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Unlike most viruses, members of Poxviridae replicate in the cytoplasm of the cell rather than the nucleus. When the virion first enters the cell via fusion with the plasma membrane, the outer proteins and lipid envelope uncoat from the virion. Then transcriptional enzymes carried in the core of the virion begin early gene expression. These early transcripts leave the core via microtubules and enter the cytoplasm to be translated by host ribosomes. Poxviruses encode their own DNA replication and transcription machineries. Proteins synthesized using the ribosomes include an RNA polymerase, a DNA polymerase, a protein kinase, an NTPase, a capping enzyme and a polyadenylation system. Other early proteins synthesized are involved in interfering with the interferon system of the host and stopping cell production of macromolecules. Once the DNA replication machinery has been synthesized, the viral genome is then able to leave the core for replication. The viral genome is made of double stranded DNA and ranges from 130 to 375 kbp long with hairpin structures at each end. The membrane of the endoplasmic reticulum forms an envelope around the site of DNA replication. Replication occurs by strand displacement synthesis and results in a long chain of concatamers that are cleaved into individual genomes. DNA replication induces late gene expression that synthesizes many of the proteins for virion structural proteins and enzymes for assembling them. Assembly of the virion occurs in stages with the formation first of a lipoprotein bilayer with spicules in the shape of a crescent. Then the particle closes and the genome and structural proteins enter and form a core. Enzymatic proteins to be carried with the virion also enter the particle. The mature virion is now covered with a membrane and moves to the cell surface. Exiting the cell via exocyotis coats the virion with an envelope and the virion can now infect other cells.
In order for viruses to successfully replicate in a host, they must employ tactics to overcome the host’s immune system. Members of Poxviridae contain many different genes within their short genomes that encode proteins specifically for this task. Often these viral proteins mimic cellular proteins or inhibit pathways necessary to the immune response. Some of the evasive tactics encoded in poxviruses include mimicry of tumor necrosis factor receptors, mimicry of interferon receptors, inhibitors of the complement system, and inhibitors of serine proteases.
One set of anti-immune genes found in poxviruses encode viral tumor necrosis factor receptors (vTNFRs) and are known as the cytokine response modifiers crmB, crmC, crmD, and crmE. Cytokines are proteins produced by a cell that bind to specific receptors on other cells to modify their behavior in response to stimuli. Tumor necrosis factor is a pro-inflammatory cytokine that is secreted by monocytes and macrophages to bind to cells when they are infected with a virus. This binding activates a signal for apoptosis (programmed death) of the cell so that the virus cannot continue replicating. When poxviruses infect the cell, they produce the vTNFRs which mimic the binding domains of the cell receptors that TNF normally binds to. The TNF is then bound up by the vTNFRs and cannot signal apoptosis of the cell, allowing the virus to finish its replication and produce more virions.
Another group of cytokines targeted by a set of viral proteins is the interferons. Inteferons are cytokines produced by infected cells and dendritic cells in response to viral infection. Interferon-α and Interferon-β are considered type I interferons (IFN) and both bind to the same receptor, IFN-α/βR. When IFNs are produced, they bind to infected cells to activate expression of genes involved in cell death. As a precaution, they also bind to uninfected cells nearby to promote death upon infection. Poxviruses have genes encoding a viral IFN-α/βR that binds up Interferon-α and Interferon-β. In Vaccinia virus, the particular gene is the B18R gene. By binding up the IFN, the virus is preventing the activation of genes for cellular death.
Poxviruses are also able to shut down the complement system which is part of both the innate and adaptive immune response. The complement system resides in the blood and is one of the first responses to viral infection. The classical, alternative, and mannan-binding pathways are the three complement pathways that lead to the production of C3 convertase. C3 convertase activates the membrane attack complex that creates holes in the membrane of the infected cell and inactivates viral particles. Proteins C3b and C4b bind to the viral particles targeting them for phagocytes and are involved in the production of C3 convertase. Poxviruses encode complement control proteins (CCPs) to interrupt this system. Examples are the Vaccinia virus Complement Control Protein (VCP) and the Smallpox Inhibitor of Complement Enzymes (SPICE) which are both comprised of CCP proteins. Both VCP and SPICE are involved in inactivating C3b and C4b, and VCP also aids in degrading the C3 convertase thereby shutting down the complement pathway.
A set of genes that directly inhibit the apoptosis of the host cell are the serpins. Serpins are serine protease inhibitors, and an example is the crmA gene found in the cowpox virus (known as SPI-2 in other poxviruses) that encodes a serpin that inhibits granzyme B. When cytotoxic T-lymphocytes and natural killer cells encounter infected cells, they directly deliver granules containing perforins and granzymes to them. The granules are taken in by endocytosis and then the perforins create holes in granules to release the granzymes. Granzyme B is known to activate both the protein Bid and the caspase pathway by proteolysis. Activated Bid directly initiates the shutdown of the mitochondria leading to cell death while the caspase pathway indirectly leads to cell death. The CrmA protein has been found to inhibit the actions of Granzyme B, thereby stopping the apoptosis.
Poxviruses carry viral epidermal growth factors (EGF) that are similar to the mammalian EGF. Mammalian EGF can bind to the ErbB receptor, a protein kinase, located in the cell membrane. This binding stimulates the ErbB to phosphorylate and activate substrates involved in induction of the mitogen activated protein kinase (MAPK) pathway. Induction of this pathway leads to cell growth and differentiation. Poxviruses may take advantage of the ErbB receptor by using it to stimulate cell proliferation which creates more cells that can be infected by the viruses. Viral EGF is thought to be important in proliferation of epithelial cells in the skin. Epithelial cells express a high number of ErbB receptors and the poxvirus encoded EGFs have been shown to induce stronger responses than the mammalian EGF. In addition, it has been seen that Vaccinia virus infects cells much more efficiently when entering through the basolateral surface of the membrane as opposed to the apical surface. Viruses entering and exiting cells through the apical surface of epithelial cells mostly cause localized infections because the apical surface faces out and away from the inside of the body. Because the basolateral surface of the epithelial cells faces the inside of the body rather than the outside, it is possibly for multiple areas of the epithelia to be infected by a virus that has replicated in other parts of the body. It is possible that poxviruses in general can access the basolateral surface of the epithelia after replication in the lymph system therefore infecting the skin over the entire body. The viral EGF and basolateral entrance are two factors that may make it easy for poxviruses to access and replicate in the skin.
The virus causing smallpox, Variola, is grouped within the family Poxviridae that contains other viruses such as cowpox, monkeypox, and Vaccinia virus. Two strains of smallpox circulated the world before its eradication. Variola major was the more severe strain with a death rate of 30-40%, while only 1% of Variola minor victims died. Due to poxviruses affinity for growing in the skin, infection with smallpox manifested as full body coverage with skin lesions that would swell with fluid teeming with virus. Though the disease usually spread through inhalation of airborne virus shed from the oropharyngeal tract, milder cases could be caused when the skin lesions burst and released the virus. In the 18th century, a practice known as variolation was adopted. It involved taking fluid from an infected person’s pustules and inoculating it under the skin of an unexposed person. This inoculation would often simply cause a mild rash, though sometimes severe disease did result. However, variolation did provide immunity to the virus. It wasn’t until Edward Jenner perfected the technique using the related cowpox that it was called vaccination. Modern smallpox vaccination utilizes the mild Vaccinia virus.
To date, the only pathogen that humans have successfully eradicated from this earth is smallpox. Several factors, including both aspects of the virus itself and the strategies used, contributed to its eradication. One important factor is the absence of a reservoir for the virus. Humans are the definitive host of smallpox, and unlike rabies or malaria it is not maintained in an animal or insect population. Thus, eliminating it from humans is removing its only chances of replicating. Smallpox is also easy to diagnose based on the unique pustular lesions. This means quick diagnosis is possible leading to quarantine and vaccination to prevent the virus from causing an outbreak. The virus also does not cause subclinical infections like poliovirus does and is not spread during the prodromal phase when diagnosis is difficult. Many people infected with poliovirus are asymptomatic, but continue to shed the virus and infect others, making it hard to eliminate. Almost all smallpox infected people develop the signature rash and are only infectious when the rash appears. Thus, there is a high rate of success in identifying those that are infected and targeting them for preventing an outbreak.
The strategies used to eliminate smallpox were very successful for multiple reasons. First of all, there is an efficient vaccine for the virus that is made from the nonpathogenic Vaccinia virus. This vaccine stimulates both cellular and humoral immunity to Variola virus allowing for long lasting immunity of about 10 years. Secondly, the use of mass and ring vaccination is possible for smallpox. Variola virus requires dense populations to spread efficiently, so efforts to mass vaccinate developed nations with 80% coverage effectively eliminated the susceptible population needed for replication. For developing nations, teams would search out cases and then vaccinate all the people around them in a ring so as to prevent its spread to those in contact with them. In regions of Nigeria, the ring vaccination program eliminated smallpox even though there was only mass vaccination of half the population. Lastly, there was a full global effort to eliminate this virus. In 1966, the World Health Assembly (part of the WHO) decided to fund a 10 year world-wide program for conquering smallpox. The two leading superpowers at the time, the Soviet Union and the United States, both donated millions of doses of vaccine to developing nations that could not afford them. With the entire world working together, it was possible to eliminate this plague of mankind.
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