(a) Photograph of Dr. Donald A. Henderson standing in front of a “Smallpox Zero” sign at an unknown location. Dr. Henderson headed the international efforts in the 1960s to eradicate smallpox and to achieve worldwide “Smallpox Zero” status, a goal that was officially reached in 1980. (b) A Royal Nepal Airlines helicopter used to transport smallpox eradication workers into the remote Gunsi village of the Ramechhap District of Nepal. (c) This female smallpox patient in Cardiff, Wales, was photographed during a 1962 smallpox epidemic.
Courtesy of the CDC.
Courtesy of David Bassett/CDC.
Courtesy of Dr. Charles Farmer Jr./CDC.
“Could it not be contrived to send the Small Pox among those disaffected tribes of Indians? We must on this occasion use every stratagem in our power to reduce them.”
—Lord Jeffrey Amherst, Commanding General of British forces in North America during the French and Indian War, July 1763
14.1 History of Poxviruses
Smallpox is a human infectious disease caused by variola virus (family Poxviridae), and it was a worldwide scourge for thousands of years. Dr. Michael T. Oster-holm of the University of Minnesota School of Public Health has called smallpox “the lion king of infectious diseases.” Smallpox killed approximately 500 million people in the 20th century—more than the 320 million deaths caused by wars, the 1918 Spanish flu pandemic, and acquired immune deficiency syndrome (AIDS) combined. Survivors of smallpox were often left blind from corneal ulcerations and badly scarred by pockmarks. Famous victims and survivors of smallpox are listed in TABLE 14-1.
Scientists speculate that smallpox emerged sometime after the first agricultural settlements, in about 10,000 BC.1 The first evidence of smallpox comes from mummified remains of the Egyptian pharaoh Ramses V. Written descriptions of the disease appeared in China (AD 340)2 and southwestern Asia (AD 910). Smallpox arrived with the explorers and traders from the Old World to the New World, where the population had no exposure or immunity against variola virus before the Europeans introduced it in the 16th century. In 1519, Spanish conquistador Hernando Cortez and his followers landed in Mexico, in the heart of the Aztec empire, followed soon after by a second group of Spanish explorers. In the second group was an African slave who was afflicted with smallpox. In the ensuing months, smallpox spread throughout the Aztec empire. Over time, the Aztec and Inca empires, and eventually the Native American Indians, were devastated by outbreaks of smallpox. Smallpox ravaged Europe in the 17th and 18th centuries as well, afflicting up to 90% of the children in Britain alone.
Table 14-1 Famous Individuals Who Suffered from Smallpox
| Individual | Age Contracted | Year Contracted | Outcome |
| Pharaoh Ramses V | 35 | 1157 BC | Died |
| Mary II, Queen of England | 32 | 1694 | Died |
| Peter II, Emperor of Russia | 15 | 1730 | Died |
| Louis XV, King of France | 64 | 1774 | Died |
| Wolfgang Amadeus Mozart | 11 | 1767 | Severe case, survived |
| George Washington | 19 | 1751 | Severe case, survived |
| Abraham Lincoln | 54 | 1863 | Mild case, survived |
| Joseph Stalin | 7 | 1886 | Severe case, survived |
Smallpox was so pervasive that it was considered unusual if someone did not have pockmarks on their face (FIGURE 14-1). In the United States, smallpox epidemics involving all age groups occurred frequently during the 18th century. Houses were converted to small hospitals referred to as “pest-houses.” The pest-houses were used to care for smallpox patients and isolate their contacts during an epidemic.
14.2 Clinical Features of Human Poxviruses
Smallpox (Variola Virus)
During the World Health Organization’s (WHO) smallpox eradication program, smallpox recognition cards were widely used by workers searching for the last remaining cases of smallpox in remote areas of India and Africa (FIGURE 14-2). Ultimately, the most effective method used to ensure prompt reporting was the offer of a reward, given to both the health worker who investigated the case and the person who reported it. At the beginning of 1974, a reward of 50 rupees ($6.25) was offered for every case found in Indian states.
By the end of 1974, the reward was increased to 100 rupees ($12.50), and by July 1975 the reward increased to 1,000 rupees ($125.00). Given that workers were sometimes paid as little as 10 rupees or less per day by their employers, this reward was a significant compensation. The reward system was also used in Bangladesh and Somalia. In 1978, the WHO increased the award to 8,000 rupees (US $1,000) for reporting a confirmed case.
Variola major and variola minor are two different strains of the same virus (variola) but cause different diseases. Variola major virus is more common and causes a severe disease, with a more extensive rash and a higher fever. Variola minor virus causes a milder disease that has a 1–2% fatality rate in unvaccinated individuals. Variola major smallpox presents as four major types:
Ordinary: The most frequent type, accounting for 89% or more of cases and with a 30% mortality rate.
Vaccine modified: Mild and rare, occurring in 2.1% of previously vaccinated persons. Not lethal.
Flat or malignant pox: Rare (only 6.7% of cases) and very severe, with a 96% mortality rate.
Hemorrhagic: Rare (2.4% of cases) and very severe, with a 96% mortality rate.
Historically, variola major smallpox has an overall mortality rate of about 30%; however, flat and hemorrhagic smallpox usually are fatal. Variola minor smallpox is a less common presentation of smallpox, and a much less severe disease, historically with death rates of 1% or less (FIGURE 14-3).
The average incubation period of smallpox is 12–14 days. Infected individuals are not contagious during the incubation period. The first symptoms of smallpox are fever of 101°–104°F (38°–40°C), a splitting headache, and a severe backache. Vomiting occurs in about half of all infected individuals, and about 10% of people experience diarrhea. Delirium and convulsions may occur in a small percentage of individuals (7–15%), usually children. During this 2- to 4-day prodromal period, the individual is too sick to carry on normal daily activities. The person may be contagious during this stage of infection. By the second or third day, the individual is feeling better. As the fever declines, a macular rash (small red spots) appears on the tongue and in the mouth (oral mucous membranes). The rash becomes papular (raised) and vesicular (blistery). It harbors large quantities of virus. As the vesicles rupture, high numbers of infectious viruses are liberated into the saliva. At this time, the person is most contagious. Lesions may line the respiratory tract, and some individuals experience a sore throat. The skin rash appears: first on the face, then spreading to the arms and legs, and finally to the hands and feet, all within a 24-hour period. The rash is described as being a centrifugal rash (it mainly covers the face and extremities as opposed to the entire body), and it is important in the diagnosis of smallpox.
FIGURE 14-1 (a) In 1751, at the age of 19, George Washington suffered from a severe case of smallpox. He had pockmarks on his face, yet the portrait artist Edward Savage portrays Washington sitting in uniform, his facial complexion smooth, free from smallpox scars. Savage painted Washington’s portrait in oil after his visit to Harvard College in October 1789. (b) While reciting one of the most famous speeches in 19th-century U.S. history, the Gettysburg Address, President Abraham Lincoln was feeling the early signs and symptoms of smallpox. Lincoln was invited in November 1863 to deliver remarks now known as the 273-word “Gettysburg Address for a Dedication of the Soldiers’ National Cemetery in Gettysburg, Pennsylvania.” He was examined by a doctor upon his return to Washington, D.C. Dr. Bibber told Lincoln he had a touch of varioloid, the old-fashioned name for smallpox. In this rare photograph of Abraham Lincoln, the crowd is shown gathered around Lincoln, who is circled in the photograph. He is facing the crowd and not wearing a top hat. Lincoln’s face had a “ghastly color.” He complained of weakness and dizziness the day before his arrival at Gettysburg. Unfortunately, there was no close-up photograph of Lincoln just before, during, or immediately after his speech. Did Lincoln cause an epidemic? Read about his illness, as described in Goldman, A. S., and Schmalstieg, F. C., Jr. 2007. “Abraham Lincoln’s Gettysburg illness.” J Med Biogr 15:104–110.
The raised rash becomes vesicular, containing an opaque and turbid fluid by the fourth or fifth day. The vesicles mature into pustules by the seventh day. By the second week, the rash begins to crust over, and the scabs separate by days 22–27. In fatal cases, death occurs between days 10 and 16 of illness. The communicability of the disease extends from the onset of the rash to the complete separation of all scabs. The reproduction number, R-nought (R0), for smallpox is about 3.
Monkeypox
Human monkeypox virus infections are rare. Before 1970, monkeypox was recognized as a disease of animals, normally occurring in the rainforests of Central and West Africa. Monkeypox virus was first isolated from infected cynomolgus monkeys in 1958. Between 1970 and 1986, the first human cases of monkeypox were reported in West Africa and the Congo Basin as smallpox disappeared. The smallpox vaccine protects or mitigates the clinical manifestations of monkeypox.
The first outbreaks of human-to-human transmission of monkeypox occurred in 13 villages in Kasai-Oriental, Zaire, from 1996 to 1997. The Zaire Ministry of Health and the WHO investigated the epidemics. It was postulated that lack of vaccination and a concurrent epidemic involving a large number of animals in close proximity to humans were factors contributing to monkeypox viruses crossing the species barrier into humans. In June 2003, the first cases of monkeypox were reported among several people in a multistate outbreak in the United States who had had contact with infected pet prairie dogs and other exotic pets. The case mortality rate of monkeypox is 1–14%.
FIGURE 14-2 (a) Front of the WHO recognition smallpox card used during the eradication program. The cards were shown by workers to query villagers for sightings of people with a similar rash. (b) Reverse side of the smallpox recognition card.
The signs and symptoms of monkeypox are similar to ordinary or modified smallpox but milder. Patients experience a fever, respiratory symptoms, and vesicular rash similar to smallpox victims (FIGURE 14-4). One difference is that lymphadenopathy (a chronic, abnormal enlargement of the lymph nodes) is more prominent in monkeypox patients during the early stages of the disease.
Molluscum Contagiosum
Molluscum contagiosum is a disease caused by molluscum contagiousum virus (MCV), a common poxvirus infection of worldwide distribution. The infectious disease has the potential to become a public health problem in areas with poor sanitary and hygienic conditions. It represents 1% of all skin infections. MCV is transmitted through direct contact, including sexual contact, or more commonly as a consequence of indirect contact through fomites, such as by sharing towels, or from swimming pools. Transmission among children is rapid in day care centers and kindergartens. MCV causes a significant opportunistic infection in patients suffering from human immunodeficiency virus (HIV) infections, especially those with severely depressed CD4+ T lymphocyte numbers. HIV patients may experience persistent and fulminating molluscum contagiosum.
FIGURE 14-3 (a) This man is suffering from ordinary smallpox caused by variola major infection. He is a patient at the Military Hospital No. 45, an isolation hospital in Gungalin, Australia. (b) The usual centrifugal pattern of smallpox maculopapular rash (fewer lesions on the trunk and more on his face and extremities) is revealed by this photograph of a Bangladeshi boy (1973). (c) The last known person in the world to have natural smallpox of any kind. At 23 years old, Ali Maow Maalin suffered from a viral infection caused by variola minor. He lived in Merka, Somalia (1977). (d) This Bangladeshi man suffered from the most severe form of smallpox (hemorrhagic) caused by variola major. He likely died from the viral illness (1975).
The incubation period for molluscum contagiosum is 2–8 weeks. The virus causes pink, pearl-like lesions (a molluscum) on the face (especially the eyelids), arms, and legs, which can be 1–5 mm in diameter (FIGURE 14-5). The molluscum usually has a dimple in the center. If the infection is sexually transmitted, the lesions can be seen on the genitals, thighs, and the lower part of the stomach. Scratching may cause the lesions to spread. MCV infection is usually self-limiting; however, it can take 6 months to 5 years for the lesions to disappear. Debate continues as to whether the lesions should be treated (e.g., by surgical removal, scraping, or freezing) or allowed to resolve spontaneously. Secondary bacterial infections are a complication of molluscum contagiosum lesions in AIDS patients. Molluscum contagiosum can serve as a marker for severe immunodeficiency.
FIGURE 14-4 Photographs of the crusted lesions present on the face of a mother and finger of her daughter. Both were infected by monkeypox virus after handling infected prairie dogs purchased at a pet “swap meet.”
Vaccinia Virus
Intradermal inoculation of vaccinia viruses propagated on the belly of calves (see Section 14.11) was directly responsible for the successful eradication of smallpox (variola). Infection with vaccinia virus provides adequate cross-protective immunity against variola and monkey-pox viruses. Although the exact origins of vaccinia virus are a mystery, genetically vaccinia virus is most similar to buffalopox. Inoculation with manufactured live vaccinia virus (Dryvax vaccine) results in a localized skin infection. In persons who are immunocompromised or who suffer from skin conditions, vaccinia may disseminate and cause severe disease (see Section 14.11 for more on adverse reactions caused by Dryvax vaccines). Routine childhood vaccination to prevent smallpox was discontinued in the United States in 1971 because the risk of contracting smallpox was reduced during the 1960s as a result of routine vaccination. In 1976, routine vaccination of healthcare workers was also discontinued. For several years all military personnel continued to be routinely vaccinated.
Recent mysterious outbreaks of an emergent pox-virus from humans and cattle in Rio de Janeiro State, Brazil, may represent a persistent vaccinia-like virus infection in nature. A 2008–2010 study of vaccinia virus infections occurring on 56 dairy farms located in the Amazon biome of Brazil concluded that animal movement on farms located along a 41-km (25-mile) highway was the major cause of vaccinia (Cantagalo strain) infections in dairy cows and workers. Cattle trading at auctions launched the spread of vaccinia virus. Migration of infected dairy cows and workers was also associated with the spread of vaccinia viruses.
FIGURE 14-5 Molluscum contagiosum lesions.
14.3 Laboratory Diagnosis of Poxvirus Infections
In the United States, the last natural case of smallpox occurred in 1949, and routine smallpox vaccination of children was discontinued in 1971. Very few U.S. physicians have seen an actual case of smallpox. Today, variola major virus is a potential biological weapon, which is why experts at the Centers for Disease Control and Prevention (CDC) have prepared information and instructions for physicians in case of a smallpox epidemic emergency. The CDC has defined a smallpox clinical case as “an illness with acute onset of fever greater than or equal to 101°F (38.3°C) followed by a rash characterized by firm, deep-seated vesicles or pustules in the same stage of development without other apparent cause.”
Typically, diagnosis of poxvirus infections is made based on the appearance of the lesions. Smallpox produces a centrifugal rash, as compared to a chickenpox rash, which is more concentrated on the trunk of the body (FIGURE 14-6). Preliminary examination of specimens such as scrapings of the pustules, blood, and scabs may be done by state health department laboratories. If variola virus or another poxvirus is suspected, scabs are also collected and analyzed by the CDC for definitive testing using electron microscopy to visualize poxvirus particles. Other methods such as virus isolation, histology, IgM and IgG enzyme-linked immunosorbent assay (ElisA), or polymerase chain reaction (PCR) techniques, followed by restriction fragment length polymorphism (RFlP) or DNA sequencing analysis, may be used to detect the causative agent.
Electron microscopy has played a major role in viral diagnosis in the past. If available, it can be a first-line method for laboratory diagnosis of poxvirus infections and may provide one of the first clues to the cause of the unknown rash illness. PCRRFLP analysis is the only way to accurately distinguish among variola virus, monkeypox virus, and vaccinia virus infections.
FIGURE 14-6 Rash distribution of smallpox versus chickenpox.
14.4 Cellular Pathogenesis
Despite the historical significance of smallpox, very little is known about the pathogenesis and virulence of variola virus. The knowledge we have comes from an era before modern advances in molecular virology and immunology. Virtually no molecular biology was applied to the study of live variola virus. Today, variola virus is stored safely in two international repositories: the State Research Center of Virology and Biotechnology (Vector) near Novosibirsk in Russia and at the CDC in Atlanta, Georgia. Variola virus can only be safely worked with in a BSL-4 (Biosafety Level 4) maximum containment facility, sometimes referred to as a “hot lab.” Some are concerned, however, that smallpox could reemerge in modern society through the use of variola virus as a bioweapon. To improve our understanding of variola pathogenesis, further research is needed to develop rapid diagnostics, antivirals, and a protective but less reactogenic and safer vaccine to prevent smallpox. To address these needs, research proposals with variola virus have been approved by the WHO. The proposals include the development of an animal model such as the use of cynomolgus monkeys to attempt to repeat some of the pathologic features of human smallpox because it is impractical and unethical to safely do smallpox experiments in humans. In nature, variola virus infections are uniquely restricted to humans, which is why surrogate primate models are needed to test vaccines and therapeutics.
14.5 Naming and Structure of Poxviruses
The name variola is derived from the Latin term varius, meaning “spotted,” or varus, which means “pimple.” The word poc or pocca—a “bag” or “pouch”—describes an exanthematous disease, a disease accompanied by skin eruption such as smallpox, and English accounts began to use the word pockes. When syphilis appeared in Europe in the late 15th century, writers began to use the prefix small to distinguish variola, the smallpox, from syphilis, the “great pox.” Syphilis is caused by the spirochete bacterium Treponema pallidum. Vaccinia virus, the “vaccine virus,” is a relative of variola virus. It is the prototype of poxviruses studied by many researchers. Determining poxvirus structure and replication is based on laboratory experiments involving cell cultures infected with vaccinia virus.
FIGURE 14-7 (a) Schematic drawing of a typical poxvirus virion. (b) Transmission electron micrograph of variola virus particles (310,000×, negative stain). (c) Transmission electron micrograph of a thin section of vaccinia viruses. The MV membrane, core, and lateral bodies are labeled.
Poxviruses are the largest of all animal viruses. They can be visualized by light microscopy. Poxvirus virions are complex-shaped. The virions are enveloped and rectangular, or “brick-shaped.” Most are 270–350 nm in length. They contain linear double-stranded DNA (dsDNA) genomes with closed ends that are 130–230 kilobase pairs (kbp) in length. The ends of the genome have inverted terminal repeat (ITR) sequences. Clinical specimens contain two different morphologies of virions: the intact M, or “mulberry,” form mainly found in the vesicular fluid and the C, or “capsule,” form associated with dried scabs. Internally, poxviruses have a dumbbell-shaped (the shape of free weights used at the gym) core and two lateral bodies surrounded by an outer membrane, as revealed by staining thin sections of infected cells using transmission electron microscopy (FIGURE 14-7). The lateral bodies contain various enzymes essential for viral replication. The core contains the viral genome with associated proteins involved in the morphogenesis of virus particles (A3L, A4L, A10L, or F17R gene products) or transcription (L4R gene product).
Broad Host Range of Poxviruses
Naturally occurring poxvirus infections affect humans and many species of animals and insects. Most poxviruses were named after the animal from which they were originally isolated (e.g., cowpox and camelpox), but the main reservoirs for the viruses may be rodents or other species (TABLE 14-2). The one large Poxviridae family contains two subfamilies: Chordopoxvirinae, which infect vertebrates, and Entomopoxvirinae, which infect insect hosts. This chapter focuses on members of the Poxviridae family that infect humans: smallpox (variola major and minor); molluscum contagiosum virus (MCV); monkeypox, a recently emerged virus in the United States transmitted as a zoonotic disease by wild rodents from Africa; and vaccinia virus (the vaccine strain use to prevent smallpox). With the exceptions of the “extinct” variola virus and MCV, all other poxvirus infections of humans today are zoonoses (TABLE 14-3).
14.6 Vaccinia Virus Replication
Vaccinia virus is the prototype of poxviruses. It can be propagated in a wide host range of tissue culture cells (e.g., monkey, rabbit, hamster, and mouse). Vaccinia virus is analogous to the Escherichia coli of the bacteriology laboratory: it can be grown to high titers in a short period of time. Within 12–24 hours, 1 × 108 or 109 infectious viruses/mL of cell culture medium can be produced. Infectious vaccinia virus particles are large (300–400 nm in diameter) and brick shaped. Lipoprotein membranes surround a complex core structure that contains a 200,000-base pair (bp) linear dsDNA genome that encodes at least 200 genes. Each end of the genome consists of a terminal hairpin loop (no free ends) consisting of inverted terminal repetitive sequences that are rich in adenine and thymine (FIGURE 14-8). The complete DNA sequence information for more than 40 poxviruses is available. The application of sequence information in order to perform mutagenesis of the poxvirus genomes, mutagenesis of poxvirus genomes, and experiments in which mutant viruses were used to infect cell cultures in order to study replication of viral mutants determined that the essential genes are located in the central part of the genome, whereas nonessential genes (i.e., not essential for growth in cell cultures) are located at the ends of the genome. Approximately 90 essential genes are highly conserved among different poxviruses. Their functions are essential for viral replication and morphogenesis.
Table 14-2 Human Poxviruses and Their Natural Hosts
| Virus | Natural Hosta/Reservoir | Disease | Geographic Distribution |
| Variola major and minor viruses | Humansa | Smallpox, rash, generalized disease | Previously worldwide; eradication in humans in 1977 |
| Monkeypox virus | Squirrels,a monkeys, rabbits, mice, rats, humans | Monkeypox, rash, generalized disease | Western and Central Africa |
| Molluscum contagiosum virus | Humansa | Molluscum contagiosum, skin nodules | Worldwide |
| Orf virus | Sheep,a dogs, goats, humans, chamois, reindeer, musk ox, Himalayan tahr, Steenbok, alpaca | Localized skin lesions | Worldwide |
| Pseudocowpox virus | European cattle,a humans | Milker’s nodules | Worldwide |
| Bovine papular stomatitis virus | European cattle,a humans | Localized skin lesions | Worldwide |
| Cowpox virus | Rodents,a cats, cows, zoo animals, humans | Localized pustular lesions | Western Asia, Europe |
| Vaccinia virus | Natural host unknown (buffalob and cattle?), humans | Buffalopox, localized pustular lesions | Asia, India, Brazil, and laboratory |
| Tanapox | Monkeys (?), rodents (?), humans | Tanapox, localized nodular lesions | Eastern and Central Africa |
| Yaba monkey tumor poxvirus | Monkeys (?), humans | Localized nodular skin lesions | Western Africa |
a Natural host. | |||
b During the WHO smallpox eradication program, water buffalo in India and cattle in Brazil were infected with the local vaccine strain of vaccinia virus, which apparently persists in these animals and occasionally infects humans. | |||
Information from White, D. O., and Fenner, F. J. Medical Virology, 4th ed. San Diego: Academic Press, 1994. | |||
Table 14-3 Members of the Poxviridae That Infect Vertebrates
| Genus | Member Viruses |
| Avipoxvirus | Canarypox, fowlpox, pigeonpox, juncopox, quailpox, turkeypox, starlingpox, sparrowpox, peacockpox, penguinpox |
| Capripoxvirus | Goatpox, sheeppox, lumpy skin disease |
| Leporipoxvirus | Hare fibroma, myxoma, squirrel fibroma, rabbit fibroma |
| Molluscipoxvirus | Molluscum contagiosum virus* |
| Orthopoxvirus | Variola (smallpox),* vaccinia, monkeypox,* buffalopox, camelpox, cowpox,* ectromelia (mousepox), volepox, raccoonpox, skunkpox, elephantpox |
| Parapoxvirus | Pseudocowpox,* orf,* bovine papular stomatitis virus,* sealpox, parapox of deer |
| Suipoxvirus | Swinepox |
| Yatapoxvirus | Tanapox,* Yaba monkey tumor* |
*Human pathogens. | |
The entry of poxviruses into cells is complicated by the existence of multiple forms of infectious particles. One form, called the mature virion (MV), contains two membranes. The MV membranes are obtained from the Golgi apparatus and the endosomes of the host cell. The second form is an extracellular virion (EV), which only contains the inner membrane derived from a Golgi-wrapping event. The outer membrane of EV is lost during fusion with the plasma membrane during exocytosis. The MVs are released upon cell lysis.
FIGURE 14-8 Typical poxvirus genome structure.
Each infectious form is thought to enter cells by a different mechanism. Vaccinia virus can enter almost every cell line tested. This means either that the receptor for vaccinia virus entry is highly conserved and ubiquitous in nature or that the virus can enter via more than one receptor. Some studies have shown that the A27L virion protein of vaccinia virus interacts with cell surface glycosaminoglycans (GAGs). GAGs are ubiquitously expressed on many different cell surfaces. The mechanism for cell entry and penetration is complex and may involve more than one mechanism.
During uncoating of EV, the particle enters the cell, losing the membrane, and the viral core passes into the cytoplasm. One of the unique hallmarks of poxviruses is that these viruses have acquired all of the functions necessary for genome replication in the cytoplasm even though they have DNA genomes. Poxviruses have a dsDNA genome that is not replicated by cellular DNA polymerases in the nucleus. Poxvirus gene expression and genome replication can occur in enucleated cells, but particle maturation is blocked. Viral enzymes associated with the core carry out the three stages of gene expression: early, intermediate, and late gene expression. The three distinct classes of viral messenger RNAs (mRNAs) are transcribed from genes containing promoters and other sequence elements that provide the basis for their programmed order of expression. Poxvirus mRNAs are not spliced.
A complete early transcription system is synthesized late during infection and is packaged into the viral core. This viral transcriptional machinery consists of a DNA-dependent RNA polymerase, a transcription factor (VETF), capping and methylating enzymes, and a poly(A) polymerase. The machinery has the capability to synthesize mRNAs that are recognized and translated by the host cell’s protein synthesis machinery. The early mRNAs encode enzymes and factors needed for transcription of the intermediate class of genes that, in turn, encode enzymes for late gene expression. Late expression occurs after genome replication. The vaccinia virus D10 gene encodes a decapping enzyme that may accelerate viral mRNA turnover and help eliminate competing host mRNAs from being translated by the translational machinery. This would allow for stage-specific synthesis of viral proteins.
After the late structural proteins are synthesized, infectious progeny particles are assembled. Assembly involves interactions with the cytoskeleton (e.g., actin-binding proteins). The MV particles are wrapped with a Golgi-derived membrane and endosomes and transported to the periphery of the cell. The EV loses a membrane as it is released from the cell, completing the virus replication cycle. EVs can initiate infection, mediating cell-to-cell spread (FIGURE 14-9).
14.7 Poxviruses and Immune Evasion
Poxviruses do not undergo a latent stage by integrating viral DNA into the host chromosome. Nor do they cause persistent infections making them invisible to the immune system. Instead, poxviruses use a number of viral proteins to evade the host’s innate (nonspecific) immune responses. They produce viroceptors and virokines. Viroceptors are altered cellular receptors that have lost their transmembrane anchor sequences. Consequently, these unanchored viral proteins are secreted from infected cells, where they sequester ligands onto the receptor portion of the protein. Virokines are secreted viral proteins that resemble host cytokines. The majority of poxvirus virokines and viroceptors were discovered by investigators who compared the viral protein sequences to the sequences of their cellular counterparts. Besides secreted viral inhibitors, poxviruses also produce intracellular proteins that interfere with signaling pathways within the infected cell. The following are examples of poxvirus inhibitors:
Secreted complement regulatory proteins
Secreted proteins that bind to interferons
Secreted interleukin 18 binding proteins
Secreted tumor necrosis factor homologs (apoptosis inhibitors)
Intracellular serpins (serine protease inhibitors)
Intracellular inhibitors of PKR, which plays a role in the cellular interferon pathway
The field of poxvirus-encoded immune modulators is more than 25 years old, and discoveries continue in this area. Even though no one specific poxvirus uses all of the strategies, all seem to target the immediate innate immune response (FIGURE 14-10).
FIGURE 14-9 Life cycle of vaccinia virus. Virions packaged with a dsDNA genome and associated enzymes and transcription factors attach to host cells and fuse with the plasma membrane. Vaccinia cores are released into the cytoplasm. Early mRNAs are synthesized and translated by the cellular machinery. Some of the early proteins are involved in intermediate transcription and DNA replication factors. The viral DNA is uncoated and the DNA is replicated. Intermediate and late genes are transcribed and translated. The late gene products are structural proteins, enzymes, and early transcription factors. Assembly involves the formation of membrane structures. Viral dsDNA is packaged into immature virions. The virus particles mature to form infectious intracellular mature virions (MVs) that are wrapped by membranes from the trans-Golgi network or endosomes. The wrapped virions (WVs) are transported to the periphery of the cell by microtubules. As the wrapped vaccinia virus particles fuse with the plasma membrane, one of the membranes is lost and extracellular virions (EVs) are released. For more detailed information about entry and membrane fusion, see Moss, B. 2006. “Poxvirus entry and membrane fusion.” Virology 344:48–54.
14.8 Human Genetics and Smallpox Resistance
In order for an infectious disease to cause selective pressure on host genetics, the disease would need to have a significant effect on morbidity and mortality before reproductive age, over a long period of time. Smallpox was endemic throughout much of the world for more than 2,000 years. In populations where epidemics occurred frequently, smallpox was a childhood disease, killing as many as 30% of its victims. Because human populations were exposed to the variola virus for generations, resistant genes should be common in smallpox survivors. In 1965 and 1966, Vogel and Chakravartti conducted a comparative study of 415 unvaccinated Indian smallpox survivors and victims. They discovered that people who were blood type A were seven times more likely to contract smallpox, three times more likely to develop a severe case of the disease, and twice as likely to die of it.
A 2003 study by Galvani and Slatkin suggested that smallpox is responsible for the genetic selection of the CCR5Δ32 deletion allele that confers resistance to HIV infection in humans. CCR5 is a chemokine coreceptor required for HIV infection. A homozygous CCR5Δ32 deletion allele provides almost complete resistance to HIV infection. A heterozygous CCR5Δ32 deletion allele confers partial resistance and a slower disease progression. Because HIV infection is relatively new, there have not been enough human generations or time for the selective rise of this resistance allele within the human genome. Researchers argue that other infectious agents are likely to have selected for this resistance allele.
FIGURE 14-10 Representation of poxvirus immune evasion proteins.
The most popular hypothesis suggests that this selective pressure was the result of bubonic plague (i.e., Black Death) pandemics in Europe. Galvani and Slatkin argue that the plague outbreaks occurred in a shorter time frame—400 years—which is not enough time to generate the resistance allele within the human population. Infections caused by variola virus (cause of smallpox) have occurred in human history for more than 2,000 years, which seems to be more consistent with the amount of time needed for selective pressure on the human genome to result in the development of the CCR5Δ32 deletion allele. The bottom line is that natural selection is a reality. The study of genetic susceptibility to infectious agents may contribute to the design of new therapeutics; for example, interference with the CCR5 chemokine receptor may have promise as a treatment for HIV infection.
14.9 Smallpox Eradication
The word eradicate comes from the Latin word eradicare, which means “to tear out by the roots.” Several diseases have been candidates for global eradication programs, including hookworm and yellow fever. Programs have also targeted the eradication of mosquito vectors that carry the malarial parasite and other disease-causing organisms. Smallpox is the only human disease that has officially been eradicated.
It was possible to eradicate smallpox for several reasons:
Smallpox has a narrow host range (variola virus only infects humans).
Smallpox does not have a long-term carrier state in humans.
Smallpox does not have an animal reservoir.
A highly effective and inexpensive freeze-dried vaccine was available.
Surveillance of the disease was easy (e.g., observation of centrifugal rash).
The WHO created a program to eradicate it.
In 1967, the WHO launched a mass vaccination pro-gram to vaccinate 100% of the human population. At that time, approximately 10–15 million cases of smallpox occurred annually, and the disease was endemic in 30 countries. Over time, the eradication strategy shifted to containment vaccination or surveillance containment around newly discovered cases or outbreaks. This new strategy was implemented after countries such as Nigeria and India experienced outbreaks originating in areas where religious groups resisted vaccination. To prevent spread of epidemics to populated areas, healthcare workers went on house-to-house searches to control local outbreaks. This was so successful in preventing the spread of the smallpox that controlling outbreaks through variations of surveillance containment were adopted (FIGURE 14-11). Evaluation and assessment procedures were modified in response to new experiences and lessons learned in the field. Important innovations, such as the use of smallpox recognition cards (Figure 14-2), rewards, bifurcated needles (FIGURE 14-12), rumor registers, and containment books, were used in the field. Every case of fever with rash was recorded, monitored, and treated as smallpox unless proven otherwise. Four watch guards were placed at homes containing infected family members and isolated contacts. All villages within 10 miles (16 km) of a case of known or suspected smallpox were searched and vaccinations were carried out. In 1977, the last natural case of smallpox was reported in Somalia. Smallpox was the first and only infectious disease to be eradicated.
14.10 Recombinant Vaccinia Viruses as Research Tools and Vaccines
Research on poxviruses did not languish after the eradication of smallpox. Interest in vaccinia virus, the prototype of poxviruses, exploded in the 1980s. Recombinant dNA technology was applied to vaccinia virus. Inserting foreign genes into vaccinia virus provided a way to study the foreign genes expressed in the cytoplasm of mammalian cells. These new recombinant vaccinia viruses have multiple applications in research, gene therapy, and vaccinology. For example, a successful recombinant vaccinia-based rabies vaccine was used to eliminate rabies in Western Europe and the United States.
FIGURE 14-11 (a) WHO smallpox eradication workers faced rugged terrain as they rode four-wheel-drive vehicles through the Bangladeshi countryside in an attempt to eradicate smallpox from the country. Off-road Land Rovers enabled the country’s eradication program to succeed, giving public health practitioners access to even the most inaccessible rural villages (1975). (b) Nigerian child being immunized during the Smallpox Eradication and Measles Control Program of West Africa (1960).
The construction of a recombinant vaccinia virus is illustrated in FIGURE 14-13. The foreign gene of interest (e.g., the rabies gene that codes for the surface glycoprotein of rabies virions) is inserted into a plasmid DNA using restriction enzymes and T4 DNA ligase. The gene is inserted into the plasmid DNA that contains a vaccinia virus promoter and a nonessential vaccinia virus gene such as the thymidine kinase (TK) gene. The presence of the vaccinia virus TK gene allows for homologous recombination of the recombinant plasmid DNA containing the foreign gene into the vaccinia virus genome. The engineered plasmid DNA is transfected into TK– tissue culture cells (cells that do not contain a TK gene) simultaneously infected with vaccinia viruses. Absence of TK activity in host cells and disruption of the TK gene in vaccinia virus renders the host cells resistant to the toxic effects of 5-bromodeoxyuridine (brdu). This selection enriches for cells that carry recombinant vaccinia viruses.
FIGURE 14-12 The bifurcated needle replaced the jet injector to administer the smallpox vaccine. The jet injector could do more than 1,000 vaccinations in an hour, but its use was not practical and it was too expensive for use for house-to-house vaccination. By 1970, the bifurcated needle replaced traditional methods and was used everywhere.
Thymidine kinase is an essential cellular enzyme that is expressed during cell division. It enables a cell to utilize an alternate metabolic pathway for incorporating thymidine into DNA. In the presence of BrdU, BrdU is incorporated into DNA instead of thymidine, causing chain termination of the replicating DNA. It causes breaks in the chromosomes of cells. It is used as an anticancer drug and is a potential antiviral drug. In the practice of creating vaccinia virus recombinants, a TK selection system was developed. Only those cells and recombinant viruses that do not contain a functional TK gene can survive the effects of BrdU. Cells or viruses with an intact TK gene will incorporate BrdU, resulting in genetic mutations and likely cell death and/or viral inactivation.
One of the major advantages of using vaccinia virus is the fact that it can package dsDNA for more than 200 genes, including foreign genes. RNA viruses have smaller genomes and a packaging size limit. RNA viruses are also more difficult to genetically engineer. Foreign genes can be introduced into nonessential parts (e.g., the TK gene) of the vaccinia genome via homologous recombination. Hence, vaccinia viruses are able to express one or more unrelated genes in a cell culture system. This makes it a very attractive tool for molecular biologists. One very important application of vaccinia virus is using its recombinants as new live vaccines against infectious agents of humans and animals.
FIGURE 14-13 Flowchart depicting the creation of recombinant vaccinia viruses using a TK selection system. When a plasmid DNA (which contains a foreign gene inserted into the vaccinia TK present in the plasmid vector) is transfected into TK-deficient cells, only those vaccinia viruses that contain the plasmid DNA will be able to replicate in the presence of BrdU. This is possible because the plasmid DNA containing the foreign gene is incorporated into the TK gene of wild-type vaccinia viruses through the homologous recombination. The transfected plasmid DNA recombines into the vaccinia TK gene (thereby inactivating TK of the wild-type vaccinia viruses), allowing only recombinant vaccinia viruses that are TK deficient to replicate in TK-deficient cells in the presence of BrdU.
A disadvantage of live vaccinia virus is the complications that can follow vaccination. To overcome this, attenuated strains of vaccinia virus have been generated. The modification of attenuated poxvirus strains has resulted in the production of safer live vaccines. It is recommended that laboratory workers who directly handle cultures or animals infected with vaccinia virus be vaccinated. Laboratory workers using highly attenuated strains of vaccinia virus, such as the modified vaccinia Ankara (MVA) virus; the New York vaccinia (NYVAC) virus; or avipox viruses, such as ALVAC (attenuated canarypox) and TROVAC (attenuated fowl-pox) do not require vaccination.
The attenuated viruses are called suicide vectors. All of them undergo abortive replication in mammalian cells. In other words, the attenuated poxviruses can infect cells, but there is a block in their replication cycle such that early viral proteins are produced but late proteins (and therefore full infectious particles) are not produced in mammalian cells. The attenuated vectors that undergo abortive infections can express high levels of a foreign antigen without being pathogenic to their host. Attenuated viruses are propagated in chick embryo fibroblasts (CEFs) or sometimes baby hamster kidney (BHK-21) cells. All of the attenuated poxviruses have a very restricted host range. Recombinant vaccinia virus or other attenuated poxvirus vectors have been genetically engineered to contain genes from a wide variety of infectious agents, including HIV and severe acute respiratory syndrome-coronavirus (SARS-CoV). During the 1990s, myxoma virus was genetically modified as an immunocontraceptive vaccine (see VIRUS FILE 14-1).
14.11 Prevention: Vaccines
Smallpox Vaccine History
Attempts to prevent the disease that caused “the speckled monster” date to practices in China beginning in the late 17th century. Two such practices were to inhale or plug the nose with powdered smallpox scabs or to put the underwear of an infected child on a healthy child for several days. Hindus in India practiced variolation during the 16th century. Variolation is the intentional inoculation of dried smallpox scabs into the skin of an uninfected individual, causing a mild form of the disease and immunity against subsequent exposure to variola virus. The practice spread from central Asia, east to China, and then west to Africa and the Ottoman Empire. The practice used the direct inoculation of smallpox into susceptible persons, so it is not surprising that 2–3% of variolated people died, as compared to 30% who died when they contracted smallpox naturally.
Lady Mary Wortley Montague (1689–1762), the wife of the British ambassador to Turkey, learned about variolation (referred to as engrafting) during her travels to Constantinople (now Istanbul) in 1717, when she observed Muslims applying the technique. Montague expressed interest regarding this practice because she had survived smallpox in 1715. The disease left her without eyelashes and a badly pockmarked face; her brother died from it. After returning to London, Montague had the embassy surgeon, Dr. Charles Maitland, engraft her 4-year-old daughter in 1721. Dr. Maitland gained permission to conduct a study in which six prisoners underwent the variolation procedure. The prisoners survived after exposure to two children with smallpox. They had a milder form of the disease and developed immunity. The study was witnessed by members of the Royal Society of London and based on the results the inoculations became the acceptable medical practice of variolation in England.
Edward Jenner (1749–1823), a physician in England, is credited with cowpox vaccination, the first attempt to control smallpox without transmitting the disease. Jenner based his experiment on folklore that milkmaids who contracted cowpox “did not take the smallpox,” and in 1796 he variolated the 8-year-old son of a local farmer with fluid from the cowpox pustules from the hand of a local dairymaid. The boy developed a slight fever and a few lesions but remained unscathed for the most part. A few months later, the boy was injected with matter from smallpox scabs and failed to develop the disease.
Jenner continued the experiments and published his findings in Inquiry into the Cause and Effects of the Variolae Vaccinae in 1798. The immediate reaction to Jenner’s work was ridicule. Critics, especially the clergy, claimed it was repulsive and ungodly to inoculate someone with material from a diseased animal. A satirical cartoon of 1802 showed people who were vaccinated sprouting cow heads. Despite the initial reactions, Jenner became famous, and the word vaccination was coined. Interestingly, about 20 years prior to Jenner’s vaccination attempts, a farmer in the county of Dorset, southwest England, inoculated family members with cowpox in a deliberate attempt to prevent smallpox. He was credited posthumously (see VIRUS FILE 14-2).
In the 1800s, the practice of arm-to-arm passage (or arm-to-arm vaccination) was used to prevent smallpox. The fluid (i.e., lymph) taken from a vesicle or pustule from a previously vaccinated person was passaged arm to arm between humans. Occasionally, the vaccine was lost and new vaccine strains were obtained from cows or horses. Arm-to-arm passage was not a safe practice. Sometimes the vaccine became contaminated with variola major or minor viruses, which sometimes resulted in the inadvertent transmission of other infectious diseases, such as syphilis and tuberculosis. At least one recorded epidemic caused by hepatitis B virus was attributed to arm-to-arm passage. Continued uncertainty about the safety of vaccination in England regarding the risk of transmitting infectious disease with human lymph led the Royal Commission to pass the Vaccination Act of 1898. The legislation banned arm-to-arm vaccination in England.
