Information

Pox virus infection process


How does Pox virus duplicate it's genome? Does it bring DNA polymerase or RNA polymerase into the host cell?


Poxviruses, such as variola (the causative agent of smallpox) and vaccinia (the active constituent of the vaccine that eradicated smallpox), are very large, complex DNA viruses that replicate in the cytoplasm, rather than the nucleus. Because of this, they must carry enzymes with them to accomplish tasks that occur in the nucleus of the eukaryotic cells they infect. This includes all the proteins necessary for mRNA synthesis (e.g., a DNA dependent RNA polymerase). The enzymes for DNA replication are encoded in the poxvirus genome and are transcribed and translated as part of the "early" stage, directly after adsorption and penetration.

So, yes, poxviruses bring their own preformed RNA polymerase with them. They don't bring their own DNA polymerase with them, but they do produce it shortly after infecting a cell.

You can read more about this in Murray Medical Microbiology Chapter 54.


Pox virus infection process - Biology

Viruses can be seen as obligate, intracellular parasites. A virus must attach to a living cell, be taken inside, manufacture its proteins and copy its genome, and find a way to escape the cell so that the virus can infect other cells. Viruses can infect only certain species of hosts and only certain cells within that host. Cells that a virus may use to replicate are called permissive . For most viruses, the molecular basis for this specificity is that a particular surface molecule known as the viral receptor must be found on the host cell surface for the virus to attach. Also, metabolic and host cell immune response differences seen in different cell types based on differential gene expression are a likely factor in which cells a virus may target for replication. The permissive cell must make the substances that the virus needs or the virus will not be able to replicate there.

Learning Objectives

  • List the steps of replication and explain what occurs at each step
  • Explain the transmission and diseases of viruses that infect animals
  • Explain the transmission and diseases of viruses that infect plants

Smallpox: Dead or Alive?

It begins with symptoms that could be caused by many diseases – pounding headaches, exhaustion, and searing fevers. Only days later, the cold has become something far worse as a telltale rash begins to spread along the patient’s body, soon swelling with pus and scabbing over to leave raised bumps over the infected skin. Even if the patient survives, the disfiguring scars can last a lifetime. This is the mark of smallpox – or it was, almost five decades ago, when the disease was still common in many parts of the world. In terms of mortality, smallpox tops the list: the smallpox virus has killed more members of the human population over the span of recorded history than all other infectious diseases combined 36 .

In most of the diseases we have studied, mankind is in a pitched evolutionary battle with the viral co-inhabitants of this planet. Advances in biomedical technology may result in victorious skirmishes, but the war is seldom won as quickly-replicating viruses race to overcome vaccines and other drugs. Smallpox is one of the few true success stories, as the 1980 announcement by the World Health Organization of the worldwide elimination of smallpox marked a true victory. While smallpox vaccination used to be commonplace and widespread, its eradication made such treatment unnecessary. Except for a few isolated laboratory strains – preserved for posterity – smallpox is just a memory.

Or is it? Those same laboratory strains have many worried. Because smallpox has not been a public health threat for decades, stockpiles of vaccine are at an all-time low. If the virus were weaponized, there would be very little that could be done in response – and that is reason to be concerned.

Poxes Small and Large:

Much of what we know about the human smallpox virus comes from studying similar viruses that infect animals. This comparison works because the smallpox virus belongs to a family of closely related pathogens known as Orthopoxviruses. The members of this family are distinguished by their host (i.e. what kind of animal they can infect) and geographical distribution. Some, like the smallpox virus, are species-specific. For example (as seen in the picture below), poxviruses like variola major (smallpox) of humans (a), mousepox virus of mice (b) or camelpox virus of camels (c) remain largely restricted to one host species and rarely, if ever, cause infections outside of that species. Other poxviruses can infect multiple host species. It is the shared characteristics of this family that allow information about smallpox to be inferred from studies of its animal infecting relatives in the viral evolutionary tree. A few of the more important members of the Orthopoxvirus family include:

Variola virus: This is the virus responsible for smallpox in humans. The name, first used to described the disease rather than the virus, is derived from the latin words varius (meaning “spotted”) and varus (meaning “pimple”), and was coined in the 6th Century in Switzerland 37 . Later, English physicians would use the term pockes, based on the word poc (meaning “pouch”) to describe diseases causing the same raised sores as smallpox 38 . The designation small came in the 15th Century, when it was necessary to distinguish the disease from the “great pox” – syphillis. The virus has two forms, called major and minor, based on the mortality rate for each strain. The classic form of smallpox is caused by V. major, while V. minor was recognized through epidemiological studies in the 20th Century 39 . Geographical variants of both V. major and V. minor also exist.

Vaccinia virus: Because variola is so dangerous to handle (requiring strict biohazard safety conditions), much of what we know about the smallpox virus actually comes from work on vaccina, a pox virus that infects several different animals including cattle 39 . This includes the cowpox virus that Jenner used in his landmark vaccination experiments (see historical section).

The Deadly Brick

The smallpox virion.

Researchers first observed pox viruses under the microscope at the end of the 19th century. Much larger than many viruses, poxes possess a bricklike appearance 39 . The genomes of the orthopox viruses are composed of DNA, not RNA 39 . Because DNA polymerase is much less error-prone than RNA polymerase, this feature has important clinical outcomes for mutation rate (discussed below). Zoom in and one can see that the brick structure is composed of three distinct components: (1) an inner “core” containing the DNA and associated proteins, (2) a protein shell that forms the outer membrane, and (3) a plasma membrane derived from the cell from which the virus budded. The virus does not always contain the outer membrane component. Sometimes, instead of budding, the host cell is broken apart and the virus exits wearing only its outer protein layer.

Schematic of smallpox virion.

While these “un-enveloped” viruses are still problematic, the lack of a cell-derived membrane has important implications in pathogenesis and vaccination 39 .

The Pox Virus Life Cycle

The cycle by which the smallpox virus replicates is similar to other viruses. First, a free-floating virion penetrates the cell membrane of its target cell. How efficiently the virus gets through the host’s membrane depends on whether or not it is coated by a plasma membrane 39 . Those pox virions with a membrane outer coating can enter a host cell more easily and, as a result, are more infectious.

Once inside, the pox virus, like HIV, sheds its outer coating and begins replicating. However, because its genome is made of DNA, the pox virus can copy itself in the cytoplasm unlike RNA viruses that must enter the nucleus of their host. The pox virus generate protein and DNA copies, recombines into a functional virion, and then buds off from the host cell, destroying it as a result. The cycle begins anew as the newly released virions go on to infect other cells.

Smallpox life cycle.

Clinical Symptoms

Even after the smallpox virus has infected a host, no symptoms are seen in the first two weeks 41 . This is a particularly dangerous time as every silently infected person can infect 10 to 15 more, who unless quarantined, go to infect 10 to 15 more themselves 41 . An infected individual initially looks and feels fine because the virus has not started actively replicating and shedding copied versions of itself throughout the body. Once this proliferation has begun in earnest, the infected individual begins to experience flu-like symptoms. Just as the patient seems to be getting better, a terrible rash develops, particularly on the face 41 . The rash worsens, becoming pus-filled bumps that eventually scab over, leaving the pitted scars that are a hallmark of smallpox 41 .

Less obvious – and even more dangerous – are the ulcers that develop inside the throat and nose of the infected individual. When the skin cells containing the virus die, they release the virus into neighboring saliva and digestive passageways. At this point, the individual’s saliva becomes contagious, able to spread the virus by coughing if precautions are not taken 39 . The virus is also freed from the confines of the ulcer, and moves throughout the body through the conduit of the digestive system to infect any organ it comes in contact with 39 . The more organs it infiltrates, the worse for the patient. Simply put, the virus overwhelms the body, killing cells of multiple organ systems.

Transmission

As noted above, transmission of the smallpox variola virus occurs exclusively from human to human with no insect intermediate or animal reservoir also containing the virus. It is mainly spread through aerosol when infected individuals cough up smallpoxlaced mucous particles 42 . Once infected, patients are only contagious once the rash has developed and remain contagious even as the rash scabs 42 . Fallen scabs containing active virus particles can collect on bed sheets or clothing and must be subjected to proper disinfection procedures 42 . In the more severe V. major, the infected are often incapacitated, and so transmission can be minimized as long as exposure by healthy individuals is kept to a minimum 42 . However, in the case of V. minor, the symptoms are so mild that infected individuals may spread the virus easily during its infectious stage 42 .

Drug Treatment?

One of the main reasons a smallpox outbreak would be so deadly is because there is no known drug treatment. The symptoms such as fever and headache can be subsided with traditional medications such as aspirin, but the virus itself cannot be killed by any drug in our current medicinal arsenal. More effective treatments may be on the horizon, though. Cidofovir, a viral DNA polymerase inhibitor used to treat cytomegalovirus (CMV) in AIDS patients, has been shown to kill smallpox virus in laboratory studies 43,44 . However, the compound must be injected intravenously and causes kidney damage in the large concentrations required to penetrate smallpox-infected cells 44 . Currently, scientists are working to re-formulate the drug in a less toxic version. Until then, vaccination remains the primary protection against smallpox. To fully appreciate the impact of vaccination on the eradication of smallpox, first we must delve into some history….

The Origins of Smallpox

Pharaoh Ramses.

The earliest records of smallpox emerged from museum collections including mummies dating from 1570 to 1085 B.C. 44 From these specimens, scientists have concluded that Ramses V (a young Pharaoh monarch who died in his early thirties) likely died of smallpox 45 . With this disease and a civil war during his reign, it seems no great wonder that he died young! Other ancient civilizations proved more fortunate. Indeed, even though the global eradication of smallpox had to await modern technologies allowing large-scale vaccine production, mankind has known for millennia that it is possible to shield oneself against the virus. Striking evidence of this comes from the subcontinent of India, where medical texts from 400 C.E. contain what may be a description of an early vaccination procedure 46 :

Take the fluid of the pock on the udder of the cow or on the arm between the shoulder and elbow of a human subject on the point of a lancer, and lance with it the arms between the shoulders and elbows until the blood appears. Then, mixing this fluid with the blood, the fever of the smallpox will be produced.

If this account is true, then it appears that Indian physicians discovered the
protective power of cowpox a millennia or more before it was known in the West 47 .
Certainly, the clinical symptoms of the disease were recognized by these early doctors, as indicated by passages such as this:

Before [smallpox] appears, fever occurs, with pain over the body, but particularly in the back . . . . When bile is deranged, in this disease, severe pain is felt in the large and small joints, with cough, shaking listlessness and langour the palate, lips and tongue are dry with thirst and no appetite. The pustules are red, yellow, and white and they are accompanied with burning pain. This form soon ripens . . . . When air, bile and phlegm are deranged, in this disease the body has a blue colour, and the skin seems studded with rice. The pustules become black and flat, are depressed in the centre, with much pain. They ripen slowly . . . this form is cured with much difficulty, and it is called Charmo or fatal form 48 .

Indeed, it appears that the disease was so well-known in India that a Hindu Goddess of Smallpox named Sitala was venerated in many regions 49 . The afflicted would pray to Sitala to rid them of the disease, using drops from the water of immortality she was said to carry with her 50 . One of the ways in which Brahmin priests venerated Sitala was to journey the countryside each spring, inoculating villagers against smallpox as they went 51 .

Jenner and Pasteur: The Birth of Vaccination

Edward Jenner.

Development of medical practices in the West comparable to India’s actions would not come until the close of the 18th century when English physician Edward Jenner made a remarkable observation in his country home 52 . The dairy maids in the surrounding farms bore the lesions of cowpox, but never acquired smallpox: they were seemingly protected from the human virus 52 . Wondering whether this immunity could be replicated, Jenner injected a young helper with pus from a cowpox lesion from one of the milkmaids, and observed that the boy developed resistance to smallpox afterwards 52 . Jenner named his discovery vaccine (after the latin vacca, meaning “cow”) though he did not

Cowpox-infected maid.

understand fully why it worked 52 . The full explanation would await modern theories of immunology, though Jenner’s technique, refined by Louis Pasteur to combat rabies, would become immensely influential nonetheless 34 .

Vaccination Takes Off

Vaccination would become increasingly widespread through the first half of the 20th century though it was never done extensively enough to eradicate smallpox. Even in countries such as England, where the disease had become uncommon, infected individuals from Africa spread a rash of cases in 1962, and only a quick surge of vaccinations prevented a full-blown epidemic. However, the vaccinations could cause harm as well, resulting in severe adverse reactions in some individuals. To avoid having to negotiate this delicate balance between the benefits and shortcomings of vaccination, the disease would have to be completely eliminated 53 .

Vaccination campaign poster.

Global Eradication

Doubtless this need for a lasting solution drove the 1950-1958 vaccination campaigns in South America, Central America, and the Caribbean, complementing earlier efforts that had pushed cases in the United States into the single digits 53 . Based on this success a global campaign was launched by the World Health Organization in 1959, though it was not until 1967 that the effort truly intensified to meet the challenge of the task 53 . At this point, consistent supply networks were established, as well as extensive monitoring systems in many countries so that the effectiveness of the vaccination campaign could be confirmed over time 54 .

Bifurcated needle.

Besides such organization, technological developments also aided this campaign. One was the bifurcated needle (shown to the right). Instead of a standard shot that punctured a vein, vaccinators employed a simple device developed in the 1960s by pharmaceutical firm Wyeth Laboratories. This horseshoe-shaped needle was dipped in vaccine then lightly tapped against a patient’s skin to make a series of small punctures. In selfless move, Wyeth allowed the needle to be used without charging patent royalties. Besides their ease of use, the bifurcated needle also had the advantage of reusability: provided they were sterilized, each instruments could vaccinate hundreds of individuals 55 . In addition to providing needles, Wyeth was also manufacturing the Dryvax smallpox vaccine used during the eradication campaign 56 .

During the campaign, field workers would scour the globe, distributing vaccine and controlling outbreaks. Their efforts eventually met with success, with the last instance of smallpox infection recorded in Somalia in 1977. Another aberrant case cropped up in 1978 when a strain escaped a laboratory in England, but besides this, no other infections were reported. In 1980 the disease was declared extinct 57-59 .

A public health victory: smallpox is eliminated!

Prior to its eradication, vaccination for smallpox was done using inactivated strains of vaccinia virus. However, only small stocks of the vaccine now remain and are not available for widespread distribution. Typically, only researchers in high security facilities are now vaccinated for smallpox 60,61 . Despite concerns about future biological warfare, the knowledge that the vaccine has side effects is a major argument against re-instituting more widespread smallpox vaccination in preparation for a bioterrorism attack. These complications usually result in skin rashes or, in more severe cases, potentially fatal tissue death. While health officials predict that only 1-2 out of every million vaccinated would actually die of such complications, the risks are still too much to reinstate vaccination on a large scale 41,42 .

A victim suffers from the adverse effects of the smallpox vaccine.

Resurrecting the Pox?

The existence of laboratory strains of the virus means there is still a possibility of it being resurrected as a bio-terror weapon. While the strains locked away at the CDC can probably be assumed safe from such abuse, the collapse of financial backing for Russian scientific research has raised concerns that private (and unfriendly) hands could now be funding work in former Soviet bioweapons facilities 62 .

Is there a benefit to maintaining smallpox stocks in the laboratory? Some researchers claim that these supplies are necessary for further research. However, this seems suspicious given that most of what we know about the virus actually comes from animal forms such as vaccinia. Nevertheless, policy makers have supported retaining samples of the human virus. Even though the World Health Organization had promised the destruction of all remaining smallpox stocks in the US and Russia by 1999, the Clinton administration decided to reverse this plan in the US, claiming the samples were needed for future anti viral research, or, in the event that the virus remerged, to develop new vaccines 53 . However, the rescued stocks have actually generated little scientific interest, with no new pharmaceutical or vaccines developed in the years since they were salvaged from their planned destruction 53 .

So how deadly would an outbreak be? Certainly, the lack of current vaccination makes smallpox a particularly dangerous threat, and since the vaccine is not used regularly in the US anymore, immunity levels among the American population are effectively nonexistent. Besides this, the virus is a tempting weapon because it can be easily grown and aerosolized 53 . Thus, because of this threat, the pros and cons of widespread vaccination are still being weighed, with the true threat of a smallpox outbreak remaining unknown.


Viruses cause many human diseases. In addition to the flu and HIV, viruses cause rabies, measles, diarrheal diseases, hepatitis, polio, cold sores and other diseases (see Figure below). Viral diseases range from mild to fatal. One way viruses cause disease is by causing host cells to burst open and die. Viruses may also cause disease without killing host cells. They may cause illness by disrupting homeostasis in host cells.

Cold Sore. Cold sores are caused by a herpes virus.

Some viruses live in a dormant state inside the body. This is called latency. For example, the virus that causes chicken pox may infect a young child and cause the short-term disease chicken pox. Then the virus may remain latent in nerve cells within the body for decades. The virus may re-emerge later in life as the disease called shingles. In shingles, the virus causes painful skin rashes with blisters (see Figure below).

Shingles. Shingles is a disease caused by the same virus that causes chicken pox.

Some viruses can cause cancer. For example, human papillomavirus (HPV) causes cancer of the cervix in females. Hepatitis B virus causes cancer of the liver. A viral cancer is likely to develop only after a person has been infected with a virus for many years.

The Flu

Influenza, or flu, is a contagious respiratory illness caused by influenza viruses. Influenza spreads around the world in seasonal epidemics. An epidemic is an outbreak of a disease within a population of people during a specific time. Every year in the United States, about 200,000 people are hospitalized and 36,000 people die from the flu. Flu pandemics can kill millions of people. A pandemic is an epidemic that spreads through human populations across a large region (for example a continent), or even worldwide. Three influenza pandemics occurred in the 20th century and killed tens of millions of people, with each of these pandemics being caused by the appearance of a new strain of the virus. Most influenza strains can be inactivated easily by disinfectants and detergents.

Emerging Viral Diseases

Modern modes of transportation allow more people and products to travel around the world at a faster pace. They also open the airways to the transcontinental movement of infectious disease vectors. One example of this occurring is West Nile Virus, which scientists believe was introduced to the United States by an infected air traveler. With the use of air travel, people are able to go to foreign lands, contract a disease and not have any symptoms of illness until they get home, possibly exposing others to the disease along the way.

Often, new diseases result from the spread of an existing disease from animals to humans. A disease that can be spread from animals to humans is called a zoonosis. When a disease breaks out, scientists called epidemiologists investigate the outbreak, looking for its cause. Epidemiologists are like detectives trying to solve a crime. The information epidemiologists learn is important to understand the pathogen, and help prevent future outbreaks of disease.

A deadly strain of avian flu virus named H5N1 has posed the greatest risk for a new influenza pandemic since it first killed humans in Asia in the 1990s. The virus is passed from infected birds to humans. Fortunately, the virus has not mutated to a form that spreads easily between people.

Several lethal viruses that cause viral hemorrhagic fever have been discovered, two of which are shown in the Figure below. Ebola outbreaks have been limited mainly to remote areas of the world. However, they have gained extensive media attention because of the high mortality rate&mdash23 percent to 90 percent&mdashdepending on the strain. The primary hosts of the viruses are thought to be apes in west central Africa, but the virus has also been isolated from bats in the same region.

The Ebola virus (left), and Marburg virus (right), are viruses that cause hemorrhagic fevers that can cause multiple organ failure and death.

People get exposed to new and rare zoonoses when they move into new areas and encounter wild animals. For example, severe acute respiratory syndrome (SARS) is a respiratory disease which is caused by the SARS coronavirus. An outbreak in China in 2003 was linked to the handling and consumption of wild civet cats sold as food in a market. In 2005, two studies identified a number of SARS-like coronaviruses in Chinese bats. It is likely that the virus spread from bats to civets, and then to humans.

Ebola is a rare and deadly disease caused by infection with a strain of Ebola virus. The 2014 Ebola epidemic is the largest in history, affecting multiple countries in West Africa, including Guinea, Sierra Leone and Liberia. Ebola is spread through direct contact with blood and body fluids of a person infected by and already showing symptoms of Ebola. Ebola is not spread through the air, water, food, or mosquitoes.


Assessment of Future Scientific Needs for Live Variola Virus (1999)

10Understanding of the Biology of Variola Virus

As noted earlier, smallpox was eradicated prior to the modem age of cell and molecular biology, virology, and immunology. Therefore, the basics of viral replication, determinants of viral virulence, and pathogenesis of the disease are not as well understood as they are for other pathogens.

Since variola virus is a pathogen that is uniquely adapted to cause severe, widespread human illness, it is highly likely that it has evolved to specifically thwart an effective immune response to infection. Poxviruses are the largest of the viruses and produce many proteins that are not necessary for virus replication, but presumably enhance the ability of the virus to cause disease. The multiple mechanisms used by poxviruses to evade host immune responses, the unique proteins these viruses produce, and their interactions with the host are just beginning to be identified. As the database expands, questions about the interactions of variola virus with human cells and immune responses and about the functions of these disease-producing variola proteins will become more obvious and pressing. The ability to identify the interactions between variola virus and host proteins would likely provide new insights into important aspects of the human immune system that would not be apparent from studies of other viruses.

Virus-Cell Interactions

Viruses adapt to their hosts in large part by evolving to interact efficiently with host cells in initiating infection and producing large amounts of virus. The vires spreads to different organs of the host and in this process causes tissue damage. Strains of a virus (e.g., variola major and variola minor) differ in their vim-

lence or ability to cause fatal disease. The differences in virulence may be due to changes in the rapidity of virus replication and spread, the amounts of vires produced, the ability to damage the cells in which the vires replicates, or the ability to evade the immune response of the host. In addition, orthopoxvirus tissue tropism genes have been identified in vaccinia vires and cowpox (C7L, K1L, and CHOhr), and the morphogenesis of the multiple forms of orthopoxvirus particles is coming better understood [43, 44]. The genetic basis of orthopoxvirus infections may thereby be revealed. Infection of human cells grown in tissue culture could begin to provide answers to some of the following questions:

  • Is there a unique molecule or series of molecules on the surface of human cells that makes them distinctly susceptible to infection with variola virus? What is the normal function of this molecule?
  • How and in what order are the many genes of the virus expressed to produce viral proteins? Do these proteins affect the infected cell by stimulating growth, by causing death, or by inhibiting death so the virus can grow for a longer period of time? Does this vary between variola major and variola minor?
  • Do any of the viral proteins provide new potential targets for antiviral drugs that can block virus replication without harming host cells? These targets may suggest new types of drugs that can be developed to treat other infections.

Finally, judging from what is known about other poxviruses, modulation of host immune responses is highly likely to contribute to the virulence of the virus. Infection of immune system cells could make it possible to assess direct effects on such cells, and incubation of human immune system cells with proteins secreted by infected cells could allow identification of potentially unique interactions between viral proteins and mediators of the antiviral immune response. These interactions could be used to identify important and potentially unique aspects of the human response to virus infections.

Virus-Host Interactions

Replication of variola virus in different types of cell cultures could provide valuable information on how this virus distinctly infects and affects human cells. It could not, however, provide information on how the virus spreads through the host or how it counteracts the host immune response.

Cultures that involve a number of cells organized into tissues and organs can currently be studied in bioreactors, SCID-hu mice, and raft cultures. These systems could allow investigators to answer some of the following questions:


Discussion

These studies were provoked by our interest in the history of Jenner’s vaccine. Contemporary accounts [6, 7] provide support for Jenner’s speculation that the vaccine probably originated as an equine disease called “grease”. Most notably Loy showed that one of two forms of grease (or horsepox) characterized by a diffuse pox reaction, provided protection against smallpox without requiring passage through cows [2]. Modern sequencing supports this hypothesis with the one known HPXV strain most closely resembling both old French [3] and early American [5] smallpox vaccines, and sharing a common origin with all known VACV [33]. Further support for a shared origin is provided by the observation that the I4L locus in a rare Dryvax clone (DPP17), encodes a block of sequence and a frameshift mutation that most closely resembles the homologous locus in HPXV [20]. Using the HPXV genome sequence [4], synthetic DNA fragments, and methods outlined elsewhere [34], we have used recombination and reactivation reactions to recreate this HPXV strain. This scHPXV resembles VACV in many of its growth properties, although it produces much smaller plaques (Fig 4a) without the secondary plaques associated with extracellular virus, which may partly explain the reduced virulence. Whether the increased infectivity and virulence of VACV reflects two centuries of selection in alternative hosts, including passage in humans, is an interesting question and because VACV-HPXV hybrids are viable (Fig 2), such strains might provide a tool for identifying some of the gene(s) responsible. Of perhaps most interest is the fact that our scHPXV strain generates immune protection against lethal virus challenge (Fig 5), and if the lower virulence in mice reflects better tolerability in humans, it supports further investigation as a vaccine and vector.

The method described here could be adapted to assemble other Orthopoxviruses, and possibly other Chordopoxviruses if the right helper virus(es) and supporting cell lines can be identified. This is timely as poxviruses offer much promise as oncolytic agents [35, 36] and better ways are needed to more rapidly and extensively engineer these agents to provoke potent anti-tumour immune responses ideally using designs based on personalized cancer neo-antigens. These synthetic approaches also provide tools for investigating certain basic features of virus genome structure (for example the mismatched and flip/flop hairpin telomeres) that are still incompletely understood 3–4 decades after they were discovered [37].

That said this is clearly an example of dual use research, and observations like these poses significant challenges for public health authorities. Most viruses could be assembled nowadays using reverse genetics, and these methods have been combined with gene synthesis technologies to assemble poliovirus [12] and other extinct pathogens like the 1918 influenza strain [13]. Given that the sequence of variola virus has been known since 1993 [38], our studies show that it is clearly accessible to current synthetic biology technology, with important implications for public health and biosecurity [34]. Our hope is that this work will promote new and informed public health discussions relating to synthetic biology, stimulate new evaluation of HPXV-based vaccines, and advance the capacity to rapidly produce next-generation vaccines and poxvirus-based therapeutics.


NIAID supports basic, preclinical, and clinical research needed to advance product development for biodefense and emerging infectious diseases. Product development goals in this arena have shifted from a “one bug-one drug” approach to a more flexible strategy that is applicable to a broad spectrum of infectious diseases. Specifically, this broad-spectrum approach is being used to develop products effective against a variety of pathogens and toxins find technologies that can be widely applied to improve multiple classes of products and establish platforms that can reduce the time and cost of creating new products. This is evident in both the treatment and vaccine research NIAID has supported for smallpox, outlined below.

To learn about risk factors for smallpox and current prevention and treatment strategies visit the MedlinePlus smallpox site.


Department of Biology

Today smallpox can only be found in deep freeze inside a few highly secured laboratories, like this one at the CDC in 1980.   Photo by CDC, CC BY

Smallpox was a terrible scourge on humanity, killing hundreds of millions of people over the centuries. But its origin remains obscure. Egyptian mummies from 3,000 to 4,000 years ago, including that of Pharaoh Ramses V, have pox-like skin lesions, but did they have smallpox? Recent genomic evidence puts that in doubt.

Whether or not ancient plagues were truly smallpox, by the 18th century the disease was endemic throughout the world. The widespread introduction of effective vaccination in the 19th century diminished – but did not eliminate – smallpox in the western world, and it persisted in many areas well into the 20th century.

I’m a microbiologist interested in how diseases jump from animals to humans and then evolve. Smallpox raged for centuries but was eradicated 40 years ago. While the idea of completely eradicating a disease has obvious appeal during the current pandemic, differences between the smallpox and SARS-CoV-2 viruses suggest that the path to ending COVID-19 pandemic will not be the same.

Wiping a virus from the face of the Earth

Smallpox is caused by a virus called variola. Although the primary route of infection is inhalation, the characteristic skin pustules are also filled with the virus. As long ago as the 10th century the Chinese knew that a person scratched with the pus from a pox sometimes gained immunity. It was, however, risky as some recipients developed smallpox and died.

This procedure, called variolation, was brought to Europe in the early 1700s. Later that century, English doctor Edward Jenner discovered that inoculation with a closely related virus, cowpox, also conferred immunity to smallpox but was much safer. Called vaccination, after “vacca,” the Latin word for cow, the process has remained essentially the same to the present.

In 1959, the World Health Organization initiated a program to eradicate smallpox worldwide, but the effort didn’t really get underway until 1967.

In 1977, a health care field worker conducts a house-to-house search for possible smallpox-infected inhabitants. Photo by CDC/Dr. Stanley Foster, CC BY

The program’s intensified vaccination efforts employed a new technique called circle vaccination, which consists of surveillance and containment when people in a community came down with the disease, health workers would try to vaccinate all their contacts to stop the spread of the virus.

Containment worked because people infected with the smallpox virus do not pass it on until they exhibit obvious symptoms. This is in contrast to SARS-CoV-2, which can be spread by infected people long before they develop symptoms, if they ever do. Surveillance and containment of cases, which was so successful for smallpox, may be less so for COVID-19 due to its asymptomatic spread.

The last known natural case of smallpox occurred in Somalia in 1977, and in May 1980 the World Health Assembly declared the disease officially eradicated. And that was the end of smallpox in the wild.

Under a magnification of 370,000X, the dumbbell-shaped viral core of the smallpox virus is visible it contains the viral DNA. Photo by CDC/Dr. Fred Murphy Sylvia Whitfield, CC BY

Deducing smallpox’s evolution from its genes

Variola is very different from SARS-CoV-2, the virus causing the current COVID-19 pandemic. The variola genome consists of double-stranded DNA, which is more stable and gets copied more accurately when the virus replicates than do single-stranded RNA genomes, such as that of SARS-CoV-2. Consequently, the rate of genetic change in variola is much lower. Although estimates vary, the evolution rate of the variola genome is at least 15-fold lower than that of SARS-CoV-2.

However, variola’s evolutionary history with humans is far longer than that of SARS-CoV-2 exactly how long is the subject of ongoing research. To reconstruct the evolutionary history of smallpox, scientists need to compare the DNA from virus samples preserved at various times.

After eradication, scientists destroyed all known existing samples of variola except for 571 live samples that had been collected in the preceding 30 years. They’re kept at two secure facilities, one in the U.S. and one in Russia.

Scientists have sequenced genomes from about 50 of these samples. They’ve also isolated variola DNA from a few accidentally preserved human remains. To date, the oldest of these is the mummified remains of a Lithuanian child who died between 1643 and 1665. It’s still unknown whether the pox-like diseases described in ancient texts or observed on Egyptian mummies were actually smallpox.

With viral DNA sequences in hand, scientists can construct genetic family trees by analyzing the small changes – mutations – that accumulated in the genes while the viruses were circulating in the human population.

These studies show that the variola strains that existed in the 20th-century cluster into two groups. One is made up of samples collected from West Africa and South America – an association probably due to the 18th-century slave trade – and the other consists of samples collected from the rest of the world.

Whether using just the DNA from the 20th-century variola strains, or including the viral DNA from the 17th-century Lithuanian mummy, researchers estimate the rate of change of variola strains to be one or two mutations per year. Previous estimates incorporating historical records had been as low as one-twentieth of this rate.

The more rapid mutation rate predicts a few things. It means the available modern variola strains shared a common ancestor only 400 years ago the two great clusters split only between 200 and 300 years ago and strains within the two clusters began to diverge about 100 years ago.

Model phylogenetic tree illustrating how an ancestor, A, could have led to the current two groups of smallpox strains, B and C. It’s possible other strains went extinct (x) and their remains are yet to be discovered. Horizontal lines represent the strain lineages and branch points represent genetic divergence. Loosely based on Duggan et al, 2016 https://doi.org/10.1016/j.cub.2016.10.061, CC BY-ND

When and how did smallpox appear in people?

If the existing variola strains appeared only a couple of centuries ago, that’s clearly inconsistent with reliable historic records of earlier smallpox outbreaks. There are two possible reasons for this discrepancy, both of which may be true.

First, the recent viral strains might have evolved more rapidly than older strains. That is, the estimated evolution rate might not be applicable across the entire history of the virus.

Second, older strains of the virus may have disappeared, leaving no progeny to be collected in the 20th century. The rise of vaccination in the 19th century may have driven these other strains to extinction. That would leave only the one strain that gave rise to the two major groups that then diverged into the modern strains.

So, how did the original strain first make it into the human population?

Variola virus belongs to a family of pox viruses that are widespread among mammals, and most with broad host-ranges. For example, cowpox infects cows, humans, cats, dogs and zoo animals, including the big cats. Variola, however, infects only humans.

At the level of their DNA, variola’s two closest relatives are camelpox, which infects only camels, and taterapox, whose only known host is an African gerbil. One interpretation of this family tree is that these three viruses emerged from a common ancestor, probably with a broad host range, about 3,000 years ago. The three viruses then independently narrowed their host range as they adapted to their new hosts, losing many of the genes that allow pox viruses to infect other host species.

Not all scientists who study the evolution of variola agree with this simplified scheme, arguing that the information gained from the available viral genomes cannot be extrapolated to the timing or the sequence of events that led to the original appearance of variola in people.

Contact tracing is much trickier for a virus like SARS-CoV-2 that doesn’t always cause obvious symptoms in those it infects. AP Photo/Rick Bowmer

Few similarities between smallpox and COVID-19

In addition to the differences between the viruses, the diseases that they cause are also very different, making the techniques used to eradicate smallpox less useful for the COVID-19 pandemic.

Smallpox is highly lethal as many as 30% of people infected with the major form of variola die, but survivors have lasting immunity. In contrast, best estimates put the death rate of COVID-19 at 1% or less, and it is not known if infection produces lasting immunity.

People infected with smallpox do not spread it until symptoms appear, at which point they are so sick as to be bedridden, whereas COVID-19 can be spread by infected people who have no symptoms. Within a community this asymptomatic spread of COVID-19 may account for more than 40% of secondary cases.

Historical records show that less virulent strains of smallpox appeared at various times, a few of which survived into the 20th century. A highly virulent pathogen may lose its virulence over time if rapidly disabling or killing its host limits its rate of transmission. However, because COVID-19 is spread by both asymptomatic and symptomatic people, there would appear to be little advantage for SARS-CoV-2 to evolve to be less virulent.

Surveillance and containment of cases, which was so successful for smallpox, also is less likely to work for COVID-19.

Our best hope is for an effective vaccine to stop the COVID-19 pandemic, just as it stopped smallpox.


Transmission

Varicella is highly contagious. The virus can be spread from person to person by direct contact, inhalation of aerosols from vesicular fluid of skin lesions of acute varicella or zoster, and possibly through infected respiratory secretions that also may be aerosolized. A person with varicella is considered contagious beginning one to two days before rash onset until all the chickenpox lesions have crusted. Vaccinated people may develop lesions that do not crust. These people are considered contagious until no new lesions have appeared for 24 hours.

It takes from 10 to 21 days after exposure to the virus for someone to develop varicella. Based on studies of transmission among household members, about 90% of susceptible close contacts will get varicella after exposure to a person with disease. Although limited data are available to assess the risk of VZV transmission from zoster, one household study found that the risk for VZV transmission from herpes zoster was approximately 20% of the risk for transmission from varicella.

People with breakthrough varicella are also contagious. One study of varicella transmission in household settings found that people with mild breakthrough varicella (<50 lesions) who were vaccinated with one dose of varicella vaccine were one-third as contagious as unvaccinated people with varicella. However, people with breakthrough varicella with 50 or more lesions were just as contagious as unvaccinated people with the disease.

Varicella is less contagious than measles, but more contagious than mumps and rubella.


Poxviridae

Species demarcation criteria in the genus

The criteria are provisional and reflect the fact that species definitions can be rather arbitrary and reflective of attempts to define natural transmission lineages. Most orthopoxviruses contain a hemagglutinin (HA) and many contain an A-type inclusion protein polymorphisms within these genes distinguish species. Species can be classified by pock morphologies and by ceiling temperature for growth on the chorioallantoic membrane of embryonated chicken eggs. Ecological niche and host range are useful in some cases, but in others ( rabbitpox virus and buffalopox virus) these can be misleading. RFLP analysis of the terminal regions of viral DNA outside of the core of common genes has aided the classification process. Detailed polymerase chain reaction (PCR) polymorphism analysis throughout the entire genome and subsequent genomic DNA sequencing studies have shown all orthopoxviruses to be unique. With genomic sequence analysis, it has become apparent that members of the species Cowpox virus are not monophyletic, as indicated by the different positions of cowpox virus GRI-90 and cowpox virus Brighton Red in the phylogenetic structure of the genus Orthopoxvirus (see Figure 4 below). This is still not reflected in the current taxonomy because the results of a wide-scale genome sequence study to clarify the issue are pending.