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At least 19 recordsLinked to original sources
PubMed2004

[Should the human smallpox virus (variola) be destroyed?].

Smallpox, caused by variola virus, was a terror for civilizations around the world for more than 3000 years. Although the disease is eradicated, hundreds of variola virus isolates are kept in two WHO-collaborating facilities, one in USA and one in Russia. In spite of several agreements on destruction, it is now doubtful that these virus isolates will be destroyed. Variola virus may exist in other places and may be used as a biological weapon in war or for terror. Further research on variola virus is thus essential in order to achieve a better understanding of the pathogenicity of the virus and to develop new anti-variola virus vaccines and antiviral drugs.

Biomedical Research↗
PubMed2004

Exploring the potential of variola virus infection of cynomolgus macaques as a model for human smallpox.

Smallpox virus (variola) poses a significant threat as an agent of bioterrorism. To mitigate this risk, antiviral drugs and an improved vaccine are urgently needed. Satisfactory demonstration of protective efficacy against authentic variola will require development of an animal model in which variola produces a disease course with features consistent with human smallpox. Toward this end, cynomolgus macaques were exposed to several variola strains through aerosol and/or i.v. routes. Two strains, Harper and India 7124, produced uniform acute lethality when inoculated i.v. in high doses (10(9) plaque-forming units). Lower doses resulted in less fulminant, systemic disease and lower mortality. Animals that died had profound leukocytosis, thrombocytopenia, and elevated serum creatinine levels. After inoculation, variola was disseminated by means of a monocytic cell-associated viremia. Distribution of viral antigens by immunohistochemistry correlated with the presence of replicating viral particles demonstrated by electron microscopy and pathology in the lymphoid tissues, skin, oral mucosa, gastrointestinal tract, reproductive system, and liver. These particles resembled those seen in human smallpox. High viral burdens in target tissues were associated with organ dysfunction and multisystem failure. Evidence of coagulation cascade activation (D dimers) corroborated histologic evidence of hemorrhagic diathesis. Depletion of T cell-dependent areas of lymphoid tissues occurred, probably as a consequence of bystander apoptotic mechanisms initiated by infected macrophages. Elaboration of cytokines, including IL-6 and IFN-gamma, contribute to a cytokine storm formerly known as "toxemia." A more precise understanding of disease pathogenesis should provide targets for therapeutic intervention, to be used alone or in combination with inhibitors of variola virus replication.

Animals↗
PubMedDate not supplied

[Comparative evaluation of test-systems for detection of Venezuelan equine encephalomyelitis virus and variola virus using dot immunoenzyme assay and solid-phase lanthanide immunofluorescent assay].

The sensitivity and specificity of 4 experimental test systems for dot enzyme immunoassay (dot-EIA) and solid-phase lanthanide immunofluorescent analysis (SP LIFA) were studied with Venezuelan equine encephalomyelitis (VEE) and variolovaccinia viruses. Test systems for SP LIFA proved to be 25 times more sensitive than those for dot-EIA. The test systems were highly specific and did not react with the heterologous viruses and proteins. Diagnostic agent for detecting VEE virus in dot-EIA was false-positive with Sindbis virus in low dilutions. The first trials of the Russian test systems for dot-EIA and SP LIFA showed that these systems rapidly and reliably detect VEE and variolovaccinia viruses in liquid samples.

Antigens, Viral↗
PubMed1996

Gene homology between orf virus and smallpox variola virus.

About 47% identity was observed between the deduced amino acid sequences of a protein encoded by a gene of the parapoxvirus orf virus (OV) strain NZ2 and a 6 kDa protein of unknown function reported to be produced by an open reading frame expressed early after infection by the orthopoxvirus Western Reserve vaccinia virus (VAC); the open reading frame is absent from VAC strain Copenhagen. Examination of sequences reported for variola virus (VAR) strains Bangladesh, India, Congo- 1970, Somalia- 1977 and Garcia- 1966 revealed each encoded a correlate 58 amino acid protein. The open reading frame was not reported in the original analyses of these sequences because a lower limit of 60 amino acids was used to identify potential encoded proteins. Inspection of partial reading frames reported for cowpox virus (CWV) and ectromelia virus (EMV) suggested that these viruses might also code for a correlate of the VAC WR protein. DNA sequencing of cloned fragments of CWV and EMV confirmed that both these orthopoxviruses encode closely related, full length variants of the VAC and VAR open reading frames. The OV homologue is coded in the OV strain NZ2 BamHI-E fragment E2L open reading frame, which we reported is transcribed early postinfection; moreover, analysis of an NZ2 variant showed E2L was absent, indicating that E2L, like the VAC cognate, is nonessential for virus replication in cell culture. The parapoxvirus and orthopoxvirus correlates have about 20% amino acid sequence resemblance to African swine fever virus DNA binding protein p10, suggesting an ancestral relation of genes.

Amino Acid Sequence↗
PubMed2002

The sequence of camelpox virus shows it is most closely related to variola virus, the cause of smallpox.

Camelpox virus (CMPV) and variola virus (VAR) are orthopoxviruses (OPVs) that share several biological features and cause high mortality and morbidity in their single host species. The sequence of a virulent CMPV strain was determined; it is 202182 bp long, with inverted terminal repeats (ITRs) of 6045 bp and has 206 predicted open reading frames (ORFs). As for other poxviruses, the genes are tightly packed with little non-coding sequence. Most genes within 25 kb of each terminus are transcribed outwards towards the terminus, whereas genes within the centre of the genome are transcribed from either DNA strand. The central region of the genome contains genes that are highly conserved in other OPVs and 87 of these are conserved in all sequenced chordopoxviruses. In contrast, genes towards either terminus are more variable and encode proteins involved in host range, virulence or immunomodulation. In some cases, these are broken versions of genes found in other OPVs. The relationship of CMPV to other OPVs was analysed by comparisons of DNA and predicted protein sequences, repeats within the ITRs and arrangement of ORFs within the terminal regions. Each comparison gave the same conclusion: CMPV is the closest known virus to variola virus, the cause of smallpox.

Animals↗
PubMed2004

Detection of orthopoxvirus DNA by real-time PCR and identification of variola virus DNA by melting analysis.

Although variola virus was eradicated by the World Health Organization vaccination program in the 1970s, the diagnosis of smallpox infection has attracted great interest in the context of a possible deliberate release of variola virus in bioterrorist attacks. Obviously, fast and reliable diagnostic tools are required to detect variola virus and to distinguish it from orthopoxviruses that have identical morphological characteristics, including vaccinia virus. The advent of real-time PCR for the clinical diagnosis of viral infections has facilitated the detection of minute amounts of viral nucleic acids in a fast, safe, and precise manner, including the option to quantify and to genotype the target reliably. In this study a complete set of four hybridization probe-based real-time PCR assays for the specific detection of orthopoxvirus DNA is presented. Melting analysis following PCR enables the identification of variola virus by the PCR product's characteristic melting temperature, permitting the discrimination of variola virus from other orthopoxviruses. In addition, an assay for the specific amplification of variola virus DNA is presented. All assays can be performed simultaneously in the same cycler, and results of a PCR run are obtained in less than 1 h. The application of more than one assay for the same organism significantly contributes to the diagnostic reliability, reducing the risk of false-negative results due to unknown sequence variations. In conclusion, the assays presented will improve the speed and reliability of orthopoxvirus diagnostics and variola virus identification.

Base Sequence↗
PubMed1984

Nucleotide sequence of the thymidine kinase gene region of monkeypox and variola viruses.

Among the orthopoxviruses variola virus induces in cells a characteristic thymidine kinase (TK) activity that can be feedback inhibited in reactions with thymidine triphosphate. Northern blot analyses of variola and monkeypox virus-infected cell extracts showed RNAs of the same molecular weight as the major (590-base) and minor (2380-base) TK transcripts described for vaccinia virus. The nucleotide sequences of 1275 bp in the TK gene region of variola and monkeypox viruses have been determined. When these sequences were compared with such sequences reported for vaccinia virus, differences were observed at 41 nucleotide positions. Examination of the putative encoded TK polypeptide for the three viruses revealed variation at eight amino acid positions. Two major differences in the amino acid composition of the variola virus TK were identified that might play a role in alteration of its kinetic properties.

Amniotic Fluid↗
PubMed1973

Differential diagnosis of variola viruses by microfocus assay.

Variola virus was identified by means of a method involving the formation of microfoci on microplate cultures of HeLa cells. It was possible to distinguish between variola major and variola minor viruses within 48 h of receipt of the specimens by determining the temperature sensitivity of focus formation.

Bacteriological Techniques↗
PubMed2003

Variola virus immune evasion proteins.

Variola virus, the causative agent of smallpox, encodes approximately 200 proteins. Over 80 of these proteins are located in the terminal regions of the genome, where proteins associated with host immune evasion are encoded. To date, only two variola proteins have been characterized. Both are located in the terminal regions and demonstrate immunoregulatory functions. One protein, the smallpox inhibitor of complement enzymes (SPICE), is homologous to a vaccinia virus virulence factor, the vaccinia virus complement-control protein (VCP), which has been found experimentally to be expressed early in the course of vaccinia infection. Both SPICE and VCP are similar in structure and function to the family of mammalian complement regulatory proteins, which function to prevent inadvertent injury to adjacent cells and tissues during complement activation. The second variola protein is the variola virus high-affinity secreted chemokine-binding protein type II (CKBP-II, CBP-II, vCCI), which binds CC-chemokine receptors. The vaccinia homologue of CKBP-II is secreted both early and late in infection. CKBP-II proteins are highly conserved among orthopoxviruses, sharing approximately 85% homology, but are absent in eukaryotes. This characteristic sets it apart from other known virulence factors in orthopoxviruses, which share sequence homology with known mammalian immune regulatory gene products. Future studies of additional variola proteins may help illuminate factors associated with its virulence, pathogenesis and strict human tropism. In addition, these studies may also assist in the development of targeted therapies for the treatment of both smallpox and human immune-related diseases.

Animals↗
PubMed1976

An antigenically atypical strain of variola virus.

The Kuwait-5-67 strain of variola virus, although indistinguishable from variola virus in its other properties, behaved atypically in immuno-diffusion tests with antivaccinia serum. The precipitation line pattern produced was similar to that of cowpox virus but lacked one of the main precipitation bands given by all other strains of variola, alastrim and vaccinia viruses tested. Trypsin treatment of cowpox virus preparations revealed the missing precipitation line but did not do so with the Kuwait-5-67 strain. The antivaccinia serum absorbed by Kuwait-5-67 strain lost its ability to interact with this strain, but still gave precipitation lines with variola, vaccinia and cowpox viruses.

Antigens, Viral↗
PubMed1982

The pathogenicity of variola virus. A comparison of the growth of standard strains of variola major and variola minor viruses in cell cultures from human embryos.

The international reference strains of variola major (Harvey) and of variola minor (Butler) were grown in cultures of skin and muscle cells from human embryos. The development of infective virus, complement-fixing antigen, haemagglutinin and cytological changes were followed at four temperatures between 35 and 40 degrees C. No significant difference was found in the amount of virus produced by Harvey or Butler viruses at any of the experimental temperatures, but Harvey attained the plateau titre at 16 h, some 4 h ahead of Butler in the cultures incubated at 38 degrees C. Harvey also produced a higher and more prolonged yield of virus in the extracellular medium of cultures, inoculated at low multiplicity and incubated at 37 degrees C. At 38 degrees C small inocula of Harvey produced foci which developed and spread till the whole culture was necrotic; Butler foci did not spread and remained relatively undeveloped at this temperature.Staining with acridine orange showed the development of cytoplasmic inclusions at all temperatures up to 39.5 degrees C, at which temperature most inclusions remained round and discrete and susceptible to digestion with DNase. The yield of virus at all temperatures correlated well with the number of cells in which the DNA cytoplasmic inclusions became irregular in outline, diffuse and insusceptible to digestion with DNase. It was concluded that elevated temperatures, up to 39.5 degrees C affected principally a maturation phase in the development of the virus.Equal amounts of complement-fixing antigen were produced at all temperatures and by either virus, but the late product, haemagglutinin was depressed at elevated temperatures much more in cultures infected with Butler than in cultures infected with Harvey. This was clearly shown by haemadsorption; in cultures, infected at high multiplicity and incubated at 39.5 degrees C Harvey gave semi-confluent haemadsorption, while only an occasional haemadsorbing cell could be found in the cultures infected with Butler virus.It is concluded that there were two ways in which temperature affected the growth of variola viruses in cultures of human embryo skin and muscle cells. The total yield of virus was reduced by inhibition of a stage in the maturation of the virus with a cut off point between 39.5 and 40 degrees C at which no cells produced infective virus, and with little observable differences between Harvey and Butler viruses. Changes at the surface of virus-infected cell, involving virus release and haemagglutinin, were affected independently of virus maturation; in these changes Butler was much more sensitive than Harvey to elevated temperatures. The relevance of these observations to the development of variola major and variola minor in man is discussed.

Cells, Cultured↗
PubMed1964

SINGLE-DOSE ASSAY TECHNIQUE FOR VARIOLA VIRUS.

A biological assay for variola virus was needed that would (i) require a minimum of time and (ii) have acceptable precision. Preliminary titrations made in 11-day-old embryonated eggs and in suckling mice (6 to 24 hr of age) demonstrated a linear relationship between the concentration of variola virus injected and the mean reciprocal time to death (MTD) of both hosts. This linear response indicated that the injection of a single dose of virus suspension and the measurement of the MTD should result in an acceptable assay method. Seven replicate samples of liquid preparations (20% chorioallantoic membrane in Heart Infusion Broth) and the freeze-dried material obtained with these suspensions were assayed in triplicate for pock infectious units and for MTD. The variance of the pock counts was far greater than was expected from a Poisson distribution, and coefficients of variation ranged from 25 to 61%. Variances obtained with the single-dilution assay were all far below that expected from a Poisson distribution, and coefficients of variations ranged from 5.2 to 13.6%. The use of the MTD assay resulted in a saving of time, a saving in the number of hosts necessary per assay, and increased precision.

Animals↗
PubMed2006

Use of internally controlled real-time genome amplification for detection of variola virus and other orthopoxviruses infecting humans.

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.

Animals↗