Chapter 11: Poxviruses

Poxviruses belong to Poxviridae family that are the largest and most complex viruses infecting humans, other mammals, birds, and even insects. Poxviruses that infect vertebrates are classified into eight genera, and four of these genera cause disease in humans, including Orthopoxvirus, Parapoxvirus, Yantapoxvirus, and Molluscipoxvirus. Orthopoxvirus genus members that cause disease in humans include variola (smallpox), cowpox, vaccinia (strain used for smallpox vaccination), and monkeypox viruses. Parapoxvirus genus members cause disease mainly in animals but sometimes also in humans, including orf and pseudocowpox viruses. Molluscipoxvirus causes molluscum contagiosum (pearl-like lesions) in humans and Yantapoxvirus comprises tanapox and yabapox viruses that mainly infect animals but may also cause mild disease in humans. The agents most important in human disease are variola (smallpox), vaccinia, monkeypox, molluscum contagiosum, orf, cowpox, and pseudocowpox (Table 11–1). Although smallpox has been eliminated, it has the potential to be used in germ warfare or in bioterrorism. In addition, monkeypox causes similar disease in humans like smallpox but usually milder. Therefore, knowledge and understanding of smallpox pathogenesis and disease is important for any future control of outbreaks of poxviral diseases.

Poxviruses are large, brick-shaped or ovoid, linear double-stranded DNA (130-300 kbp) containing core within a double membrane and a lipoprotein envelope carrying virions measuring approximately 350 × 270 nm (vaccinia virus) (Figures 11–1 and 11–2). The core is flanked by two lateral bodies containing several viral enzymes and proteins, including DNA-dependent RNA polymerase and transcription factors required for viral replication. The poxvirus genome encodes all essential enzymes, proteins, and factors needed for viral replication in the cytoplasm of infected cells, including transcription, DNA synthesis, and virus assembly. The envelope is acquired in the cytoplasm either from the Golgi apparatus or other cellular organelles, but not by budding from the plasma membrane and may not be essential for viral infectivity.

Poxvirus replication is unique among DNA viruses in that the viral replication cycle takes place in the cytoplasm of the infected cell (Figure 11–3). The viral replication cycle starts with attachment, rapid adsorption to receptors followed by viral entry, and release of cores in the cytoplasm. Viral DNA-dependent RNA polymerase in the cores initiate early transcription to synthesize several proteins, including DNA and RNA polymerases, transcription factors, growth factors, and immune defense molecules. The uncoating of the cores uses viral DNA to synthesize concatemeric DNA molecules, which are eventually resolved into viral DNA genomes for progeny viruses. The late mRNAs synthesize viral structural proteins required for virus assembly and early transcription factors for packaging in the virions. Assembly of the progeny viruses begins with the formation of membrane structures followed by maturation of intracellular mature virions (IMV). The virions are further wrapped by membranes from the Golgi apparatus that are lost upon the release of extracellular enveloped virions (EEV).

VIROLOGY

Generally, two types of viruses are known: Variola major and variola minor (alastrim). Although the viruses are indistinguishable antigenically, their fatality rates differ considerably (greater than 1% for variola minor, 3-40% for variola major). The high replicative fidelity of variola DNA polymerase enzyme limited its ability to significantly mutate and adapt to the humans, which preserved the antigenic cross-reactivity with other orthopoxviruses such as vaccinia virus that was used for vaccination. There is no known animal reservoir for variola virus.

SMALLPOX

Smallpox has played a significant role in world history with respect to both the serious epidemics recorded since antiquity and the sometimes dangerous measures taken to prevent infection. Smallpox virus is highly contagious and can survive well in the extracellular environment. Acquisition of infection by infected saliva droplets or by exposure to skin lesions, contaminated articles, and fomites has been well documented. Variola caused a severe systemic illness when inhaled but a milder disease when inoculated into the skin.

In 1967, the World Health Organization (WHO) launched an ambitious program aimed at eradication of smallpox. This goal was considered realistic for two major reasons: (1) no extra-human reservoir of the virus was known to exist, and (2) asymptomatic carriage apparently did not occur. The basic approach included intensive surveillance for clinical cases of smallpox, prompt quarantine of such patients and their contacts, and immunization of contacts with vaccinia virus (vaccination) to prevent further spread. A tremendous amount of effort was involved, but the results were astonishing: The last recorded case of naturally acquired smallpox occurred in Somalia in 1977. Global eradication of smallpox was confirmed in 1979 and accepted by WHO in May 1980. Since then, the virus has been solely secured in two WHO-restricted laboratories: One at the United States Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, and the other at a similar facility in Moscow, Russia.

Unfortunately, the dramatic world events that occurred in 2001 have raised the chilling possibility that clandestine virus stocks of smallpox may exist elsewhere and could be effectively used for major bioterrorist attacks. Reasons for such concern include: (1) smallpox is one of the most stable viruses; (2) it can remain stable for a long time, if freeze-dried; (3) it is unaffected by environmental conditions; (4) scab forms are stable for 1 year at room temperature and in one case it has been found to be stable for 13 years in a laboratory; (5) it has high infectivity among humans; (6) it is associated with high susceptibility among populations (routine vaccination against smallpox ended in 1972, and current vaccine supplies are limited); (7) there is a risk that health care providers may not promptly recognize and respond to early cases; and (8) there is an absence of specific antiviral treatment.

A response plan and guidelines for such threats is posted on a CDC website (www.cdc.gov/smallpox) and is updated at regular intervals.

Continuing surveillance also includes studies of poxviruses of animals (eg, buffalopox, monkeypox), which are antigenically somewhat similar to smallpox. Some virologists remain legitimately concerned that an animal poxvirus, such as monkeypox, could mutate to become highly virulent to humans—a further reminder that complacency could be dangerous.

PATHOGENESIS

The virus enters the mucous membranes of the upper respiratory tract through inhalation followed by viral replication at the site of entry and infection of mononuclear phagocytic cell in the regional lymph nodes. Viremia allows the virus to be transported to liver, spleen, and other tissues. At the end of the incubation period, inflammatory mediators are released causing fever and other symptoms. In variola, a secondary viremic phase has been demonstrated. The virus spreads through the capillaries to the skin followed by viral replication and evolution of rash. The virus further spreads cell-to-cell or through the mid and basal layers of skin causing necrosis and vesicles. The orthopoxviruses as a group cause a dramatic effect on host cell macromolecular function, leading to a switch from cellular to viral protein synthesis, changes in cell membrane permeability, and cytolysis. Eosinophilic inclusions, called Guarnieri bodies, can be seen in the cytoplasm. Multiple viral proteins, such as complement regulatory and immunomodulatory proteins are encoded by the virus that can interfere with induction or activities of multiple host mononuclear cell cytokines, chemokines and other immune mediators. This serves to impair the host innate defenses that are important in early control of infection. Some immunomodulatory proteins interfere with the T_H1 response, causing depressed cell-mediated immunity in controlling primary infection. Enormous inflammatory responses were also accountable for main characteristics of illness. In some patients, high levels of circulating virus caused hemorrhagic disease that resembled septic shock. Although variola was found in several tissues of infected patients, the lesions are limited to skin and oropharyngeal mucosa because the virus produces a homolog of epidermal growth factor that proliferates keratinocytes, followed by virus replication and spread.

CLINICAL ASPECTS

MANIFESTATIONS AND DIAGNOSIS

The incubation period of smallpox is usually 12 to 14 days, although in occasional fulminating cases it can be as short as 4 to 5 days. The typical onset is abrupt, with fever, chills, and myalgia, followed by a rash 3 to 4 days later. The rash evolves to firm papulovesicles that become pustular over 10 to 12 days, then crust and slowly heal. Only a single crop of lesions (all in the same stage of evolution) develop; these lesions are most prominent over the head and extremities (Figure 11–4). Some cases are fulminant, with a hemorrhagic rash (“sledgehammer” smallpox). Death can result from the overwhelming primary viral infection or from bacterial superinfection. Diagnostic methods use vesicular scrapings and include culture, electron microscopy, gel diffusion, and polymerase chain reaction.

PREVENTION

The first major step toward modern prevention and subsequent eradication of smallpox can be credited to Edward Jenner, who noted that milkmaids who develop mild cowpox lesions on their hands appeared immune to smallpox. In 1798, he published evidence indicating that purposeful inoculation of individuals with cowpox material could protect them against subsequent infection by smallpox. The concept of vaccination gradually evolved, with the modern use of live vaccinia virus, a poxvirus of uncertain origin to be discussed later, which produced specific immunity.

Vaccinia virus is serologically related to smallpox, although its exact origin is unclear. Some virologists believe it is a recombinant virus derived from smallpox and cowpox, and others suggest it originated from a poxvirus of horses. The virus is usually propagated by dermal inoculation of calves, and the resultant vesicle fluid (“lymph”) is lyophilized and used as a live virus vaccine in humans. The vaccine is inoculated into the epidermis and produces a localized lesion, which indicates successful immunization. The lesion becomes vesicular, then pustular, followed by crusting and healing over 10 to 14 days. The local reaction is sometimes severe and accompanied by systemic symptoms such as fever, rash, and lymphadenopathy. Patients who are immunocompromised may experience severe reactions, such as progressive vaccinia. Vaccinia-produced immunity to smallpox was believed to wane rapidly after 3 to 5 years, and the duration of long-term immunity beyond that time was uncertain. However, several studies, including the Baltimore Longitudinal Study of Aging suggested that the immunity (IgG neutralizing antibody) to smallpox vaccine (vaccinia) persisted for decades.

There has been a resurgence of scientific interest in vaccinia as a possible vector for active immunization against other diseases, such as hepatitis B, herpes simplex, and human immunodeficiency virus. It has been shown that gene sequences coding for specific immunogenic proteins of other viruses can be inserted into the vaccinia virus genome, with subsequent expression as the virus replicates. For example, a recombinant vaccinia strain carrying the gene sequence for hepatitis B surface antigen (HbsAg) can infect cells, lead to production of HbsAg, and stimulate an antibody response to it. Theoretically, gene sequences coding for a variety of antigens could be packaged in a single viable vaccinia virus, thus allowing simultaneous active immunization against multiple agents. It has been suggested that use of other poxviruses of animal or avian origin, such as canarypox, may be even safer, yet effective vectors for use in humans. These vectors are being used to develop gene therapy approaches and vaccines for HIV and other infections. Whether such approaches become routinely applicable to clinical medicine remains to be seen.

Monkeypox was first reported in laboratory monkeys in 1958 and in humans in 1970. The primary reservoir for monkeypox is not monkeys but Central and West African rodents. However, two other African viruses classified in the Yatapoxvirus genus (tanapox and yabapox) have subhuman primates as their primary reservoirs. All these three viruses can spread to humans by direct contact producing generally mild illness that, in more severe cases, may be confused with smallpox. While the symptoms of monkeypox in human are milder than smallpox, another difference is that there is swelling of lymph nodes in monkeypox infection. Monkeypox causing infection in humans is also referred to as human monkeypox.

The first case of human monkeypox was first identified in 1970 Democratic Republic of Congo (DRC) followed by majority of cases occurring in this region. There were several major outbreaks reported in the same region of Central Africa. Between 1970 to 2007, 1378 cases of monkeypox in humans were reported in DRC, including 830 cases in 2005-2007. Sporadic cases have been reported in West African countries. Two strains of monkeypox virus, the Central African strain and West African strain, have been found. In 2003, at least 34 cases of human monkeypox occurred in the Midwestern United States. There were no fatalities. The contact sources were ill pet prairie dogs that had been housed with various exotic rodents imported from Ghana. In 2005, human monkeypox was reported in Sudan.

Direct transmission to humans occurred by close contact with the ill animals, including direct contact with blood, bodily fluids, or rashes of infected animals. Secondary, human-to-human transmission occurs from close contact to respiratory secretions, lesions, or droplet nuclei of infected people. However, the transmission efficiency is far less than smallpox virus. Monkeypox could be transmitted through the placental route.

After transmission, there is an incubation period of 6 to 16 days, in which the virus replicates in the lymphatic system followed by viremia and transportation of the virus to all body organs, including multiplication within the epithelial cells of the skin. Human monkeypox infection can be divided into two phases, the invasion phase and the skin eruption phase. In the invasion phase that lasts from 0 to 5 days after incubation period, fever, severe headache, lymphadenopathy (swelling of the lymph nodes), back pain, myalgia, and an extreme asthenia (lack of energy) are noted. The skin eruption phase is characterized by appearance of maculopapular rash on the face in about 95% of the cases, on the palms of the hands and soles of the feet in 75% of the cases, and on the body concurrently. The maculopapular rash develops into vesicles, pustules, followed by crusts in about 10 days, which is generally eliminated in about 3 weeks. The symptoms of human monkeypox last about 12 to 14 days. One of the characteristics of monkeypox is that there may be severe lymphadenopathy in some patients before the development of rash, unlike smallpox or chickenpox. The fatality is less than 10%.

The clinical diagnosis may be confusing because of similarities with smallpox, chickenpox, measles, and other rash-like diseases. Therefore, laboratory diagnosis by ELISA (antibody), antigen detection, PCR (genome amplification), or virus isolation by cell culture must be performed.

No specific treatment is available for monkeypox. However, smallpox vaccine (vaccinia virus) provides more than 85% protection.

Molluscum contagiosum is a benign, cutaneous poxvirus disease of humans, spread by direct contact with infected cells. It is usually acquired by inoculation into minute skin abrasions; events that commonly lead to transmission include “roughhousing” in shower rooms and swimming pools, sharing of towels, bathing sponges, pool equipment, toys, and sexual contact. Most infection occurs in children over 1 year of age. Patients with AIDS are especially prone to develop widespread lesions.

After an incubation period between 2 weeks and 6 months, nodular, pale, firm (pearl-like) lesions known as Mollusca that are usually 2 to 5 mm in diameter develop in the epidermis. These lesions are painless, flesh colored papules, and umbilicated in appearance (Figure 11–5). They may become itchy, red, sore, and swollen. A cheesy material may be expressed from the pore at the center of each lesion. Local trauma may cause spread of lesions in the involved skin area. Since the virus is localized in the epidermis, there is no viremia and no spread to other body organs. The lesions are not associated with systemic symptoms, and they disappear in 6 to 12 months without treatment but may take as long as 4 years. Specific treatment, if desired, is usually by curettage or careful removal of the central core by expression with forceps. Some oral (cimetidine for pediatric cases) and topical (podophyllotoxin) agents are also available. HIV/AIDS and other immunocompromised patients may develop lesions more than 15 mm in diameter that do not respond to therapy.

Pathologic findings, which are limited to the epidermis, include hyperplasia, ballooning degeneration, and acanthosis. The diagnosis, made on clinical grounds, can be confirmed by demonstration of large, eosinophilic cytoplasmic inclusions (molluscum bodies) in the affected superficial epithelial cells (Figure 11–6).

People who recover from molluscum infection are not protected against new infections. The virus does not persist indefinitely. The smallpox (vaccinia) vaccine does not provide any protection either.

Orf is an old Saxon term for a human infection caused by a parapoxvirus of sheep and goats. Synonyms for the infection in animals include contagious pustular dermatitis, ecthyma contagiosum, pustular ecthyma, and “scabby mouth.” Humans usually acquire the infection by close contact with infected animals and accidental inoculation through cuts or abrasions on the hands or wrists. The typical skin lesion is solitary; it begins as a vesicle and evolves into a nodular mass that later develops central necrosis (Figure 11–7). Regional lymphadenopathy sometimes develops. Dissemination is rare. The average duration of the lesion is 35 days, followed by complete resolution. The diagnosis is usually made on the basis of clinical appearance and occupational history. Serologic confirmation or electron microscopy of the lesion can be performed but is rarely necessary.

Milker’s nodules (pseudocowpox) constitute a cutaneous parapoxvirus disease of cattle, distinct from cowpox, which can cause local skin infections similar to those of orf in exposed humans. Healing of the skin lesions may take 4 to 8 weeks. There is no cross-immunity to cowpox. Cowpox is now very rare in the United States. It produces a vesicular eruption on the udders of cows and similar, usually localized, vesicular skin lesions in humans (referred as cowmaid blisters) who are accidentally exposed.