BW-ch11.pdf

Smallpox and Related Orthopoxviruses

Chapter 11

SMALLPOX AND RELATED

ORTHOPOXVIRUSES

PETER B. JAHRLING, P h D * ; JOHN W. HUGGINS, P h D † ; M. SOFI IBRAHIM, MS c , P h D ‡ ; JAMES V. LAWLER, MD § ; a n d

JAMES W. MARTIN, MD, FACP ¥

INTRODUCTION

AGENT CHARACTERISTICS

Classification

Morphology

Phylogenetic Relationships

Replication

Pathogenesis

ORTHOPOXVIRUSES AS BIOLOGICAL WARFARE AND BIOTERRORISM

THREATS

CLINICAL ASPECTS OF ORTHOPOXVIRUS INFECTIONS

Smallpox

Monkeypox

Other Orthopoxviruses Infecting Humans

DIAGNOSIS

Clinical Diagnosis

Laboratory Diagnosis

Phenotypic Diagnosis

Immunodiagnosis

Nucleic Acid Diagnosis

MEDICAL MANAGEMENT

Prophylaxis

Treatment

SUMMARY

* Director, National Institute of Allergies and Infectious Diseases, Integrated Research Facility, National Institutes of Health, 6700A Rockledge Drive,

Bethesda, Maryland 20897; formerly, Senior Research Scientist, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort

Detrick, Maryland

† Chief, Viral Therapeutics Branch, US Army Medical Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

‡ Lieutenant Colonel, Medical Service Corps, US Army Reserve; Microbiologist, Division of Virology, US Army Medical Research Institute of Infectious

Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

§ Lieutenant Commander, Medical Corps, US Navy Reserve; Director for Biodefense Policy, Homeland Security Council, The White House, Washington,

DC 20502; formerly, Infectious Diseases Physician, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick,

Maryland

¥ Colonel, Medical Corps, US Army; Chief, Operational Medicine Department, Division of Medicine, US Army Medical Research Institute of Infectious

Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

215

Medical Aspects of Biological Warfare

INTRODUCTION

Variola, the virus that causes smallpox, is one of

the most significant bioterrorist threat agents. During

the 20th century, smallpox is estimated to have caused

over 500 million human deaths. 1 Yet the disease and

the naturally circulating virus itself were eradicated

by the World Health Organization’s (WHO) global

eradication campaign, which was declared a success

in 1980. 2 This program, which involved vaccinating

all humans in a ring surrounding every suspected

case of variola infection, was successful in part be-

cause smallpox is solely a human disease; there are

no animal reservoirs to reintroduce the virus into

the human population. The impact of a smallpox

virus attack in the human population would be even

more catastrophic now than during the 20th century,

because most vaccination programs were abandoned

worldwide in the 1970s, the prevalence of immunosup-

pressed individuals has grown, and mobility, including

intercontinental air travel, has accelerated the pace of

viral spread. Smallpox virus is stable, highly infectious

via the aerosol route, and highly transmissible from

infected to susceptible persons, and it has a relatively

long asymptomatic incubation period, making contact

tracing difficult. 3 Mathematical models of a variola

reintroduction into contemporary human populations

indicate dire consequences. 4 Public health experts have

argued that a significant portion of the population

should be prevaccinated to blunt the impact of such

an attack. 5 However, the vaccine is associated with

significant adverse events, 6 which are more serious in

persons who are immunocompromised, and prerelease

vaccination is contraindicated for a significant portion

of the population.

Recent revelations that the former Soviet Union

produced ton quantities of smallpox virus as a strategic

weapon 3 and conducted open-air testing of aerosolized

variola on Vozrozhdeniye Island in the Aral Sea have in-

creased the plausibility of variola being used as a bioter-

rorism agent. 7 Considerable investment is being made in

biopreparedness measures against smallpox and related

orthopoxviruses, including emergency response plans

for mass immunization and quarantine, 8 as well as de-

velopment of improved countermeasures such as new

vaccines and antiviral drugs. 9 These countermeasures

are also needed to respond to the public health threat

of the closely related monkeypox virus, which occurs

naturally in western and central Africa and produces

a disease in humans that closely resembles smallpox.

Alibek claimed that monkeypox virus was weaponized

by the former Soviet Union. 10 Monkeypox virus was

imported inadvertently into the United States in 2003

via a shipment of rodents originating in Ghana, where,

in contrast to the significant morbidity and mortality

seen in the Democratic Republic of Congo, little mor-

bidity was associated with infection. Over 50 human

infections were documented in the United States as a

result, demonstrating the public health importance of

this agent and the potential bioterrorist threat. 11,12

AGENT CHARACTERISTICS

Classification

Poxviruses infect most vertebrates and invertebrates,

causing a variety of diseases of veterinary and medical

importance. The poxvirus family is divided into two

main subfamilies: (1) the Chordopoxvirinae , which infects

vertebrates; and (2) the Entomopoxvirinae , which infects

insects. Subfamily Chordopoxvirinae is divided into eight

genera, one of which is Orthopoxvirus , which includes

the human pathogens variola (Figure 11-1), monkeypox

virus, and other species that infect humans such as cow-

pox and vaccinia viruses. Members of the Orthopoxvirus

genus are mostly zoonotic pathogens, and a few of these

viruses produce disease in humans (Table 11-1).

Morphology

Orthopoxviruses are oval, brick-shaped particles

with a geometrically corrugated outer surface. Their

size ranges from 220 nm to 450 nm long and 140 nm

Fig. 11-1. A transmission electron micrograph of a tissue

section containing variola viruses.

Photograph: Courtesy of FA Murphy, University of Texas

Medical Branch, Galveston, Texas.

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Smallpox and Related Orthopoxviruses

TABLE 11-1

POXVIRUSES THAT CAUSE HUMAN DISEASE

Genus

Species

Animal Reservoir

Orthopoxvirus

Variola virus

None

Vaccinia virus

Unknown (none?)

Cowpox virus

Rodents

Monkeypox virus

Rodents

Parapoxvirus

Bovine popular stomatitis virus

Cattle

Orf virus

Sheep

Pseudocowpox virus

Cattle

Seal parapoxvirus

Seals

Parapoxvirus

Tanapox

Rodents (?)

Yabapox virus

Monkeys (?)

Molluscipoxvirus

Molluscum contagiosum virus

None

to 260 nm wide. The outer envelope consists of a lipo-

protein layer embedding surface tubules and enclosing

a core described as biconcave because of an electron

microscopy fixation artifact. The core contains the viral

DNA and core fibrils, and it is surrounded by the core

envelope and a tightly arranged layer of rod-shaped

structures known as the palisade layer. Between the

palisade layer and the outer envelope are two oval

masses known as the lateral bodies (Figure 11-2). Two

infectious forms of orthopoxviruses (described next)

result from the replication cycle.

185,000-kilobase (kb) genome.

As anticipated from the genomic homologies,

members of the Orthopoxvirus genus are antigenically

related. Serum absorption and monoclonal antibody

studies have identified cross-reacting and species-

specific neutralizing antigens. 15 Nine neutralizing

epitopes have been identified among the intracellular

Phylogenetic Relationships

The evolutionary relationships among the poxvi-

ruses have been facilitated by the recent availability

of complete DNA sequences for over 30 species. Phy-

logenetic analysis reveals that variola and camelpox

viruses are more closely related to each other than

any other members of the genus, and vaccinia is most

closely related to cowpox virus strain GRI-90. 13,14

Cowpox virus strain GRI-90 appears to be less closely

related to cowpox virus strain Brighton, indicating that

at least two separate species are included under the

name cowpox virus. Monkeypox virus does not group

closely with any other orthopoxvirus, which indicates

that it diverged from the rest of the genus members

long ago. Yet vaccination prevents monkeypox. Minor

modifications to the camelpox virus genome might

result in a virus with variola attributes. Virulence or

attenuation may hinge on a few genetic determinants.

For example, variola major (associated with a 30%

fatality rate) and variola minor ( < 1% fatality rate)

are greater than 98% identical over the length of the

Fig. 11-2. Thin section of smallpox virus growing in the cy-

toplasm of an infected chick embryo cell of infected person.

Intracellular mature virions (brick-shaped) and immature

virions (spherical) are visible. Magnification is approximately

x 25,000.

Photograph: Courtesy of FA Murphy, University of Texas

Medical Branch, Galveston, Texas.

217

Medical Aspects of Biological Warfare

mature virion (IMV) particles of different species of

orthopoxviruses 16 ; additional epitopes, believed to

be critical in protection against infection in vivo, ex-

ist on extracellular enveloped viral particles. 17,18 Viral

envelope proteins are important in protective antibody

responses: envelope antigens were absent from virion

suspensions used for inactivated smallpox vaccines

that proved to be ineffective. 19,20

tion. The central region of the genome contains highly

conserved genes that are essential for viral replication,

and the terminal regions contain less conserved genes

that are important for virus-host interactions. The vi-

rus contains a number of virus-encoded enzymes, in

particular a DNA-dependent RNA polymerase that

transcribes the viral genome. 21 Replication occurs in

cytoplasmic factories referred to as B-type inclusions,

in which virions at various stages of assembly are seen.

Whether host cell nuclear factors are involved in viral

replication or maturation is unclear. Cells infected

with some poxviruses (eg, cowpox, avian poxviruses)

also contain electron-dense A-type inclusions, usually

containing mature virions; A-type inclusions are easily

seen by light microscopy (Figure 11-3).

Replication

Orthopoxvirus genomes are linear, double-stranded

DNA approximately 200 kb long. The genomes encode

about 176 to 266 proteins, including enzymes and fac-

tors that are necessary for self-replication and matura-

Fig. 11-3. Cytoplasmic inclusion bodies in cells infected with

orthopoxviruses. ( a ) B-type (pale-red, irregular) inclusion, or

Guarnieri, bodies, and A-type (large eosinophilic, with halo)

inclusion bodies in ectodermal cells of the chorioallantoic

membrane, in a pock produced by cowpox virus. A number

of nucleated erythrocytes are in the ectoderm and free in the

mesoderm, and the surface of the pock is ulcerated. Hematoxylin-eosin stain. ( b ) This section of the skin of a patient with

hemorrhagic-type smallpox shows Guarnieri bodies and free erythrocytes below an early vesicle. Hematoxylin-eosin stain.

Reproduced with permission from Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and Its Eradication . Geneva,

Switzerland: World Health Organization; 1988: 85.

218

Smallpox and Related Orthopoxviruses

Viral replication begins with attachment of viral

particles to the host cell surface, most likely through

cell receptors, and involves expression of early, in-

termediate, and late genes. 21 Initial uncoating occurs

during entry, followed by synthesis of early mRNAs,

which are translated to facilitate further uncoating and

transcription of intermediate mRNAs. Intermediate

mRNAs, in turn, are translated to allow transcription

of the late mRNAs. The late mRNAs are translated into

structural and nonstructural proteins of the virions.

These proteins, along with DNA concatemers that

are formed during the early phase of replication, are

assembled into genomic DNA and packaged into im-

mature virions, which then evolve into brick-shaped

infectious IMVs. IMVs are infectious only when they

are released by cell lysis. IMV particles, which can

acquire a second membrane from an early endosomal

component to form the intracellular enveloped virion

(IEV), migrate to the cell surface via microtubules and

fuse with the cell membrane to form cell-associated

virions (CEVs). CEVs induce polymerization of actin

to form filaments that affect the direct transfer of CEVs

to adjacent cells. If CEVs become dissociated from the

cell membranes, they are called extracellular envel-

oped virions (EEVs). Although IMVs are produced

in greatest abundance in cell culture and are the most

stable to environmental degradation, CEVs and EEVs

probably play a more critical role in cell-to-cell spread

in the intact animal. 22

Many of the Orthopoxvirus gene products, known as

virokines and viroceptors, interact with and modulate

essential functions of the host cells and immune pro-

cesses. 21,23 The limited host range of variola may relate

to the unique association of viral gene products with

various host signaling pathways. Therefore, strategies

that block such key pathways in the replication and

maturation of poxviruses provide potential targets for

therapeutic intervention. 24

including regional lymphatics, spleen, and tonsils. A

second, brief viremia transports the virus to the skin

and to visceral tissues immediately before the prodro-

mal phase. In humans, the prodrome is characterized

by an abrupt onset of headache, backache, and fever,

and usually sore throat resulting from viral replication

in the oral mucosa. Characteristic skin lesions develop

following viral invasion of the capillary epithelium of

the dermal layer. The virus may also be present in urine

and conjunctival secretions. 30 At death, most visceral

tissues contain massive virus concentrations.

In a review of all pathology reports published in

English over the past 200 years, 31 Martin suggested

that generally healthy patients who died of smallpox

usually died of renal failure, shock secondary to vol-

ume depletion, and difficulty with oxygenation and

ventilation as a result of viral pneumonia and airway

compromise, respectively. Degeneration of hepatocytes

might have caused a degree of compromise, but liver

failure was not usually the proximate cause of death.

Much of the pathogenesis of smallpox remains

a mystery because of the limited tools that were

available when it was an endemic disease. Detailed

analysis of the pathophysiology of the disease course

using the monkeypox and variola primate models and

in comparison with limited clinical and pathology

data from human smallpox victims suggests a role

for dysregulation of the immune response involv-

ing the production of proinflammatory cytokines,

lymphocyte apoptosis, and the development of co-

agulation abnormalities. High viral burdens, which

were identified in numerous target tissues in the

animal models, were probably associated with organ

dysfunction and multisystem failure. Immunohisto-

chemistry studies showing the distribution of viral

antigens as well as electron microscopy evidence of

the replicating virus correlated with pathology in the

lymphoid tissues, skin, oral mucosa, gastrointestinal

tract, reproductive system, and liver. Apoptosis was

a prominent observation in lymphoid tissues, with

a striking loss of T cells observed. The cause of this

widespread apoptosis remains unknown. However,

strong production of proinflammatory cytokines at

least in part likely contributed to the upregulation

of various proapoptotic genes. The strong upregula-

tion of cytokines may also have contributed to the

development of a hemorrhagic diathesis. The detec-

tion of D-dimers and other changes in hematologic

parameters in monkeys that developed classical or

hemorrhagic smallpox suggests that activation of the

coagulation cascade is a component of both disease

syndromes. In human populations, however, the oc-

currence of hemorrhagic smallpox was approximately

1% to 3% of the total cases observed.

Pathogenesis

Most knowledge about smallpox pathogenesis is

inferred from animal studies of mousepox, 25,26 rab-

bitpox, 26 and monkeypox 27,28 in their respective hosts,

and from vaccinia in humans. Studies using primates

infected with variola 29 corroborate these findings and

lend further insight into human smallpox and monkey-

pox infections. In both natural and experimental infec-

tions, the virus is introduced via the respiratory tract,

where it first seeds the mucous membranes, including

membranes of the eye, and then passes into local lymph

nodes. The first round of replication occurs in the lymph

nodes, followed by a transient viremia, which seeds tis-

sues, especially those of the reticuloendothelial system,

219

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