Table of Contents:

• Taxonomy Information
• Lifecycle Information
• Genome Information
• Biosafety Information
• Culturing Information
• Epidemiology Information
• Diagnostic Tests Information
• Infected Hosts Information
• Phinet: Pathogen-Host Interaction Network
• Lab Animal Pathobiology & Management
• References
• Data Provenance and Curators

1. Species:
   1. Variola virus (Website 4):
      1. Common Name: Smallpox
      2. GenBank Taxonomy No.: 10255
      3. 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).
      4. Variant(s):
         • Variola major virus (Website 5):
           • Common Name: Smallpox
           • GenBank Taxonomy No.: 12870
           • Parents: 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):
           • Common Name: Alastrim
           • GenBank Taxonomy No.: 52358
           • Parents: 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).

1. Vatiola virus virion information
   1. Stage Information:
      1. Virion(Henderson et al., 1999):
         • Size: The virus particles are brick-shaped to ovoid and measure approximately 300 by 200 by 100 um.
         • Shape: The virus particles are brick-shaped to ovoid and measure approximately 300 by 200 by 100 um.
         • 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.

1. Genome of Variola virus
   1. 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).
   2. Variola virus Genome Information(Website 7, Website 11)
      1. GenBank Accession Number: NC_001611, X69198
      2. Size: 185578 bp(Website 7, Website 11).
      3. Gene Count: There are 187 putative proteins identified from the sequencing of variola virus(Website 1).
      4. 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).
2. Genome of Variola major virus(Massung et al., 1994)
   1. 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).
   2. Variola major virus Bangladesh-1975 Genome Information(Website 8)
      1. GenBank Accession Number: L22579
      2. Size: 186103 bp(Website 8).
      3. Gene Count: 187(Massung et al., 1994).
      4. 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).
3. Genome of Variola minor(Shchelkunov et al., 2000)
   1. 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).
   2. Variola minor Garcia-1966 Genome Information(Website 9, Website 10)
      1. GenBank Accession Number: X72086, Y16780
      2. Size: 186986 bp(Shchelkunov et al., 2000).
      3. 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).
      4. 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).

1. Biosafety information for Variola virus
   1. Level: Laboratory examination requires high-containment (BL-4) facilities and should be undertaken only in designated laboratories with the appropriate training and equipment.
   2. 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).

1. Variola virus Culturing Methods :
   1. 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).
   2. 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).
   3. 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).

1. Outbreak Locations:
   1. 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).
2. Transmission Information:
   1. From: Human(Fenner et al., 1988C). , To: Human(Fenner et al., 1988C). , With Destination:Human(Fenner et al., 1988C). (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).
3. Environmental Reservoir:
   1. Currently no environmental reservoir information is available.
4. Intentional Releases:
   1. Intentional Release Information:
      1. 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).
      2. 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).
      3. 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).
      4. 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).

1. Organism Detection Test:
   1. Variola virus Particles Detection :
      1. Time to Perform: minutes-to-1-hour
      2. 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).
   2. Variola virus Electron Microscopy :
      1. Time to Perform: 1-hour-to-1-day
      2. 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).
      3. 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).
   3. Gel diffusion or gel precipitation :
      1. 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).
      2. 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).
      3. 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).
   4. Inoculation of the chorioallantoic membrane :
      1. 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).
      2. 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).
      3. 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).
2. Nucleic Acid Detection Test: