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34 C H A P T E R Poxviruses...

34 C H A P T E R Poxviruses Poxviruses are the largest and most complex of viruses infect- coding regions, so some genes are present at both ends of the ing humans. The family encompasses a large group of agents genome. The DNA is rich in adenine and thymine bases. that are similar morphologically and share a common nucleo- The chemical composition of a poxvirus resembles that protein antigen. Infections with most poxviruses are charac- of a bacterium. Vaccinia virus is composed predominantly terized by a rash, although lesions induced by some members of protein (90%), lipid (5%), and DNA (3%). More than 100 of the family are markedly proliferative. The group includes structural polypeptides have been detected in virus particles. variola virus, the etiologic agent of smallpox—a viral disease A number of the proteins are glycosylated or phosphorylated. that has affected humans throughout recorded history. The lipids are cholesterol and phospholipids. Although smallpox was declared globally eradicated in The virion contains a multiplicity of enzymes, including 1980 after an intensive campaign coordinated by the World a transcriptional system that can synthesize, polyadenylate, Health Organization, there is concern that the virus could be cap, and methylate viral mRNA. reintroduced as a biologic weapon. There is a continuing need to be familiar with vaccinia virus (used for smallpox vacci- nations) and its possible complications in humans, as well Classification as other poxvirus diseases that may resemble smallpox and Poxviruses are divided into two subfamilies based on must be differentiated from it by laboratory means. Also, vac- whether they infect vertebrate or insect hosts. The vertebrate cinia virus is under investigation as a vector for introducing poxviruses fall into nine genera, with the members of a given actively immunizing genes for a variety of viral diseases of genus displaying similar morphology and host range as well humans and domestic animals. as some antigenic relatedness. Most of the poxviruses that can cause disease in humans are contained in the genera Orthopoxvirus and Parapoxvirus; PROPERTIES OF POXVIRUSES there are also several that are classified in the genera Yatapoxvirus and Molluscipoxvirus (Table 34-2). Important properties of the poxviruses are listed in Table 34-1. The orthopoxviruses have a broad host range affecting several vertebrates. They include ectromelia (mousepox), camelpox, cowpox, monkeypox, vaccinia, and variola (small- Structure and Composition pox) viruses. The last four are infectious for humans. Vaccinia Poxviruses are large enough to be seen as featureless par- virus differs only in minor morphologic respects from variola ticles by light microscopy. By electron microscopy, they and cowpox viruses. It is the prototype of poxviruses in terms appear to be brick-shaped or ellipsoid particles measur- of structure and replication. Monkeypox can infect rodents, ing about (300–400) × 230 nm. Their structure is complex monkeys, and humans and may resemble smallpox clinically. and conforms to neither icosahedral nor helical symmetry. Some poxviruses have a restricted host range and infect The external surface of particles contains ridges. An outer only rabbits (fibroma and myxoma) or only birds. Others lipoprotein membrane, or envelope, encloses a core and infect mainly sheep and goats (sheeppox, goatpox) or cattle two structures of unknown function called lateral bodies (pseudocowpox, or milker’s nodule). (Figure 34-1). Parapoxviruses are morphologically distinctive. Compared The core contains the large viral genome of linear double- with the orthopoxviruses, parapoxviruses are somewhat stranded DNA (130–375 kbp). The complete genomic sequence smaller particles (260 × 160 nm), and their surfaces exhibit a is known for several poxviruses, including vaccinia and vari- crisscross pattern (Figure 34-2). Their genomes are smaller ola. The vaccinia genome contains about 185 open reading (∼135 kbp) and have a higher guanine plus cytosine content frames. The DNA contains inverted terminal repeats of vari- (63%) than those of the orthopoxviruses. able length, and the strands are connected at the ends by termi- All vertebrate poxviruses share a common nucleoprotein nal hairpin loops. The inverted terminal repeats may include antigen in the inner core. There is serologic cross-reactivity 499 Riedel_CH34_p499-p510.indd 499 05/04/19 4:56 PM  500   SECTION IV  Virology TABLE 34-1 Important Properties of Poxviruses factors may be involved in transcription and virion assembly. Poxviruses are further distinguished from all other animal Virion: Complex structure, oval or brick shaped, 300–400 nm in length × 230 nm in diameter; external surface shows ridges; viruses by the fact that the uncoating step requires a newly contains core and lateral bodies synthesized, virus-encoded protein. Composition: DNA (3%), protein (90%), lipid (5%) Genome: Double-stranded DNA, linear; size, 130–375 kbp; has A. Virus Attachment, Penetration, and Uncoating terminal loops; has low G + C content (30–40%) except for Virus particles establish contact with the cell surface and fuse Parapoxvirus (63%) with the cell membrane. Some particles may appear within Proteins: Virions contain more than 100 polypeptides; many vacuoles. Viral cores are released into the cytoplasm. Among enzymes are present in core, including transcriptional system the several enzymes inside the poxvirus particle, there is a viral Envelope: Virion assembly involves formation of multiple RNA polymerase that transcribes about half the viral genome membranes into early mRNA. These mRNAs are transcribed within the Replication: Cytoplasmic factories viral core and are then released into the cytoplasm. Because the necessary enzymes are contained within the viral core, early Outstanding characteristics: Large and complex viruses; very resistant to inactivation transcription is not affected by inhibitors of protein synthe- Virus-encoded proteins help evade host immune defense system sis. The “uncoating” protein that acts on the cores is among Smallpox was the first viral disease eradicated from the world the more than 50 polypeptides made early after infection. The second-stage uncoating step liberates viral DNA from the cores; it requires both RNA and protein synthesis. The synthe- among viruses within a given genus but very limited reactiv- sis of host cell macromolecules is inhibited at this stage. ity across genera. Consequently, immunization with vaccinia Poxviruses inactivated by heat can be reactivated either virus affords no protection against disease induced by other by viable poxviruses or by poxviruses inactivated by nitro- genera of poxviruses. gen mustards (which inactivate the DNA). This process is called nongenetic reactivation and is caused by the action of the uncoating protein. Heat-inactivated virus alone can- Poxvirus Replication not cause second-stage uncoating because of the heat labil- The replication cycle of vaccinia virus is summarized in ity of the RNA polymerase. Apparently, the heat-killed virus Figure 34-3. Poxviruses are unique among DNA viruses in provides the template, and the second virus provides the that the entire multiplication cycle takes place in the cyto- enzymes needed for transcription. Any vertebrate poxvirus plasm of infected cells. It is possible, however, that nuclear can reactivate any other vertebrate poxvirus. A B FIGURE 34-1 Electron micrographs of vaccinia (Orthopoxvirus) virions. A: Negatively stained particle showing ridges or tubular elements covering the surface (228,000×). (Reproduced with permission from Dales S: The uptake and development of vaccinia virus in strain L cells followed with labeled viral deoxyribonucleic acid. J Cell Biol 1963;18:51.) B: Thin section of vaccinia virion showing a central biconcave core, two lateral bodies, and an outer membrane (220,000×). (Reproduced with permission from Pogo BGT, Dales S: Two deoxyribonuclease activities within purified vaccinia virus. Proc Natl Acad Sci USA 1969;63:820.) Riedel_CH34_p499-p510.indd 500 05/04/19 4:56 PM  CHAPTER 34 Poxviruses   501 TABLE 34-2 Poxviruses Causing Disease in Humans Genus Virus Primary Host Disease Orthopoxvirus Variola (major and minor) Humans Smallpox (now eliminated) Vaccinia Humans Localized lesion; used for smallpox vaccination Buffalopox Water buffalo Human infections rare; localized lesion Monkeypox Rodents, monkeys Human infections rare; generalized disease Cowpox Cows Human infections rare; localized ulcerating lesion Parapoxvirus Orf Sheep Human infections rare; localized lesion Pseudocowpox Cows Bovine papular stomatitis Cows Molluscipoxvirus Molluscum contagiosum Humans Many benign skin nodules Yatapoxvirus Tanapox Monkeys Human infections rare; localized lesion Yabapox Monkeys Human infections very rare and accidental; localized skin tumors B. Replication of Viral DNA and Synthesis of viral-coded enzymes. Viral DNA replication starts soon after Viral Proteins the release of viral DNA in the second stage of uncoating. It Among the early proteins made after vaccinia virus infection occurs 2–6 hours after infection in discrete areas of the cyto- are enzymes involved in DNA replication, including a DNA plasm, which appear as “factories” or inclusion bodies in elec- polymerase and thymidine kinase. Viral DNA replication tron micrographs. The number of inclusion bodies per cell is occurs in the cytoplasm and appears to be accomplished by proportionate to the multiplicity of infection, suggesting that each infectious particle can induce a “factory.” High rates of homologous recombination occur within poxvirus-infected cells. This has been exploited experimentally to construct and map mutations. The pattern of viral gene expression changes markedly with the onset of replication of viral DNA. The synthesis of many of the early proteins is inhibited. There is a small inter- mediate class of genes whose expression temporally precedes the expression of the late class of genes. Late viral mRNA is translated into large amounts of structural proteins and small amounts of other viral proteins and enzymes. C. Maturation The assembly of the virus particle from the manufactured components is a complex process. Some of the particles are released from the cell by budding, but the majority of poxvi- rus particles remain within the host cell. About 10,000 virus particles are produced per cell. How the multiple components of the transcription system are incorporated within the core of the assembling virus particle is unknown. D. Virus-Encoded Host Modifier Genes A polypeptide encoded by one of the early genes of vaccinia virus is closely related to epidermal growth factor and to trans- forming growth factor-α. Production of growth factors simi- lar to epidermal growth factor by virus-infected cells could account for the proliferative diseases associated with mem- FIGURE 34-2 Electron micrograph of orf virus (Parapoxvirus). bers of the poxvirus family such as Shope fibroma (rabbits), Note the distinctive crisscross pattern of the surface of the virion Yaba tumor (monkeys), and molluscum contagiosum viruses (200,000×). (Courtesy of FA Murphy and EL Palmer.) (humans). Riedel_CH34_p499-p510.indd 501 05/04/19 4:56 PM  502   SECTION IV  Virology Envelope Core Cytoplasm Intermediate mRNA 6 DNA Attachment Entry Uncoating Replication Late transcription factors 1 2 4 5 tion 7 se mera n s crip 3 oly tra Late mRNA Ap ate s RNA polymerase DN m edi factor ase r r Concatemer resolution Transcription factor Early mRNA Inte me Capping enzyme p oly DNA packaging A Late enzymes Poly(A) polymerase RN Early transcription factors Growth factors Structural proteins Immune defense Nuclear factors? 9 8 Assembly molecules Golgi Exit wrapping Maturation 12 11 10 FIGURE 34-3 Outline of replication cycle of vaccinia virus. The replication of this large DNA virus occurs in the cell cytoplasm. (1) Virus particles attach to cells, (2) fuse with the cell membrane and release viral cores into the cytoplasm. (3) The cores produce early mRNAs using viral enzymes and transcription factors contained within the cores; these mRNAs are translated into numerous viral proteins having replication functions. (4) The cores are uncoated, and (5) viral DNA is replicated. (6 and 7) Intermediate and late genes are transcribed; products include viral structural proteins. (8–10) Assembly of infectious virions occurs on membrane structures. (11) Envelopes are acquired at the Golgi and plasma membrane, leading to (12) the release of enveloped progeny virions. (Reproduced with permission from Moss B: Poxviridae: The viruses and their replication. In Fields BN, Knipe DM, Howley PM [editors-in-chief]. Fields Virology, 3rd ed. Lippincott-Raven, 1996.) Several poxvirus genes resemble mammalian genes for year. The last Asian case occurred in Bangladesh in 1975, proteins that would inhibit host defense mechanisms. Exam- and the last natural victim was diagnosed in Somalia in 1977. ples include tumor necrosis factor receptor, interferon-γ Smallpox was officially declared eliminated in 1980. There receptor, interleukin-1 receptor, and a complement-binding were several reasons for this outstanding success: There is a protein. These poxvirus-encoded host defense modifiers single serotype of virus; most infections are clinically appar- presumably counter the complement and cytokine networks ent; the vaccine was easily prepared, stable, and safe; the vac- important in the host immune response to viral infection, cine could be given simply by personnel in the field; and mass allowing enhanced virus replication and, perhaps, facilitat- vaccination of the world population was not necessary. Cases ing virus transmission. of smallpox were traced, and contacts of the patient and those in the immediate area were vaccinated. Even though there has been no evidence of smallpox transmission anywhere in the world, the World Health Orga- POXVIRUS INFECTIONS IN HUMANS: nization coordinated the investigation of 173 possible cases VACCINIA AND VARIOLA of smallpox between 1979 and 1984. All were diseases other than smallpox, most commonly chickenpox or other illnesses Control and Eradication of Smallpox that produce a rash. Even so, a potential case of smallpox Control of smallpox by deliberate infection with mild forms becomes a public health emergency and must be promptly of the disease was practiced for centuries. This process, called investigated by means of clinical evaluation, collection of variolation, was dangerous but decreased the disastrous laboratory specimens for diagnosis, and patient isolation. effects of major epidemics, reducing the case-fatality rate The presence of stocks of virulent smallpox virus in labo- from 25% to 1%. Edward Jenner introduced vaccination with ratories is of concern because of the danger of laboratory infec- live cowpox virus in 1798. tion and subsequent spread into the community. Variola virus In 1967, the World Health Organization introduced a stocks supposedly were destroyed in all laboratories except worldwide campaign to eradicate smallpox. Epidemiologic two World Health Organization collaborating centers (one in features of the disease (described later) made it feasible to Atlanta and the other in Moscow) that pursue diagnostic and attempt total eradication. At that time, there were 33 coun- research work on variola-related poxviruses. However, in the tries with endemic smallpox and 10–15 million cases per 1990s, it was learned that the former Soviet Union had used Riedel_CH34_p499-p510.indd 502 05/04/19 4:56 PM  CHAPTER 34 Poxviruses   503 smallpox virus in its biologic warfare program, and it is pos- throughout the body; (3) a secondary phase of multiplication sible that it was transferred to other countries as well. Small- in those cells, leading to (4) a secondary, more intense vire- pox virus is considered to be a dangerous potential biothreat mia; and (5) the clinical disease. agent, and it is theoretically possible that virus frozen in per- In the preeruptive phase, the disease was barely infec- mafrost could reinfect the human population. Because of the tive. By the sixth to ninth day, lesions in the mouth tended worldwide eradication of variola virus and subsequent discon- to ulcerate and discharge virus. Thus, early in the disease, tinuation of vaccination programs, today’s human population infectious virus originated in lesions in the mouth and upper possesses low or nonexistent smallpox immunity and thus is respiratory tract. Later, pustules broke down and discharged highly susceptible to infection with smallpox virus. virus into the environment of the smallpox patient. Research scientists may obtain portions of the variola Histopathologic examination of the skin showed prolif- virus genome from the collaborating centers but not a com- eration and cytoplasmic inclusions of the stratum spinosum. plete genome. The distribution, synthesis, and handling of There was infiltration with mononuclear cells, particularly variola virus DNA are governed by recommendations from around dermal vessels. Epidermal cells became swollen the World Health Organization. through distention of cytoplasm and underwent “balloon- ing degeneration,” with enlargement of cytoplasmic vacuoles. The cell membrane broke down and coalesced with neigh- Comparison of Vaccinia and boring, similarly affected cells, resulting in the formation of Variola Viruses vesicles. The vesicles enlarged and then became filled with Vaccinia virus, the agent used for smallpox vaccination, is a white cells and tissue debris. All of the layers of the skin were distinct species of Orthopoxvirus. Restriction endonuclease involved, with subsequent dermal necrosis. Thus, scarring maps of the genome of vaccinia virus are distinctly differ- occurred after variola infection. Similar histopathology is ent from those of cowpox virus, which was believed to be its seen with vaccinia, although vaccinia virus ordinarily causes ancestor. At some time after Jenner’s original use of “cowpox” localized pustular lesions only at the site of inoculation. virus, the vaccine virus became known as “vaccinia virus.” Vaccinia virus may be the product of genetic recombination, a new species derived from cowpox virus or variola virus by Clinical Findings serial passage, or the descendant of a now extinct viral genus. The incubation period of variola (smallpox) was 10–14 days. Variola has a narrow host range (only humans and The onset was usually sudden. One to 5 days of fever and mal- monkeys), but vaccinia has a broad host range that includes aise preceded the appearance of the exanthems, which began rabbits and mice. Some strains of vaccinia can cause a severe as macules, then papules, then vesicles, and finally pustules. disease in laboratory rabbits that has been called rabbitpox. These formed crusts that fell off after about 2 weeks, leaving Vaccinia virus has also infected cattle and water buffalo, and pink scars that faded slowly. In each affected area, the lesions the disease in buffalo has persisted in India (buffalopox). were generally found in the same stage of development (in Both vaccinia and variola viruses grow on the chorioallantoic contrast to chickenpox). membrane of the 10- to 12-day-old chick embryo, but the lat- A “Smallpox Recognition Card” prepared by the World ter produce much smaller pocks. Both grow in several types Health Organization shows the typical rash (Figure 34-4). of chick and primate cell lines. Lesions were most abundant on the face and less so on the The nucleotide sequences of variola (186 kb) and vac- trunk. In severe cases, the rash was hemorrhagic. The case- cinia (192 kb) are similar, with the most divergence in ter- fatality rate varied from 5% to 40%. In the less common and minal regions of the genomes. Of 187 putative proteins, 150 more benign variola minor strain (also eradicated), or in vac- were markedly similar in sequence between the two viruses; cinated persons, the mortality rate was under 1%. the remaining 37 diverged or were variola-specific and may represent potential virulence determinants. The sequences do not reveal variola virus origins or explain its strict human Immunity host range or its particular virulence. All viruses within the Orthopoxvirus genus are so closely related antigenically that they cannot be easily differenti- ated serologically. Infection with one induces an immune Pathogenesis and Pathology of Smallpox response that reacts with all other members of the group. Although smallpox has been eradicated, the pathogenesis of Survival of an attack of smallpox gave complete protec- the disease (described here in the past tense) is instructive for tion against reinfection. Vaccination with vaccinia induced other poxvirus infections. immunity against variola virus for at least 5 years and some- The portal of entry of variola virus was the mucous mem- times longer. Antibodies alone are not sufficient for recov- branes of the upper respiratory tract. After viral entry, the ery from primary poxvirus infection. In the human host, following are believed to have taken place: (1) primary mul- neutralizing antibodies develop within a few days after the tiplication in the lymphoid tissue draining the site of entry; onset of smallpox but do not prevent progression of lesions, (2) transient viremia and infection of reticuloendothelial cells and patients may die in the pustular stage with high antibody Riedel_CH34_p499-p510.indd 503 05/04/19 4:56 PM  504   SECTION IV  Virology FIGURE 34-4 Smallpox rash. A “Smallpox Recognition Card” from the World Health Organization illustrates the distribution and nature of the typical rash of smallpox in an unvaccinated child. (Reproduced by permission of WHO, from Fenner F et al: Smallpox and Its Eradication. Geneva: World Health Organization, 1988.) levels. Cell-mediated immunity is probably more important Polymerase chain reaction (PCR) tests that are specific than circulating antibody. Patients with hypogammaglobu- for various poxviruses are the primary methods available linemia generally react normally to vaccination and develop for detection and identification purposes, generally in public immunity despite the apparent absence of antibody. Patients health laboratories. who have defects in both cellular immune response and anti- Direct examination of clinical material in the electron body response develop a progressive, usually fatal disease microscope can be used for rapid identification of virus upon vaccination. particles (∼1 hour) and can readily differentiate a poxvirus Production of interferon (see Chapter 30) is another infection from chickenpox (caused by a herpesvirus). Ortho- possible immune mechanism. Irradiated animals without poxviruses cannot be distinguished from one another by detectable antibody or delayed hypersensitivity response electron microscopy because they are similar in size and recovered from vaccinia infection as rapidly as untreated morphology. However, they can be easily differentiated from control animals. tanapox virus and parapoxviruses. Viral antigen can be detected by immunohistochemistry in tissues and in material collected from skin lesions. Many Laboratory Diagnosis antigens are cross-reactive and identify orthopoxviruses as a Several tests are available to confirm the diagnosis of small- group. The use of PCR or restriction enzyme cleavage of viral pox. Now that the disease is presumably eradicated, it is DNA or the analysis of polypeptides in poxvirus-infected important to diagnose any cases that resemble smallpox. The cells can demonstrate distinct characteristics for variola, vac- tests depend on identification of viral DNA or antigen from cinia, monkeypox, and cowpox. the lesion, direct microscopic examination of material from Cell cultures can be used for virus isolation, though cul- skin lesions, recovery of virus from the patient, and, least ture of variola virus is only attempted in Biosafety Level 4 importantly, demonstration of antibody in the blood. facilities. The orthopoxviruses grow well in cultured cells; parapoxviruses and tanapox virus grow less well; and mol- A. Isolation and Identification of Virus luscum contagiosum virus cannot be cultured. Skin lesions are the specimen of choice for viral detection and Virus isolation can also be carried out by inoculation of isolation. Poxviruses are stable and remain viable in speci- vesicular fluid onto the chorioallantoic membrane of chick mens for weeks even without refrigeration. embryos. This test can distinguish cases of smallpox from Riedel_CH34_p499-p510.indd 504 05/04/19 4:56 PM  CHAPTER 34 Poxviruses   505 generalized vaccinia because the lesions produced by these program. The source of each outbreak was determined, and viruses on the membrane differ markedly. In 2–3 days, vac- all susceptible contacts were identified and vaccinated. cinia pocks are large with necrotic centers; variola pocks are much smaller. Cowpox and monkeypox produce distinc- tive hemorrhagic lesions. The parapoxviruses, molluscum Vaccination with Vaccinia contagiosum virus, and tanapox virus do not grow on the Vaccinia virus for vaccination is prepared from vesicular membrane. lesions (“lymph”) produced in the skin of calves, or it can be grown in chick embryos. The final vaccine contains 40% glyc- B. Serology erol to stabilize the virus and 0.4% phenol to destroy bacteria. World Health Organization standards require that smallpox Antibody assays can be used to confirm a diagnosis of poxvi- vaccines have a potency of no fewer than 108 pock-forming rus infection. Antibodies appear after the first week of infec- units per milliliter. A new cell culture-produced live vaccinia tion that can be detected by hemagglutination inhibition, vaccine was approved for use in the United States in 2007. The neutralization, enzyme-linked immunosorbent assay, radio- vaccinia vaccine does not contain smallpox (variola) virus. immunoassay, or immunofluorescence tests. None of these Smallpox vaccination was associated with a definite mea- tests will distinguish among the orthopoxviruses. surable risk. In the United States, the risk of death from all complications was 1 per million for primary vaccinees and 0.6 per million for revaccinees. For children less than 1 year of Treatment age, the risk of death was five per million primary vaccinations. Treatment of smallpox is primarily supportive. Vaccinia Severe complications of vaccination occurred in conjunction immune globulin has not shown a survival benefit for estab- with immunodeficiency, immunosuppression, malignancies, lished disease. and pregnancy or early childhood (Figure 34-5). Those condi- Methisazone is a chemotherapeutic agent historically tions are contraindications for vaccinia vaccine use, as well as evaluated against poxviruses. It is effective as prophylaxis but eczema, allergy to a vaccine component, and living in a house- is not useful in treatment of established disease. Cidofovir, a hold with someone having a vaccination contraindication. nucleotide analog, shows activity against poxviruses in vitro The success of smallpox eradication has meant that routine and in vivo. It has been used to treat molluscum contagiosum vaccination is no longer recommended. Routine smallpox vac- and orf virus infections. cination of children in the United States was stopped in 1971. Vaccinia virus is used in research and has resulted in laboratory-acquired infections. Current recommendations Epidemiology are that laboratory workers who handle cultures or animals Transmission of smallpox occurred by contact between cases. infected with vaccinia or other orthopoxviruses that infect Smallpox was highly contagious. The virus was stable in the humans should be vaccinated at least every 10 years. Recent extracellular environment but was most commonly transmit- concerns about a possible terrorist attack involving smallpox ted by respiratory spread. The dried virus in crusts from skin have resulted in limited scale vaccination for military person- lesions could survive on clothes or other materials and result nel and emergency health care responders. in infections; this property was occasionally used in early biological warfare. Patients were most highly infectious during the first week of rash after the fever had begun. Respiratory droplets were infectious earlier than skin lesions. The following epidemiologic features made smallpox ame- nable to total eradication: There was no known nonhuman reservoir. There was an effective vaccine, leading to immunity to both variola major and variola minor. Subclinical infectious cases did not occur. Chronic asymptomatic carriage of the virus did not occur. Because virus in the environment of the patient derived from lesions in the mouth and throat (and later in the skin), patients with infection sufficiently severe to transmit the disease were likely to be so ill that they quickly reached the attention of medical authorities. The close contact required for effective spread of the disease generally made for ready identi- fication of a patient’s contacts so that specific control measures FIGURE 34-5 Eczema vaccinatum in a young child with eczema. could be instituted to interrupt the cycle of transmission. The disease developed after exposure to a newly vaccinated family The World Health Organization was successful in member. (Courtesy of AE Kaye; Centers for Disease Control and eradicating smallpox by using a surveillance–containment Prevention Public Health Image Library.) Riedel_CH34_p499-p510.indd 505 05/04/19 4:56 PM  506   SECTION IV  Virology MONKEYPOX INFECTIONS lesions that cowpox virus produces on the chorioallantoic membrane of the chick embryo. Monkeypox virus is a species of Orthopoxvirus. The disease The natural reservoir of cowpox seems to be a rodent, was first recognized in captive monkeys in 1958. Human and both cattle and humans are only accidental hosts. infections with this virus were discovered in the early 1970s Domestic cats also are susceptible to cowpox virus. More in West Africa and central Africa after the eradication of than 50 cases in felines have been reported from the United smallpox from those regions. Kingdom, but transmission from cats to humans is believed The disease is a rare zoonosis that has been detected to be uncommon. Cowpox is no longer enzootic in cattle, in remote villages in tropical rain forests, particularly in although bovine and associated human cases occasionally the Congo basin countries of Africa and perhaps in West occur. Feline cowpox is sporadic, and transmission is prob- Africa. It is probably acquired by direct contact with wild ably from a small wild rodent, including field voles. Human animals killed for food and skins. The primary reservoir cases (with hemorrhagic skin lesions, fever, and general mal- host is not known, but squirrels, rabbits, and rodents can aise) may occur without any known animal contact and may be infected. not be diagnosed. There is no specific treatment. The clinical features of human monkeypox are similar to those of smallpox, but usually less severe. Pronounced lymphadenopathy occurs in most patients, a feature not seen BUFFALOPOX INFECTIONS with smallpox or chickenpox. Complications are common and often serious. These are Buffalopox virus is a derivative of vaccinia virus that has per- generally pulmonary distress and secondary bacterial infec- sisted in India in water buffalo since smallpox vaccination tions. In unvaccinated patients, the fatality rate can reach was discontinued. The disease in buffalo—and occasionally about 10%. Vaccination with vaccinia either protects against in cattle—is indistinguishable from cowpox. Buffalopox can monkeypox or lessens the severity of disease. Since the cessa- be transmitted to humans, and localized pox lesions develop. tion of smallpox vaccination, the number of human monkey- There is some concern that human-to-human transmission pox cases is markedly increasing in tropical Africa. may also occur. Human monkeypox infection is generally believed not to be easily transmitted from person to person. Previous esti- mates were that only about 15% of susceptible family contacts ORF VIRUS INFECTIONS acquired monkeypox from patients. However, an outbreak The orf virus is a species of Parapoxvirus. It causes a disease in Zaire in 1996 and 1997 suggested a higher potential for in sheep and goats that is prevalent worldwide (Figure 34-6C). person-to-person transmission. The disease is also called contagious pustular dermatitis or The first outbreak of monkeypox in the Western Hemi- sore mouth infection. sphere occurred in the United States in 2003. More than 80 Orf is transmitted to humans by direct contact with an human cases (no deaths) were diagnosed, mostly in midwest- infected animal. It is an occupational disease of sheep and goat ern states. The source was traced to an exotic pet store where handlers. Recent reports from the United States emphasized apparently an imported African rat spread the virus to pet the temporal association between human lesions and recent prairie dogs and they transmitted it to humans. It is likely flock vaccination with live orf virus. Infection by orf virus is that the isolate of monkeypox virus introduced was a natu- facilitated by skin trauma. Infection of humans occurs usually rally attenuated virus from West Africa that was less patho- as a single lesion on a finger, hand, or forearm (Figure 34-6F) genic in humans than isolates from central Africa. but may appear on the face or neck. Lesions are large nodules, rather painful, with surrounding inflamed skin. The infection is seldom generalized. Healing takes several weeks. COWPOX INFECTIONS Electron microscopy can confirm a parapoxvirus infec- tion, but only nucleic acid testing methods can definitively Cowpox virus is another species of Orthopoxvirus. This disease identify a parapoxvirus as orf virus. of cattle is milder than the pox diseases of other animals, the lesions being confined to the teats and udders (Figure 34-6A). Infection of humans occurs by direct contact during milk- MOLLUSCUM CONTAGIOSUM ing, and the lesion in milkers is usually confined to the hands (Figure 34-6D). The disease is more severe in unvaccinated Molluscum contagiosum is a benign epidermal tumor that persons than in those vaccinated with vaccinia virus. occurs only in humans (although there is evidence of a closely Cowpox virus is similar to vaccinia virus immunologi- related virus in horses). The causative agent is classified as the cally and in host range. It is also closely related immunologi- sole member of the Molluscipoxvirus genus. cally to variola virus. Jenner observed that those who have had The virus has not been transmitted to animals and has cowpox are immune to smallpox. Cowpox virus can be dis- not been grown in tissue culture. It has been studied in the tinguished from vaccinia virus by the deep red hemorrhagic human lesion by electron microscopy. The purified virus is Riedel_CH34_p499-p510.indd 506 05/04/19 4:56 PM  CHAPTER 34 Poxviruses   507 A D B E C F FIGURE 34-6 Cowpox, pseudocowpox, and orf in animals and humans. A: Cowpox ulcer on the teat of a cow 7 days after onset of signs. B: Pseudocowpox (milker’s nodule virus) on the teat of a cow. C: Scabby mouth in a lamb, caused by orf virus. D, E, F: Hand lesions caused by these viruses. D: Cowpox. E: Milker’s nodule (pseudocowpox). F: Orf. (A and B courtesy of EPJ Gibbs; C courtesy of Dr. Anthony J. Robinson; D courtesy of AD McNae; E and F courtesy of J Nagington.) oval or brick shaped and measures about 230 nm by 330 nm; virus (∼190 kbp) is known. It contains at least 163 genes, it resembles vaccinia. Antibodies to the virus do not cross- about two-thirds of which resemble genes of smallpox and react with any other poxviruses. cowpox viruses. The viral DNA resembles that of vaccinia virus with The lesions of this disease are small, pink, wart-like respect to terminal cross-linking and inverted terminal tumors on the face, arms, back, and buttocks (Figure 34-7). repeats. It has an overall G + C content of about 60%. The They are rarely found on the palms, soles, or mucous mem- sequence of the entire genome of molluscum contagiosum branes. The disease occurs throughout the world in both Riedel_CH34_p499-p510.indd 507 05/04/19 4:56 PM  508   SECTION IV  Virology tumor poxvirus is smaller (145 kbp; 32.5% G + C). The viruses grow only in cultures of monkey and human cells, with cytopathic effects. They do not grow on the chorioal- lantoic membrane of embryonated eggs. Tanapox begins with a febrile period of 3–4 days and can include severe headache and prostration. There are usu- ally only one or two skin lesions; pustulation never occurs (Figure 34-8). Healing may take 4–7 weeks. Yaba monkey tumor poxvirus causes benign histiocyto- mas 5–20 days after subcutaneous or intramuscular adminis- tration to monkeys. The tumors regress after about 5 weeks. Intravenous administration of the virus causes the appear- ance of multiple histiocytomas in the lungs, heart, and skeletal muscles. True neoplastic changes do not occur. The virus is easily isolated from tumor tissue, and characteristic inclusions FIGURE 34-7 Lesions of molluscum contagiosum in humans. (Courtesy of D Lowy.) sporadic and epidemic forms and is more frequent in chil- dren than in adults. It is spread by direct and indirect contact (eg, by barbers, common use of towels, or swimming pools). The incidence of molluscum contagiosum as a sexually transmitted disease in young adults is increasing. It is seen also in some patients with AIDS. The skin of late-stage AIDS patients may be covered with many papules. Although the typical lesion is an umbilicated papule, lesions in moist geni- tal areas may become inflamed or ulcerated and may be con- fused with those produced by herpes simplex virus (HSV). The incubation period may extend for up to 6 months. Lesions may itch, leading to autoinoculation. The lesions may persist for up to 2 years but eventually regress sponta- neously. The virus is a poor immunogen; about one-third of A patients never produce antibodies against it. Second attacks are common. The diagnosis of molluscum contagiosum can usually be made clinically. However, a semisolid caseous material can be expressed from the lesions and used for laboratory diagnosis. PCR can detect viral DNA sequences, and electron micros- copy can detect poxvirus particles. TANAPOX AND YABA MONKEY TUMOR POXVIRUS INFECTIONS Tanapox is a fairly common skin infection in parts of Africa, mainly in Kenya and in the Democratic Republic of Congo. Its natural host is probably monkeys, although it is possible that there is another reservoir and that monkeys are only incidental hosts. The mode of transmission is not known. Tanapox and Yaba monkey tumor viruses are serologi- cally related to each other but are distinct from all other pox- B viruses. They are classified in the Yatapoxvirus genus. They FIGURE 34-8 Lesions produced by tanapox virus. A: Ten are morphologically similar to orthopoxviruses. The tanapox days after first appearance of the lesion. B: Thirty-one days after virus genome is 160 kbp in size, and that of Yaba monkey appearance of the lesion. (Courtesy of Z Jezek.) Riedel_CH34_p499-p510.indd 508 05/04/19 4:56 PM  CHAPTER 34 Poxviruses   509 are found in the tumor cells. Monkeys of various species and 4. A 7-year-old boy has pox-like lesions on his left hand and arm. humans are susceptible to the cellular proliferative effects of He has a pet rodent imported from West Africa. Monkeypox is the virus, but other laboratory animals are insusceptible. Yaba diagnosed in the boy and the rodent. Which of the following virus infections have been seen in animal handlers in Africa. statements about monkeypox virus is most correct? (A) Clinical disease resembles smallpox. (B) Human infections are never fatal. (C) Smallpox vaccination is not protective. CHAPTER SUMMARY (D) Infections are readily transmitted among family members. Poxviruses are large and complex viruses that contain (E) Virus particles can be distinguished from smallpox virus by electron microscopy. many enzymes, including a transcriptional system. Family Poxviridae includes variola virus, the agent of 5. Which of the following best describes the currently licensed smallpox vaccines? smallpox, the first viral disease eradicated from the earth. Poxviruses encode proteins that inhibit the host immune (A) Live attenuated smallpox virus (B) Inactivated smallpox virus defense system. (C) Live vaccinia virus Vaccinia virus is used for smallpox vaccination and as a (D) Inactivated vaccinia virus laboratory model for poxviruses. (E) Reassortant vaccine containing both vaccinia and small- There is a risk with smallpox vaccination in association pox viruses with immunodeficiency, immunosuppression, malignan- 6. Which of the following does not apply to vaccinia virus replica- cies, and pregnancy. tion in cultured cells? Most poxvirus infections are accompanied by a vesicular (A) Viral replication cycle takes place in the cytoplasm of rash. infected cells. Smallpox virus is a potential biothreat agent because the (B) The uncoating step leading to release of the viral genome human population today has little or no immunity. requires a newly synthesized viral protein. Several animal poxviruses, including monkeypox, cowpox, (C) Early transcription of more than 50 viral genes occurs orf, and tanapox, can infect humans. within viral cores and precedes viral DNA replication. Patients with symptoms suggestive of pox viruses need (D) Newly formed virus particles mature by budding through to be isolated with supportive treatment until smallpox is the nuclear membrane. excluded diagnostically. 7. Which feature of the variola virus makes it an extreme bioter- Molluscum contagiosum causes benign epidermal tumors. rorism threat? (A) Wide availability of the virus (B) Weaponized strains present in several laboratories REVIEW QUESTIONS (C) Limited immunity in present population (D) Low stockpiles of effective drugs for treatment 1. A patient presents to the emergency room with vesicular lesions (E) Potential emergence from animal reservoir on both hands potentially resembling smallpox. A public health 8. A patient presents with skin lesions similar in appearance to investigation is begun to rule out smallpox. The patient is an molluscum contagiosum. How is diagnosis of this condition immigrant working as a shepherd in several states. What is the typically made? most likely cause of his skin lesions? (A) Viral culture (A) Vaccinia virus (B) Rapid antigen test (B) Variola virus (C) PCR for viral DNA (C) Monkeypox virus (D) Clinical appearance (D) Tanapox virus (E) Inoculation of chorioallantoic membrane of chick embryos (E) Orf virus 9. Which of the following does not fulfill the criteria for exposure 2. An emergency services worker is considering smallpox vaccina- to vaccinia? tion because of the potential for bioterrorism. Which one of the (A) Smallpox vaccination following conditions is not a contraindication for the use of vac- (B) Close contact with a recent smallpox vaccine cinia (smallpox) vaccine under routine nonemergency conditions? (C) Intrauterine exposure (A) Immunosuppression (D) Injection of vaccinia immune globulin (B) Severe allergy to a component of the vaccine 10. A researcher wishes to obtain a full-length genome of variola (C) Household contact with a person with eczema virus for vaccine studies. Which of the following is the appro- (D) Pregnancy priate source of the viral DNA? (E) Previous smallpox vaccination (A) The Centers for Disease Control and Prevention. 3. Which of the following poxviruses infects only humans? (B) A World Health Organization collaborating center. (A) Monkeypox (C) The American Type Culture Collection. (B) Molluscum contagiosum (D) A colleague with a variola virus clone. (C) Tanapox (E) Distribution of a full-length viral genome is prohibited. (D) Cowpox (E) Yaba tumor virus Riedel_CH34_p499-p510.indd 509 05/04/19 4:56 PM  510   SECTION IV  Virology 11. Laboratory scientists who work with vaccinia virus-infected Answers cultures or animals are at risk of accidental exposure to the 1. E 6. D 11. B virus. Which of the following procedures by the laboratory 2. E 7. C 12. B worker is of least benefit in protecting against inadvertent infection with vaccinia virus? 3. B 8. D 13. D 4. A 9. D 14. A (A) Proper use of personal protective equipment such as gloves and goggles 5. C 10. E (B) Cleaning of laboratory work space before experimentation (C) Smallpox vaccination (D) Safe needle-handling practices REFERENCES (E) Use of biosafety hoods Li Y, Carroll DS, Gardner SN, et al: On the origin of smallpox: 12. Vaccinia virus has all of the following attributes except Correlating variola phylogenics with historical smallpox (A) Can cause severe localized or disseminated disease. records. Proc Natl Acad Sci USA 2007;104:15787. (B) Is a live, attenuated smallpox virus. MacNeil A, Reynolds MG, Damon IK: Risks associated with (C) Can induce immunity that lasts only a few years. vaccinia virus in the laboratory. Virology 2009;385:1. (D) Has been in use for more than 200 years. McFadden G: Poxvirus tropism. Nat Rev Microbiol 2005;3:201. (E) Gene sequences coding for other viral proteins can be Moss B: Genetically engineered poxviruses for recombinant gene inserted into its genome. expression, vaccination, and safety. Proc Natl Acad Sci USA 13. The eradication of smallpox was facilitated by several features 1996;93:11341. of the virus. Which one of the following contributed least to Casey C, Vellozzi C, Mootrey GT, et al: Surveillance guidelines eradication? for smallpox vaccine (vaccinia) adverse reactions. MMWR Recomm Rep 2006;55(RR-1):1–16. (A) It has one antigenic type. Rotz LD, Dotson DA, Damon IK, et al: Vaccinia (smallpox) (B) Inapparent infection is rare. vaccine: Recommendations of the Advisory Committee on (C) Administration of live vaccine reliably induces immunity. Immunization Practices (ACIP), 2001. MMWR Recomm Rep (D) It multiplies in the cytoplasm of infected cells. 2001;50(RR-10):1–25. 14. Vaccination with the vaccinia (smallpox) vaccine protects Wharton M, Strikas RA, Harpaz R, et al: Recommendations for against infections by the following poxviruses except using smallpox vaccine in a pre-event vaccination program: (A) Molluscum contagiosum Supplemental recommendations of the Advisory Committee on (B) Variola Immunization Practices (ACIP) and the Healthcare Infection (C) Cowpox Control Practices Advisory Committee (HICPAC). MMWR (D) Monkeypox Recomm Rep 2003;52(RR-7):1. WHO recommendations concerning the distribution, handling and synthesis of variola virus DNA, May 2008. World Health Org Wkly Epidemiol Rec 2008;83:393. 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