger grades do several times a week. There are also many enrichment programs offered before and after school such as mad science, computer club, spanish, various musical classes, art and many others. I have had three children over the past eight years enrolled in the school and found that each one has excelled. I love this school for my children!
Wonderful school! We were so happy with the teachers, how the school was run, the parent involvement and all the extras the school offered (music performance programs, foreign languages etc). My children are now in high school and college so it has been a few years, but they have both done well since leaving Lenski. I am sure due to the great foundation the school provided.
We have been very pleased with Lenski and their academic focus. Lenski's principal Dr. DeSpain has a remarkable reputation and delivers excellence. The parent involvement and volunteer participation is very high. Fundraising is hugely successful and helps support some great programs. I would highly recommend this gem to any family seeking a top notch school in the area.
Lenski is a small school with a great community and fabulous teachers! I think that the students do lots of hands on projects, music, art, and theater, and accelerated learning programs. Wonderful school!
They are children centric and have helped keep my daughter engaged and challenged.
Great school; involved parents; access to GT programming at early ages. Caring and committed teaching staff. PE and outdoor activities.
I have a kindergartner at Lois Lenski Elementary. This is a school with many high achieving children and extremely high parental involvement. Excellent music and art classes in curriculum. Many extra-curricular activites available like Spanish, Geography Club, Science, Art, etc. One huge disappointment is class size. Although they promote a 19:1 ratio, there are 26 kids in my son's kindergarten class with one teacher, and no aide. Class sizes of 25+ are the norm. The individual attention is not there, but somehow test scores are the highest in the district. Most parents and children are happy with the school despite large class sizes.
The quality of academic programs is superior. The teachers are highly educated, and they create a great learning environment for children. A sense of commorodory and sensitivity is taught to students. Before and after-school programs are abundant, providing opportunities to fulfill your child's full potential.
Excellent school choice! It's big for an elementary school, but that allows additional resources to be available for all types of learners. Incredible parent support in and out of classrooms. Great extra-curricular options. They teach to students abililities, challenging students with extra projects or advanced grade levels when needed. Teacher turnover is low, and kids come from out of district, just to attend Lenski. Super school!
This is a fabulous school. Lenski got my children back on track, academically and emotionally after a less than perfect charter school experience. Teachers here have high expectations for work and behaviour and it shows. Just come and walk the halls. Happy kids, great work displayed all over the walls!!The teachers and support staff are very accessible and friendly. I am willing to drive long distances to have my children in this school.
Community ratings and reviews do not represent the views of GreatSchools nor does GreatSchools check their accuracy or verify the reviewers' identities. Use your discretion when evaluating these reviews.
The Community Rating is the school's average rating from its community members (e.g., parents, students, and school staff). The highest possible rating is five stars; the lowest is one star.
6350 South Fairfax Way
Centennial, CO 80121
Phone: (303) 347-4575
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Impact Liberia Missions is the governing board serving alongside the Danny Buegar family in Liberia, Africa. This group of individuals and supporting churches hold Danny and his family very close in their hearts, as Danny works with the Liberian community to bring the hope of Jesus.


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A few years ago, Greg C. founded a coat drive to gather warm clothes for Bakersfield's needy residents. He shares that he got the idea from his Mom, Juanita, a local baker who'd always put out a table of slightly-worn clothing that she'd collected from family members. Greg had thought that the items were for sale. When his Mom passed away, he learned that Juanita had been giving the clothing away. Each year, Greg and his siblings organize a multi-location One Warm Coat drive in her memory. This week, we honor Greg C., his family and their work to keep their Mom's legacy alive in the community. Our hearts are warmed by this touching tribute and we know yours will be, too.
One Warm Coat: blog
Taking Inspiration from the Three "Rs" of Earth Day
Earth Day (Tuesday, April 22nd) is one of our favorite days! Founded 44 years ago by Sen. Gaylord Nelson (WI) as an environmental "teach-in" for Americans of all ages, the first Earth Day provided the clever motto "Reduce-Reuse-Recycle" to remind us of how easy it is to make a difference.
The first teach-in brought together homemakers, educators, church and religious organizations, professional groups and conservation groups across the U.S. Each lent their strength to the important work of cleaning up their communities. (Sounds very familiar…) Today, Earth Day is a worldwide movement in over 125 countries!
Here's why the annual celebration is a favorite for the One Warm Coat team: Our friends can reduce material in landfills by donating their extra warm coats. We know that someone in need will gratefully reuse them! When each of us recycles our warmth in this way, we improve our environment in important ways all across the land. We invite you to access our Coat Drive Locator map or locate a One Warm Coat community service Agency and recycle your extra warm coat today!
Celebrating Success!
It's time to celebrate your success! Whether a coat drive collects one coat or 100, we "measure" success by the warmth shared in physical terms (number of coats) and in community relationships. Today, we are happy to tell you about Kathy B. from Chester County (PA) and her coat drive success.
Three years ago, Kathy contacted One Warm Coat for assistance with organizing a coat drive in memory of her late sister-in-law, Amy Dwyer. This year, Kathy and her team collected over 1,000 coats—and she received personal recognition from her County's Women's Commission! Congratulations, Kathy and team! And thank you for making One Warm Coat part of your drive to bring the warmth of good coats and good community relationships to Chester County. (Organizers: Why not share your success through our website today? http://onewarmcoat.org/organize-a-drive/share-your-success/)
Happiness = Your Thinking + What You Do!
Happiness is a goal that we all share. Experience and research show us that the "secret" to achieving it is simple: do something. Here's another "secret": doing something for someone else makes both of us happy! The website mylivehappy.com says, "What you do matters. True joy comes from spreading happiness to those around you." In San Francisco last week, our National Founder and Board Chair, Sherri Wood, attended the annual International Day of Happiness celebration, and shared this photo taken while she added her commitment to the thousands made that day! Today, we at One Warm Coat thank every coat drive Organizer, Agency, and Sponsor who joined us this season to do something to improve the lives of others. Your friendship and your partnership in this important work make us very happy!
Celebrating Women Then and Now
This month recognizes the significant work American women have done—and continue to do–on behalf of people in need. We invite you to join us in honoring their contributions to communities here and beyond our borders. Here are some who come to mind today.
* Jane Addams (1860-1935), founder of one of the original settlement houses for disadvantaged citizens.
* Dr. Justina Ford (1871-1952), a physician who often provided services to low-income and indigent people.
* Melinda Gates (current), whose work with the Bill & Melinda Gates Foundation advances health care and reduces extreme poverty.
* And our own National Founder, Sherri Lewis Wood (current), whose compassion and skill built One Warm Coat from a one-city coat drive into a nationally-known organization that warms the lives of millions each winter.
If you have a woman whose community work you wish to honor this month, we invite you to visit our Facebook page and leave us a post. We would enjoy learning about her!
A Salute to Girl Power! (Girl Scouts, that is!)
This week, we join our friends, Girl Scouts of the USA, in celebrating Girl Scout Week. Each year, Girl Scout, Brownie and Daisy troops across the country hold and help with coat drives that bring life-giving warmth to people in need. For over 100 years, Girl Scouts of the USA has helped young girls to develop courage, confidence and character. This week, we salute their spirit of service. We are thankful for their contributions and very proud that they are our friends!
There's Still Time – and There's Always a Need
It's nearly Spring, when the wind blows warmer, the days grow longer and the jackets get lighter. Right? Well, not at the moment. Weather conditions like the "polar vortex" are still with us—as is the need for warm coats. Warming centers from Maine to California have remained opened to handle the unusual number of "Code Blue" nights. The good news? There's still time to hold a coat drive! Spring will arrive eventually. In the meantime, we've got plenty of Promo Packs ready to ship, to help you and your teams hold a coat drive and spread life-giving warmth to your neighbors in need. Why not register yours here today?
This week's "holidays" include Tell a Fairy Tale Day! We love fairy tales, because although they need not start with "Once upon a time," they almost always end with "…and they lived happily ever after." Here's our tale:
Once upon a time, during a winter that was very cold, a small group of friends noticed that some of their neighbors had no coats to keep them warm. This worried the friends a great deal. Then, one of them had an idea. They would gather all their extra coats and give them to their neighbors in need! The idea grew and grew. Soon, almost everyone who could help did so—across the whole country! In the end, everyone who needed a warm coat could get one, free of charge! The communities became much warmer places to live, and they all lived happily ever after. (To make this fairy tale a reality, please visit One Warm Coat today. Thank you!)
Winter Holiday Fun – Brought to You by Warm Coats Everywhere
For many, President's Day (today) is a holiday from work and school. As our friends from north to south dig out from recent snowstorms, we thought we'd share some ideas of what to do with all that time off—and all that snow.
Note: This fun was made possible by warm coats everywhere! Why not donate your extra warm coat today and share the fun in your community?
The Heart of the Matter
It's 7 degrees in Milwaukee today; in Pittsburgh, tonight's low will be 2 degrees. Charleston, WV is expecting nighttime temperatures in the teens, and the frigid forecast goes on and on. If you have heard the phrase "the heart of the matter," you know that it means to focus on the most important part of something. For people living through this winter with little, the need for a warm coat is the heart of the matter. Whether you can donate your extra warm coat or can support a way to make warm coats available to others, we hope you'll take action today. Your support will get to the heart of the matter, to warm the life of someone in need.


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Tuesday, November 25, 2014
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Good Hood Cop
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Well people we as a community have lost a great man, good brother and a Black urban professional from the hood by the name of Kenneth Garner. For many years I have seen this man in restaurants and ...
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Hearing Thunder with Harriet Tubman
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05 March 2009
To talk of Harriet Tubman is to speak of one of those special persons who serve as sacred sources and cultural anchors of our expansive self-understanding and whose lives are the precious and heavy ...
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The Sacrifice And Chosen Status Of Rev. Dr. Jeremiah Wright: Part 2
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05 March 2009
A New Perspective On His Role In The 2008 Presidential Election (Part II) I recently had a chance to hear a sermon delivered by Rev. Dr. Jeremiah Wright at Bethel AME Church in Los Angeles. ...
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The Audacity of Hope
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05 March 2009
Right now, some people are feeling that the end is near. The economy is the worst that it has been in decades and many people are losing everything. Some people are losing homes, jobs, cars and at ...
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Expansion or Contraction
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05 March 2009
We are facing the worst economic crisis since the Great Depression, and Republicans have chosen political posturing over cooperation. Republicans gave Obama's recovery plan exactly zero votes in ...
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Global Gloom to World Doom?
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05 March 2009
This piece could have easily been entitled, "One Pastor's Precise Prediction." Why? Because Pastor Charles Taze Russell, the second president of the Zion's Watch Tower Tract ...
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The Station I Hate to Love: KFI 640AM
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05 March 2009
I hate Los Angeles talk show radio station KFI 640 AM. Really I do. I once wrote an article about how their morning show host Bill Handel, who I think is as a racist as they come, "could kiss ...
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Wal-Mart Continues to Exploit Workers Worldwide
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05 March 2009
Wal-Mart periodically announces policy changes that turn out to be nothing more than camouflage to deflect criticism of its discriminatory employment practices. But its transgressions don't often ...
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The Republican Leadership-They Just Don't Get It
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26 February 2009
It is completely unconscionable how far the Republican leadership has strayed from anything resembling responsible governance. While they thrive on their claim of patriotism, their petulant obstructionism ...
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State of Foster Care
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26 February 2009
Hello World, hope all is well with you and yours. This past 17th of February I was fortunate to participate as a panelist during an open discussion regarding children who age out of foster care. The ...
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I Need Help!
Written by Malik Spellman
26 February 2009
Dear beloved homies behind the wall I really need your help with stopping the killing of our youth out here in these streets. I would like to use all the juice that I have to help stop the killing ...
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Keeping It Real: There's Nothing Funny in the New York Post
Written by Rev. Al Sharpton
26 February 2009
Last week the New York Post published a cartoon that was troubling at best, given the racist attacks throughout history that have made African-Americans synonymous with monkeys. Upon seeing it, one ...
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The Sacrifice And Chosen Status Of Rev. Dr. Jeremiah Wright
Written by Mshujaa Komoyo
26 February 2009
(Part I) I recently had a chance to hear a sermon delivered by Rev. Dr. Jeremiah Wright at Bethel AME Church in Los Angeles. His selection was titled "Looks Can Be Deceiving", and just as ...
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Yes On Los Angeles City Measure B
Written by Rev. Eric Lee & Forescee Hogan-Rowles
26 February 2009
On Tuesday, March 3, South LA Voters finally have an opportunity to vote on a measure which will create jobs, develop clean energy and improve the air we breathe. We strongly urge support for Measure ...
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Revisiting the Radical Evil Racism
Written by Dr. Maulana Karenga (Columnist)
26 February 2009
If we want to critically understand American or any society, we cannot simply accept or mindlessly mouth its self-congratulatory narratives-neither those masquerading as curriculums in colleges and ...
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Abolishing Black History Month - An Incomplete Argument
Written by Darryl James (Columnist)
26 February 2009
We are now at the end of another Black History Month. We have some Black History in the making, especially with the new Black President. However, some of us are also in the middle of silly arguments ...
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Murdoch's Monkey Business
Written by Dr. Firpo W. Carr (Columnist)
26 February 2009
Last week the New York Post, owned by multi-billionaire Australian-born Rupert Murdoch, carried a highly inflammable cartoon that shows two White cops just after they shot and killed a chimpanzee that ...
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The Elected Obama
Written by Larry Aubry (Columnist)
26 February 2009
Barack Obama's election is mega change from George W. Bush's calamitous imperialism. However, those who believe he is a flawless icon will be sorely disappointed. Not only is Obama mortal, ...
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God's plans for our lives
Written by Pamela Mason (Special to Sentinel)
19 February 2009
Many of us relish those special scriptures that encourage us from time to time, especially when we are being challenged in certain areas of our lives. One of my personal favorites is Jeremiah 29:11, ...
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Obama's Stimulus Package Is Just What The Doctor Ordered
Written by Angela J. Reddock (Trustee, L.A. Community College Board of Trustees)
19 February 2009
For Community College Students Seeking New Careers in Health If all goes as planned, the stimulus signed by President Barack Obama this week will result in an unprecedented, multibillion-dollar ...
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The Republican Rape of America
Written by Eric L. Wattree Sr. (Columnist)
19 February 2009
The Republican leadership and their corporate cronies have systematically brutalized and raped America. Now they're insisting that we give them a bonus instead of treating our wounds. Those are ...
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Honoring Black History
Written by Alfonzo Tucker (Columnist)
19 February 2009
Hello world, with this article I would like to pay homage to our forefathers who suffered so as to allow all people of color the right to live as free individuals and claim honorable achievement in ...
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Dialoging with DuBois on History
Written by Dr. Maulana Karenga (Columnist)
19 February 2009
The relevance of Black History Month is not determined by our debatable changed or changing position in American society, nor by unfounded assumptions that diversity is divisive, and that in the interest ...
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Black History - Cops Gone Wild
Written by Darryl James (Columnist)
19 February 2009
It has been more than fifteen years since the now infamous riots that ripped the City of Angels apart over a serious incident that revealed problems with police and the people that were felt across ...
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Word: harmanlamak
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This is the final week of business for the East Broadway location of Pushcart Coffee, whose owners tell the Lo-Down that high rent is to blame. It's not the end for Pushcart, however: The company, which takes a curatorial approach to its coffee by serving selections from a wide variety of small-batch roasters, has two other locations in Manhattan, and is otherwise committed to the neighborhood. Were not quitting the Lower East Side," says co-owner Jamie Rogers. "We love it here." [The Lo-Down]
Pushcart Coffee Closing Lower East Side ShopBy Hugh Merwin Follow @hughmerwin
"We're not quitting the Lower East Side," a co-owner says.


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You guys rock! Your customer service is great. I ordered a package of about $62 with 3 special order items in it on a Monday, and you guys e-mailed...Read More ->
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text: As soon as there is more definite information it will be posted on the Skorragrdr website. I was told to hold off until we had more specifics. Lady Isobel de Kirkbryde leanbh a Úna Guild Head of the Guild of St. Camillus de Lellis, Kingdom of Ansteorra V-Scribe Canton of Skorragardr Deputy Northern Regional Scribe Member of Clann Lochlan Member of Clann Haddock Member of House MOO __________________________________________________ Do You Yahoo!? Tired of spam? Yahoo! Mail has the best spam protection around http://mail.yahoo.com -------------- next part -------------- An HTML attachment was scrubbed... URL: http://lists.ansteorra.org/pipermail/namron-ansteorra.org/attachments/20071021/ee90192e/attachment.htm


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I am a recent Fine Art Graduate who specialised in Sculpture in 2006. My work also involves drawing, digital technology, research and walking! I am interested in the environment, how it effects us in our daily lives, the ongoing conflicts of living in the city and the clash between rural and urban spaces. I have exhibited mainly in London and won an Award in the 2nd year of my Degree at London Metropolitan University in 2005. One of the loves of my life is walking, absorbing the surroundings of my travels and then going back to the studio to make miniatures of the streets, trees, walls, ruins e.t.c. I then take photographs of my art and the images give the impression they are real places when in fact they are models.
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Gives the frame its character and provides the perfect setting for an artwork to be fully enjoyed.
Protects the artwork from dust and wilting so that it can be admired for many years.
Adds depth to an image and works with the moulding to enhance and protect the artwork.
Bonds to the artwork ensuring it is perfectly smooth and holds the whole frame together.
We partner with the world's top museums and galleries to bring you exclusive prints of the highest quality. Our teams of designers ensure the colours are accurate, papers are well suited and the best frames are suggested.
In the printing room, artworks are printed on state-of-the-art machines with a team of technicians checking colour and quality every step of the way.
After being cut down to size, our team carefully finish any stray edges, check measurements and prepare the prints for mounting and framing.
We have a team of master framers who work with high-quality, responsibly-sourced wood to create our vast range of framing combinations - each frame is bespoke and made to order for every print.
Our selection of hand-finished frames are painted or stained by hand in a variety of colours, and finished with a layer of wax - the end result is a uniquely crafted, beautiful frame that is made to last.
In this workshop everything comes together - the print, the frame and the glass - in a seamless and stream-lined process.
The artwork is carefully mounted in preparation for framing.
Once mounted, the print is ready to be covered by glass or perspex - a delicate procedure but expertly done with not a fingerprint in sight.
After a final, thorough check, the framed print is ready to be carefully packaged up and shipped to the customer.
For those who order their art as canvas prints, the same amount of attention and care goes into the process. Here, the print is being transferred to a wooden frame.
As with the framed prints, our canvases are all hand-finished in the workshop - a labour of love from start to finish.


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DEFINITION of 'Toggle Note'
A payment-in-kind bond, where the issuer has the option to defer an interest payment by agreeing to pay an increased coupon in the future. With toggle notes, all deferred payments must be settled by the bond's maturity.
INVESTOPEDIA EXPLAINS 'Toggle Note'
Toggle notes provide firms with a way to raise debt while staying afloat during times of strained cash flow. When cash is at a minimum, the firm can use the toggle to defer an interest payment.
While this seems like an attractive option for the firm, it does come at a cost. The increased interest rate provides ample incentive to not miss an interest payment.
Cash Flow1. A revenue or expense stream that changes a cash account over ...
CouponThe interest rate stated on a bond when it's issued. The coupon ...
Interest1. The charge for the privilege of borrowing money, typically ...
Payment-In-Kind - PIK1. The use of a good or service as payment instead of cash. 2. ...
Payment-In-Kind BondsA type of bond that pays interest in additional bonds rather ...
Deferred InterestThe amount of interest that is added to the principal balance ...
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Saul David was born in Monmouth in 1966 and educated at Ampleforth College and Edinburgh and Glasgow Universities (History MA and PhD).
An expert in the wars of the Victorian period, he began writing his first history book when he was twenty-five and has since completed eleven more (one edited). They include: The Homicidal Earl: The Life of Lord Cardigan (1997), a critically-acclaimed biography of the man who led the Charge of the Light Brigade; The Indian Mutiny:1857 (2002), shortlisted for the Westminster Medal for Military Literature; Zulu: The Heroism and Tragedy of the Zulu War of 1879 (2004), a Waterstone's Military History Book of the Year; Victoria's Wars: The Rise of Empire (2006); and All the King's Men: The British Redcoat in the Era of Sword and Musket. In 2007 he signed a three book deal with Hodder & Stoughton to write a series of historical novels set in the late Victorian period. The Times described the first, Zulu Hart (2009), as a 'rattling good yarn' with 'a compelling, sexy hero who could give Cornwell's Sharpe a run for his money'. His latest history – 100 Days to Victory: How the Great War was Fought and Won – will be published in September 2013.
An experienced broadcaster, Saul appears often on British radio and television. His TV presenting credits include the Escape From… series for Five (2004), Sky One's genealogy series So You Think You're Royal? (2007) and three programmes for the BBC's flagship history series Timewatch: The Queen's Lover (2001), The Greatest Knight (2008) and the Queen Elizabeth's Lost Guns (February 2009). He was an on-screen expert on BBC2's virtual battle series Time Commanders (2004) and is a regular contributor to history programmes on all channels. Two of his books – Mutiny at Salerno and Zulu – have been made into documentaries. More recently he presented Filton's Fabulous Flying Machines for BBC1 (June 2010) and How The Rest Got Home, a 70th anniversary programme about the British soldiers who escaped from France after Dunkirk, for Radio 4 (June 2010). His 3 x 1 hour series on the history of military logistics – Bullets, Boots and Bandages: How Wars are Really Won – was aired on BBC4 in February 2012. In the last year he has guest presented on two ITV1 flagship series, Britain's Secret Treasures (July 2012) and Britain's Secret Homes (June 2013), and this autumn is due to present a Revealed special for Channel Five/National Geographic Channel on The First Great Escape from Holzminden POW camp in Germany in July 1918.
Saul gives frequent talks about military history and has spoken at all the major literary festivals. He has also lectured at the Security Service (MI5), the National Army Musuem and the Imperial War Museum. He has led history tours to India and the Crimea, and has appeared as a guest speaker on The World ship.
Saul is Professor of War Studies at the University of Buckingham and Programme Director for Buckingham's London-based MPhil in Military History.
He lives in Somerset with his wife and three daughters.