most people with the late-stage form die within eight to 10 days of illness. among the few who recover, the hemorrhagic lesions gradually disappear after a long period of convalescence. the case fatality rate for late hemorrhagic smallpox is around 90–95%. pregnant women are slightly more likely to experience this form of the disease, though not as much as early hemorrhagic smallpox. cause variola virus this transmission electron micrograph depicts a number of smallpox virions. the "dumbbell-shaped" structure inside the virion is the viral core, which contains the viral dna; mag. = ~370,000× virus classification (unranked): virus realm: varidnaviria kingdom: bamfordvirae phylum: nucleocytoviricota class: pokkesviricetes order: chitovirales family: poxviridae genus: orthopoxvirus species: †variola virus smallpox was caused by infection with variola virus, which belongs to the family poxviridae, subfamily chordopoxvirinae, and genus orthopoxvirus. evolution the date of the appearance of smallpox is not settled. it most probably evolved from a terrestrial african rodent virus between 68,000 and 16,000 years ago. the wide range of dates is due to the different records used to calibrate the molecular clock. one clade was the variola major strains (the more clinically severe form of smallpox) which spread from asia between 400 and 1,600 years ago. a second clade included both alastrim (a phenotypically mild smallpox) described from the american continents and isolates from west africa which diverged from an ancestral strain between 1,400 and 6,300 years before present. this clade further diverged into two subclades at least 800 years ago. a second estimate has placed the separation of variola virus from taterapox (an orthopoxvirus of some african rodents including gerbils) at 3,000 to 4,000 years ago. this is consistent with archaeological and historical evidence regarding the appearance of smallpox as a human disease which suggests a relatively recent origin. if the mutation rate is assumed to be similar to that of the herpesviruses, the divergence date of variola virus from taterapox has been estimated to be 50,000 years ago. while this is consistent with the other published estimates, it suggests that the archaeological and historical evidence is very incomplete. better estimates of mutation rates in these viruses are needed. examination of a strain that dates from c. 1650 found that this strain was basal to the other presently sequenced strains. the mutation rate of this virus is well modeled by a molecular clock. diversification of strains only occurred in the 18th and 19th centuries. virology variola virus is large and brick-shaped and is approximately 302 to 350 nanometers by 244 to 270 nm, with a single linear double stranded dna genome 186 kilobase pairs (kbp) in size and containing a hairpin loop at each end. four orthopoxviruses cause infection in humans: variola, vaccinia, cowpox, and monkeypox. variola virus infects only humans in nature, although primates and other animals have been infected in an experimental setting. vaccinia, cowpox, and monkeypox viruses can infect both humans and other animals in nature. the life cycle of poxviruses is complicated by having multiple infectious forms, with differing mechanisms of cell entry. poxviruses are unique among human dna viruses in that they replicate in the cytoplasm of the cell rather than in the nucleus. to replicate, poxviruses produce a variety of specialized proteins not produced by other dna viruses, the most important of which is a viral-associated dna-dependent rna polymerase. both enveloped and unenveloped virions are infectious. the viral envelope is made of modified golgi membranes containing viral-specific polypeptides, including hemagglutinin. infection with either variola major virus or variola minor virus confers immunity against the other. variola major the more common, infectious form of the disease was caused by the variola major virus strain. variola minor variola minor virus, also called alastrim, was a less common form of the virus, and much less deadly. although variola minor had the same incubation period and pathogenetic stages as smallpox, it is believed to have had a mortality rate of less than 1%, as compared to smallpox's 30%. like variola major, variola minor was spread through inhalation of the virus in the air, which could occur through face-to-face contact or through fomites. infection with variola minor virus conferred immunity against the more dangerous variola major virus. because variola minor was a less debilitating disease than smallpox, people were more frequently ambulant and thus able to infect others more rapidly. as such, variola minor swept through the united states, great britain, and south africa in the early 20th century, becoming the dominant form of the disease in those areas and thus rapidly decreasing mortality rates. along with variola major, the minor form has now been totally eradicated from the globe. the last case of indigenous variola minor was reported in a somali cook, ali maow maalin, in october 1977, and smallpox was officially declared eradicated worldwide in may 1980. variola minor was also called white pox, kaffir pox, cuban itch, west indian pox, milk pox, and pseudovariola. genome composition the genome of variola major virus is about 186,000 base pairs in length. it is made from linear double stranded dna and contains the coding sequence for about 200 genes. the genes are usually not overlapping and typically occur in blocks that point towards the closer terminal region of the genome. the coding sequence of the central region of the genome is highly consistent across orthopoxviruses, and the arrangement of genes is consistent across chordopoxviruses the center of the variola virus genome contains the majority of the essential viral genes, including the genes for structural proteins, dna replication, transcription, and mrna synthesis. the ends of the genome vary more across strains and species of orthopoxviruses. these regions contain proteins that modulate the hosts' immune systems, and are primarily responsible for the variability in virulence across the orthopoxvirus family. these terminal regions in poxviruses are inverted terminal repetitions (itr) sequences. these sequences are identical but oppositely oriented on either end of the genome, leading to the genome being a continuous loop of dna components of the itr sequences include an incompletely base paired a/t rich hairpin loop, a region of roughly 100 base pairs necessary for resolving concatomeric dna (a stretch of dna containing multiple copies of the same sequence), a few open reading frames, and short tandemly repeating sequences of varying number and length. the itrs of poxviridae vary in length across strains and species. the coding sequence for most of the viral proteins in variola major virus have at least 90% similarity with the genome of vaccinia, a related virus used for vaccination against smallpox. gene expression gene expression of variola virus occurs entirely within the cytoplasm of the host cell, and follows a distinct progression during infection. after entry of an infectious virion into a host cell, synthesis of viral mrna can be detected within 20 minutes. about half of the viral genome is transcribed prior to the replication of viral dna. the first set of expressed genes are transcribed by pre-existing viral machinery packaged within the infecting virion. these genes encode the factors necessary for viral dna synthesis and for transcription of the next set of expressed genes. unlike most dna viruses, dna replication in variola virus and other poxviruses takes place within the cytoplasm of the infected cell. the exact timing of dna replication after infection of a host cell varies across the poxviridae. recombination of the genome occurs within actively infected cells. following the onset of viral dna replication, an intermediate set of genes codes for transcription factors of late gene expression. the products of the later genes include transcription factors necessary for transcribing the early genes for new virions, as well as viral rna polymerase and other essential enzymes for new viral particles. these proteins are then packaged into new infectious virions capable of infecting other cells. research two live samples of variola major virus remain, one in the united states at the cdc in atlanta, and one at the vector institute in koltsovo, russia. research with the remaining virus samples is tightly controlled, and each research proposal must be approved by the who and the world health assembly (wha). most research on poxviruses is performed using the closely related vaccinia virus as a model organism. vaccinia virus, which is used to vaccinate for smallpox, is also under research as a viral vector for vaccines for unrelated diseases. the genome of variola major virus was first sequenced in its entirety in the 1990s. the complete coding sequence is publicly available online.the current reference sequence for variola major virus was sequenced from a strain that circulated in india in 1967. in addition, there are sequences for samples of other strains that were collected during the who eradication campaign. a genome browser for a complete database of annotated sequences of variola virus and other poxviruses is publicly available through the viral bioinformatics resource center. genetic engineering the who currently bans genetic engineering of the variola virus. however, in 2004, a committee advisory to the who voted in favor of allowing editing of the genome of the two remaining samples of variola major virus to add a marker gene. this gene, called gfp, or green fluorescent protein, would cause live samples of the virus to glow green under fluorescent light. the insertion of this gene, which would not influence the virulence of the virus, would be the only allowed modification of the genome. the committee stated the proposed modification would aid in research of treatments by making it easier to assess whether a potential treatment was effective in killing viral samples. the recommendation could only take effect if approved by the wha. when the wha discussed the proposal in 2005, it refrained from taking a formal vote on the proposal, stating that it would review individual research proposals one at a time. addition of the gfp gene to the vaccinia genome is routinely performed during research on the closely related vaccinia virus. controversies the public availability of the variola virus complete sequence has raised concerns about the possibility of illicit synthesis of infectious virus. vaccinia, a cousin of the variola virus, was artificially synthesized in 2002 by nih scientists. they used a previously established method that involved using a recombinant viral genome to create a self-replicating bacterial plasmid that produced viral particles. in 2016, another group synthesized the horsepox virus using publicly available sequence data for horsepox. the researchers argued that their work would be beneficial to creating a safer and more effective vaccine for smallpox, although an effective vaccine is already available. the horsepox virus had previously seemed to have gone extinct, raising concern about potential revival of variola major and causing other scientists to question their motives. critics found it especially concerning that the group was able to recreate viable virus in a short time frame with relatively little cost or effort. although the who bans individual laboratories from synthesizing more than 20% of the genome at a time, and purchases of smallpox genome fragments are monitored and regulated, a group with malicious intentions could compile, from multiple sources, the full synthetic genome necessary to produce viable virus. transmission smallpox was highly contagious, but generally spread more slowly and less widely than some other viral diseases, perhaps because transmission required close contact and occurred after the onset of the rash. the overall rate of infection was also affected by the short duration of the infectious stage. in temperate areas, the number of smallpox infections was highest during the winter and spring. in tropical areas, seasonal variation was less evident and the disease was present throughout the year. age distribution of smallpox infections depended on acquired immunity. vaccination immunity declined over time and was probably lost within thirty years. smallpox was not known to be transmitted by insects or animals and there was no asymptomatic carrier state. transmission occurred through inhalation of airborne variola virus, usually droplets expressed from the oral, nasal, or pharyngeal mucosa of an infected person. it was transmitted from one person to another primarily through prolonged face-to-face contact with an infected person. some infections of laundry workers with smallpox after handling contaminated bedding suggested that smallpox could be spread through direct contact with contaminated objects (fomites), but this was found to be rare. also rarely, smallpox was spread by virus carried in the air in enclosed settings such as buildings, buses, and trains. the