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Genome Composition
The genome of Variola major 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 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 have at least 90% similarity with the genome of Vaccinia, a related virus used for vaccination against smallpox.

Gene Expression
Gene expression of Variola 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 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 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 was first sequenced in its entirety in the 1990s. The complete coding sequence is publicly available online. The current reference sequence for Variola major 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 and other poxviruses is publicly available through the Viral Bioinformatics Resource Center.

Genetic Engineering
The WHO currently bans genetic engineering of the smallpox 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 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 Variola's complete sequence has raised concerns about the possibility of illicit synthesis of infectious virus. Vaccinia, a cousin of the smallpox 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.