Chapter 3: Plus-Strand RNA Viruses - Introduction - Family Picornaviridae

Plus-Strand RNA Viruses

the relationships of these viruses to the plant viruses and what

INTRODUCTION

this means in terms of virus evolution.

The plus-strand RNA [(+)RNA] viruses comprise a very

large group of viruses belonging to many families. Among

FAMILY PICORNAVIRIDAE

these are viruses that cause epidemic disease in humans,

including encephalitis, hepatitis, polyarthritis, yellow fever,

The picornaviruses are so named because they are small

dengue fever, poliomyelitis, and the common cold. The

(pico = small), RNA-containing viruses. Nine genera of

number of cases of human disease caused by these viruses

picornaviruses, five of which contain human pathogens, are

each year is enormous. As examples, dengue viruses infect an

currently recognized (Table 3.2), and more will probably

estimated 50 to 100 million people each year; most humans

be recognized as further studies of the known viruses occur

suffer at least one rhinovirus-induced cold each year, with the

and as new viruses are described. A dendrogram that illus-

cases therefore numbering in the billions; and most humans

trates the relationship of the nine genera to one another,

during their lifetime will suffer several episodes of gastro-

as well as the relationships of a number of viruses within

enteritis caused by astroviruses or caliciviruses. In terms of

the various genera, is shown in Fig. 3.1. This dendrogram

frequency and severity of illness, the (+)RNA viruses con-

makes clear that all picornaviruses are closely related.

tain many serious human pathogens, and we will begin our

They share significant nucleotide and amino acid sequence

description of viruses with this group.

identity and form a well-defined taxon. The dendrogram

The human (+)RNA viruses belong to seven families

also illustrates the rationale for grouping these viruses into

(Table 3.1). These seven families also contain numerous

nine genera.

viruses that infect other vertebrates, of which many are

As described in Chapter 2, the structures of several picor-

important pathogens of domestic animals. Large numbers of

naviruses have been solved to atomic resolution by X-ray

(+)RNA viruses that infect plants are also known; in fact, most

crystallography. The picornavirus virion is composed of 60

plant viruses contain (+)RNA genomes. The plant viruses,

copies of each of four different proteins (called VP14) that

however, belong to different families and are currently classi-

form an icosahedral shell having T=3 symmetry (or pseudo-

fied by the International Committee on Taxonomy of Viruses

T=3) and a diameter of approximately 30 nm (see Figs. 2.1,

(ICTV) into nine families plus many unassigned genera.

2.5, 2.7, and 2.8).

Because of their importance as disease agents of domestic

crops, much is known about these viruses. Other families of

(+)RNA viruses include two families of bacterial viruses,

Organization and Expression of the Genome

one of fungal viruses, and four families of insect viruses (the

The structure of the genome of poliovirus and comparison

nodaviruses, in particular, have been intensively studied).

of it with the genomes of the other genera are shown in Fig.

Thus, the (+)RNA viruses have evolved into many distinctly

3.2. The picornaviral genome is a single RNA molecule of

different families and must have arisen long ago. In this chap-

about 7.5 kb. It contains one open reading frame (ORF) and

ter, the seven families of viruses that include human viruses

is translated into one long polyprotein. This polyprotein is

as members are considered, followed by a brief discussion of

TABLE 3.1

Families of Plus-Strand RNA Viruses That Contain Human Pathogens

Family

Size of genome (nucleotides)

Other vertebrate hosts

Representative human pathogens

∼7500

Picornaviridae

Cattle, primates, mice

Poliovirus

Human rhinovirus

Hepatitis A

∼7500

Caliciviridae

Rabbits, swine, cats

Norwalk

Hepeviridae

7200

Primates, swine

Hepatitis E

Astroviridae

68007900

Cattle, ducks, sheep, swine

Human astrovirus

∼11,600

Togaviridae

Mammals, birds, horses

Semliki Forest, Ross River, WEE, VEE, EEE,

Mayaro, rubella

Flaviviridae

950012,500

Swine, cattle, primates, birds

Dengue, yellow fever, JE, MVE, TBE, WNV,

hepatitis C

Coronaviridae

20,00030,000

Mice, birds, swine, cattle, bats

SARS coronavirus

Virus name abbreviations: WEE, VEE, EEE, Western, Venezuelan, Eastern equine encephalitis viruses; JE, Japanese encephalitis virus; MVE, Murray Valley

encephalitis virus; TBE, tick-borne encephalitis virus; WNV, West Nile virus; SARS, severe acute respiratory syndrome.

TABLE 3.2 Picornaviridae

Virus name abbreviationa

Genus/members

Usual host(s)

Transmission

Disease

World distribution

Enterovirus

Human enterovirus A

HEV-A

Humans

Oralfecal, contact

See Table 3.5

Worldwide

Human enterovirus B

HEV-B

Humans

Oralfecal, contact

See Table 3.5

Worldwide

Human enterovirus C

HEV-C

Humans

Oralfecal, contact

See Table 3.5

Worldwide

Human enterovirus D

HEV-D

Humans

Oralfecal, contact

See Table 3.5

Worldwide

Poliovirus (Types 1,

Humans

Oralfecal, contact

See Table 3.5

Originally worldwide,

2, and 3)

extirpated in Americas

Bovine enterovirus, porcine enteroviruses A and B; Unassigned enteroviruses of humans and monkeys

Rhinovirus

Human rhinoviruses

HRV-A, HRV-B

Humans

Aerosols, contact

Common cold

Worldwide

(>100 serotypes)

Cardiovirus

Encephalomyocarditis

EMCV

Mice

Oralfecal, contact

Encephalitis,

Worldwide

Theilovirus

myocarditis

Aphthovirus

Foot and mouth disease

FMDV

Cattle, swine

Oralfecal, contact

Lesions on

Worldwide (except

mouth and feet

United States,

Australia)

Equine rhinitis A

ERAV

Horses

Worldwide

Hepatovirus

Hepatitis A

HAV

Humans

Oralfecal

Hepatitis

Endemic worldwide

Parechovirus

Human parechovirus

HPeV

Humans

Oralfecal

Gastroenteritis

Worldwide

Erbovirus

Equine rhinitis B

ERBV

Horses

Kobuvirus

Aichi

AiV

Humans

Oralfecal

Gastroenteritis

Isolated in Japan (oysters)

Teschovirus

Porcine teschoviruses

PTV-1

Swine

Oralfecal

Paralysis, porcine

Britain, central and

(10 species recognized)

encephalomyelitis Eastern Europe

Standard abbreviations are given for either the virus listed (such as poliovirus) or for the type member of the genus.

Hepatovirus

Hepatitis A

Parechovirus

Human parechovirus 1

Human enterovirus B

Human enterovirus D

Human enterovirus C

Enterovirus

Poliovirus type 1

Bovine enterovirus 1

Porcine enterovirus 1

Human enterovirus A

Human rhinovirus 14

Rhinovirus

Human rhinovirus 2

Kobuvirus

Aichi

Equine rhinitis A

Aphthovirus

Foot and mouth disease

Encephalomyocarditis

Cardiovirus

Theilovirus

Erbovirus

Equine rhinitis B

Teschovirus

Porcine teschovirus

Evolutionary Distance

0.5

FIGURE 3.1 Relationships between 18 representative picornaviruses. The viruses shown have been classified into the 9

recognized genera. This tree was generated from the amino acid sequences of the 3Dpol proteins. Adapted from Yamashita et

al. (2003), and updated with the taxonomy found in Fauquet et al. (2005). Evolutionary distance, calculated by the UPGMA

(unweighted pair group method with averages) method, is the number of residue substitutions that have occurred between

two sequences since their divergence from a common ancestor and is defined as D = number of base mismatches/total

alignment length in nucleotides.

cleaved by one or more virus-encoded proteinases to form

of RNA synthesis. VPg is normally removed from RNA that

more than 25 different polypeptides, including processing

serves as mRNA by a cellular enzyme, but its removal is not

required for its translation. The 3′ end of the RNA is poly-

intermediates (not all of which are shown in the figure) as well

adenylated. As described in Chapter 1, the 5′ nontranslated

as final cleavage products. The ORF in the genome contains

three regions, called P1 (the 5′ region), P2 (the middle region),

region of a picornaviral RNA possesses an IRES (internal

and P3 (the 3′ region). Region 1 encodes the structural proteins

ribosome entry site) and the RNA is translated by a cap-inde-

and regions 2 and 3 encode proteins required for RNA replica-

pendent mechanism. The translation of picornaviral RNA is

tion. The genome organization of all picornaviruses is similar,

greatly favored in the infected cell because picornaviruses

but each genus differs in important details. For example, the

interfere with host cell macromolecular synthesis and, in

aphthoviruses and the cardioviruses have a poly(C) tract near

particular, interfere with host protein synthesis. Infection

the 5′ end of the RNA that is important for virus replication.

with entero-, rhino-, and aphthoviruses leads to proteolytic

These two genera also have a leader polypeptide that precedes

cleavage of a cellular protein called eIF4G that is a compo-

the structural protein region. The aphthovirus leader peptide is

nent of the cap-binding complex. Cleavage of this protein by

2Apro of entero- and rhinoviruses or by the leader protease

a papain-like protease that cleaves itself from the polyprotein

and has a role in the shutoff of cellular protein synthesis. The

of aphthoviruses results in inhibition of the translation of

function of the cardiovirus leader is not known. Hepatitis A

RNAs that require the cap-binding protein complex, that is,

virus, Aichi virus, and echovirus 22, representatives of three

capped host cell mRNAs. The cardioviruses, which are also

other genera, also have leaders.

cap independent, interfere with translation of host mRNAs

The picornaviral genome has a small protein, VPg, cova-

in a different way, by interfering with phosphorylation of

lently bound to the 5′ end, which is the primer for initiation

cap-binding protein. Poliovirus also interferes with host

protein synthesis by cleavage of poly(A)-binding protein by

illustrated for poliovirus in Fig. 3.2. Some cleavages occur in

the viral 3Cpro, but the mechanism by which this interference

cis and some in trans. The crystal structures of 3Cpro of polio-

operates on cellular protein synthesis and not viral protein

virus and of a rhinovirus have been solved to atomic resolu-

synthesis, since poliovirus RNA is also polyadenylated, is

tion, and their core structure resembles that of chymotrypsin

not yet clear. In addition, 3Cpro of apthoviruses cleaves ini-

(Fig. 1.19A). The catalytic center has the same geometry as

that of chymotrypsin, but in 3Cpro the catalytic serine has

tiation factors eIF4A and eIF4GI, and it is thought that these

cleavages lead to a decrease in the level of viral protein syn-

been replaced by cysteine. Moreover, in many, but not all,

thesis later in infection, which facilitates packaging of the

picornaviruses the aspartic acid in the catalytic triad has been

replaced by glutamic acid. Thus, 3Cpro is related to cellular

viral RNA.

The viral 3Cpro and its precursor 3CDpro make multiple

serine proteases and may have originated by the capture of a

cleavages in the polyprotein translated from the genome, as

cellular serine protease during the evolution of the viruses.

2Apro

3Cpro

P2A-3AB

P3CD

2Apro

pro

CDpro

3C pro

2Apro

VP3

VP1

VP0

3Cpro

VPg

VP4

VP2

3Dpol

1 2A 2B

3A 3C

Entero- and Rhinovirus

L 4

1 2A 2B

3A 3C 3D

Hepatovirus

Cardiovirus

4 2

2A 2B

3A 3C

3B 3B

Aphthovirus

3C 3D

VP0

2C 3A 3C

Kobuvirus

3A 3B

Parechovirus

1 2A 2B 2C

VP0

Viral Proteases

Replicase Motifs

Coding Domains

Cleavages Enzymes

3Cpro

Nonstructural proteins

Polymerase (GDD)

3CDpro

Structural proteins

2Apro

Helicase

Poly(C) tract

Vpg

FIGURE 3.2 Genome organization of the Picornaviridae. (A) Genome organization of poliovirus showing the proteolytic

processing steps. Both the 3Cpro and 2Apro proteases are "serine-type proteases" with cysteine in the catalytic site. (B)

Comparative genome organizations of representatives of seven of the nine genera of Picornaviridae. The key to the different

shadings of coding domains and the symbols for various enzymatic motifs used in both (A) and (B) is given below. Adapted

from Murphy et al. (1995) p. 300 and Yamashita et al. (1998).

that this protease is unrelated to 2Apro of polioviruses, how-

Protease 3Cpro is present in all picornaviruses whereas pro-

ever. Thus, these two proteases in closely related

tease 2Apro is present in only a subset of picornaviruses. In

viruses have different origins, and the viruses have solved

poliovirus, 2Apro, like 3Cpro, is a serine protease in which the

the prob- lem of how to separate regions 1 and 2 in the

active site serine has been replaced by cysteine. 2Apro cata-

polyprotein in different ways. Furthermore, this finding

lyzes one essential cleavage in the polyprotein of poliovirus,

illustrates that recombination to introduce new functions into

that between P1 and P2. This cleavage occurs in cis. The pro-

viral genomes has been important in the evolution of these

teolytic activity of 2Apro is also required for other functions

viruses, a theme to which we will refer many times in this

topic. Still another solution to the problem of separating

during poliovirus replication, the nature of which have not

been established. An interesting experiment is illustrated in

regions 1 and 2 has been adopted by the cardio- and

Fig. 3.3 because it illustrates the power of molecular genetics

aphthoviruses. Protein 2A is not a protease in these viruses.

and the tricks that modern virologists can play with viruses.

Indeed, 2A is only 18 resi- dues long, and cleavage between

This experiment will serve as a prelude to the discussion of

P1 and P2 is catalyzed by 3Cpro. Another interesting feature

the uses of viruses as vectors in Chapter 11. A poliovirus was

of these viruses is that the cleavage at the 2A2B junction

constructed in which a stop codon was placed after the struc-

occurs spontaneously during translation, catalyzed by the

tural protein domain (region 1), so that 2Apro was not needed

specific amino acid sequence at the scissile bond. This

to remove P1 from the polyprotein precursor. The stop codon

cotranslational cleavage occurs only during translation on

was followed by an IRES and a new AUG start codon, so that

eukaryotic ribosomes, and it has been proposed that no

P2A and the rest of the genome could be translated from the

cleavage actually occurs, but that the 2A sequence

polycistronic RNA. This virus was viable. However, when

synthesis of the specific peptide bond at

prevents

the 2A proteinase was inactivated by changing the active site

the 2A2B junction.

In addition to these cleavages catalyzed by 2Apro, 3Cpro,

cysteine, the resulting virus was dead, showing that the prote-

olytic activity of P2A is required not only to separate regions

and the leader protease of aphthoviruses, VP0 is cleaved

1 and 2 of the polyprotein but also for other function(s).

during virion maturation to VP2 and VP4 in most, but not

Protein 2A of rhinoviruses is also a protease. The crystal

all, picornaviruses. Available evidence suggests that this

structure of protein 2A of human rhinovirus type 2 reveals

cleavage is not catalyzed by a protease.

Construct

2A Protease

Viable

Activity

virus

3 NTR

IRES

pT7PVXL2

++++

Large plaque

1 2A 2B

3A 3C

(wt)

EMCV

3 NTR

IRES

Not

Small plaque

4 2

2A 2B

3A 3C 3D

pT7PVE2A

applicable

AUG

EMCV

3 NTR

IRES

pT7PVE2AX

None

Dead

2A 2B

3A 3C

AUG

C109A

FIGURE 3.3 Diagramatic illustration of constructs used to unravel the functions of protein 2A in poliovirus replication.

cDNA copies of the virus RNA can be manipulated by genetic engineering to insert IRES elements or make specific

mutations. RNA can be transcribed from the clones in vitro and used to infect cells, which is possible for plus-strand RNA

viruses because the first event after infection is translation of the genomic RNA. The wild-type construct pT7PVXL2 is

shown in the top line. The 2A proteolytic activity normally cleaves the bond between domains P1 and P2 of the translated

polyprotein. If this function is rendered nonessential, as in construct pT7PVE2A, by the insertion of a stop codon at the

C terminus of P1 (solid diamond), followed by a second IRES and an initiation AUG at the beginning of 2A, virus is still

produced, but forms small plaques. Thus separation of the structural region and the nonstructural region in this way results

in viable virus. However, if the proteolytic activity of 2A is inactivated by mutation of the catalytic cysteine to alanine as in

pT7PVE2AX, no virus is produced, demonstrating that the proteolytic activity of 2Apro is necessary for other functions in

addition to the P1/P2 cleavage. The pink hexagon is the VPg encoded in 3B and linked to the 5′ end of the RNA. Adapted

from Lu et al. (1995) and Molla et al. (1993).

RNA viruses. The 3A protein has hydrophobic sequences

Functions of the Picor navirus Proteins

that may be involved in this association. During replication, a

The cleavage product P1 consists of a polyprotein pre-

full-length complementary copy of the genomic RNA is pro-

cursor for the four structural proteins of the virus, VP14.

duced that serves as a template for the synthesis of genomic

P1 is first cleaved in trans to VP0, VP1, and VP3 by 3CDpro

RNA (illustrated schematically in Fig. 1.11A). This com-

(Fig. 3.2A). VP0 is later cleaved to VP2 and VP4 during

plementary RNA template has been variously called minus-

assembly of most picornaviruses.

strand RNA [abbreviated (-)RNA], antigenomic RNA, or

The cleavage products of P2 and P3 are required for

virion-complementary (vc) RNA. Much more (+)RNA than

RNA replication. 2Apro has been described. Protein 2B from

(-)RNA is produced, since (+)RNA is needed for translation

a Coxsackie virus has been shown to induce the influx of

and encapsidation into progeny as well as for replication,

extracellular Ca2+ and the release of Ca2+ from stores in the

whereas (-)RNA is needed only as a template for making

endoplasmic reticulum, and it is proposed that this protein

(+)RNA. It is probable that disproportionate amounts of (+)

induces lesions in cellular membranes that allow release of

and (-) strands are synthesized because the promoters in the

progeny virions. 2CATPase has been shown to be an ATPase,

viral RNA recognized by the viral replication machinery for

not a GTPase, and contains sequence motifs characteristics

(+) and (-)RNA synthesis (which might also be called ori-

of helicases. Many, but not all, RNA viruses encode heli-

gins of replication) differ in their strength, but other mecha-

cases to unwind duplex RNA during replication, and it is

nisms are known to be used in at least some RNA viruses.

assumed that 2CATPase performs such a function. The precur-

The RNA-dependent RNA polymerase 3Dpol is strictly

sor to 2B and to 2CATPase, a protein called 2BCATPase, has a dif-

primer dependent. In the presence of template, 3Dpol can uri-

ferent role in RNA replication. It is required for proliferation

dylate VPg on a specific tyrosine residue. This nucleotidyl

of membranous structures in poliovirus-infected cells that

peptide, VPgpU or VPgpUpU, then functions as a primer for

serve as sites for RNA replication.

the initiation of RNA synthesis. It is of interest that several

Region 3 encodes VPg, 3CDpro/3Cpro, and the viral RNA

viruses belonging to other families, such as hepatitis B virus

polymerase 3Dpol. Cleavages effected by 3Cpro are illustrated

(a virus that uses reverse transcription during the replica-

in Fig. 3.2. 3Cpro may also have a regulatory role in the virus

tion of its genome) and adenovirus (a DNA virus), have also

life cycle, because the cleavage intermediate 3CDpro, which

adopted the strategy of using a protein primer for initiation

is fairly long lived, has properties that differ from 3Cpro. One

of nucleic acid synthesis.

function of 3CDpro is to bind the viral RNA in conjunction

The nature and function of the promoters in the poliovirus

with 3AB, the precursor for VPg, or with a cellular protein,

genome that are involved in the initiation of RNA replica-

poly(C)-binding protein. Formation of a complex with the

tion are incompletely understood. One essential element has

viral RNA is essential for its replication, and differential

been called a cis-acting replication element, abbreviated cre,

cleavage of the 3C3D bond during the infection cycle may

or 3B-uridylation site, abbreviated bus. cre is a stem-loop

regulate replication. A strategy in which precursor proteins

structure that contains a motif in the loop, AAACA, that is

perform different functions than those performed by the final

conserved in all picornaviruses. This motif serves as a tem-

cleavage products, such as those illustrated by 2BCATPase and

plate for the uridylation of VPg described before, which is

3CDpro, allows the virus to optimize the coding capacity of

required for the initiation of RNA synthesis. The description

its small genome, because a given sequence is used for more

of this element as cis acting is a misnomer because the ele-

than one function.

ment can act in trans, and there is a pool of VPgpUpU within

poliovirus-infected cells that can be used to initiate RNA

synthesis. The cre element is found in different locations in

Replication of Picor naviruses

different picornaviruses. In poliovirus it is found in the cod-

The replication of poliovirus has been particularly well

ing sequence for 2C, in rhinovirus in the coding region for

VP1, and in FMDV it is in the 5′ NTR. Furthermore, the ele-

studied and the virus has served as a model for the replica-

tion of eukaryotic RNA viruses. All nonstructural poliovi-

ment can be moved to other regions within a viral genome

rus proteins, including cleavage intermediates, have been

and still function normally.

purified and studied for their possible function as enzymes

A second sequence element required for RNA replica-

or RNA-binding proteins. These studies have been comple-

tion, present in polioviruses and rhinoviruses if not in all

picornaviruses, is located within the 5′-terminal NTR. This

mented by studies of replication complexes isolated from

infected cells, studies using replicons in which the luci-

element forms a cloverleaf that binds protein complexes

ferase gene replaces the P1 coding region (see, for example,

containing 3CDpro.

Fig. 3.3 and Chapter 11), and studies of processes that

In addition to the various viral proteins just described, a

occur in infected cells.

number of cellular proteins are also required for viral RNA

Replication of poliovirus RNA is associated with cellular

replication. In fact, cellular proteins appear to be required

membranes, as appears to be true of all eukaryotic plus-strand

for replication of all (+)RNA virus RNAs, but the identity

of these proteins and their function in viral RNA replica-

suggested that the virus may be a natural pathogen of these

tion is only poorly understood. One such protein in the

monkeys but it is unlikely that nonhuman primates con-

case of poliovirus is a cellular protein called heterogeneous

stitute a reservoir for it, which is important in relation to

nuclear ribonucleoprotein C1 (hnRNP C1), which interacts

efforts spearheaded by the World Health Organization to

with RNA synthesis initiation complexes and appears to be

eradicate poliovirus globally.

required for the initiation of positive-strand RNA. A sec-

The classification of human enteroviruses has recently

ond protein is the poly(A)-binding protein. Efficient repli-

undergone extensive revision, based upon the wealth

cation of polio RNA requires a poly(A) tract at the 3′ end

of sequence information that is increasingly available.

of the RNA that is at least 20 residues in length, and it is

Previously, classification was based upon the symptomology

believed that the poly(A)-binding protein binds this poly(A)

of disease caused or upon the characteristics of the growth

tract and participates in the initiation of minus-strand RNA

of a virus in experimental animals or in cultured cells.

synthesis.

Poliovirus has been known for more than a century as the

It has been possible to achieve a complete replication

causative agent of epidemic poliomyelitis. It was first shown

cycle of poliovirus in an extract of uninfected HeLa cells.

to be a filterable virus in 1908. However, early experiments

RNA from poliovirus virions added to such an extract will

could only be conducted in monkeys, because the virus will

direct the synthesis of all the poliovirus proteins, and these

only infect primates. Thus, the amount of information that

in turn will replicate the input RNA and encapsidate the

could be obtained was limited, but such studies eventually

progeny genomes. This cell-free, de novo synthesizing sys-

showed that more than one poliovirus serotype existed. The

tem for poliovirus, is as yet unique in virology.

development of methods for the cultivation of viruses in cell

In cell culture, most picornaviruses complete their rep-

culture in the 1940s made it possible to screen human stool

lication cycle in about 6 hours. The infection is cytolytic,

samples in an effort to type poliovirus isolates, which was

and large quantities of virus are produced. An exception is

necessary if a vaccine was to be produced. Such screening

hepatitis A virus, which establishes chronic infections in cell

resulted not only in the identification of three serotypes of

culture and grows to very low titers.

poliovirus, but also in the discovery of many other entero-

viruses as well. The study of virology in the United States

owes much to the campaign to develop a vaccine against

Genus Enterovirus

poliomyelitis. This campaign generated a great deal of pub-

Enteroviruses replicate primarily in the enteric tract where

lic support, which led to funding through private as well as

they usually cause only mild disease. More serious entero-

governmental agencies, and the successful development of a

viral disease may develop after spread to other organs, such

vaccine reinforced this support.

as the central nervous system or the heart. Enteroviruses are

The first of these other enteroviruses to be found were two

normally contracted though ingestion of the virus, either

Coxsackie viruses, found by screening patients in Coxsackie,

in contaminated food or water or by exposure to the virus

New York, who were suffering from paralysis during a polio

through contacts with individuals that are excreting the

epidemic. Coxsackie viruses will infect mice and are clas-

virus. The epidemiology of poliovirus has been the most

sified into two subgroups, called A and B, which differ in

intensively studied among the enteroviruses. Poliovirus is

their biological properties in mice. They were simply given

present in oropharyngeal secretions early after infection and

serial numbers in the order of their isolation--23 Coxsackie

is excreted in feces over a period of weeks following infec-

A viruses and 6 Coxsackie B viruses are now recognized.

tion. The virus spreads readily and rapidly through house-

Another series of enteroviruses that were first identified in

holds, which demonstrates the importance of close contacts

these early studies were called echoviruses (enteric cyto-

in virus spread. The virus also has the ability to persist in the

pathic human orphan virus), because these viruses infected

external environment for weeks under favorable conditions,

the enteric tract of humans, caused cytopathology in cul-

and this may represent another source of infection during

tured cells, and were orphans, not known to cause disease.

epidemics. Sewage surveys, for example, have been used to

Echoviruses were distinguished from Coxsackie viruses by

follow poliovirus epidemics, and poliovirus has been found

their inability to infect suckling mice. Currently 29 echo-

in lakes and swimming pools.

viruses are recognized in the genus Enterovirus. The latest

In general, enteroviruses have a fairly narrow host

human viruses to be isolated are now simply called entero-

range. Most of the well-studied viruses are human viruses,

viruses and given serial numbers. The first four such viruses

because humans take a particular interest in the viruses

to be recognized were thus called human enterovirus 68, 69,

that cause them the most trouble, but enteroviruses of non-

70, and 71. Numbering started with 68 because at the time

human primates, pigs, cattle, and insects are known. The

there were thought to be 67 polio, Coxsackie, and echovi-

more than 65 known human enteroviruses, many of which

ruses. However, 5 of these (one Coxsackie A virus and 4

are important pathogens, normally infect only humans,

echoviruses) were subsequently found to be misidentified,

but poliovirus will infect Old World monkeys. It has been

and one (echovirus 22) is sufficiently distinct that it has been

renamed human parechovirus and classified into the genus

Polioviruses

Parechovirus (Table 3.2).

The best known of the enteroviruses are the three sero-

Thus, from such studies, a total of 65 human enterovi-

types of poliovirus. These viruses are the causative agents of

ruses was isolated and, as indicated, classified according to

poliomyelitis, a disease characterized by the death of motor

their biological properties. As the genomes of these vari-

neurons in the spinal cord. Most poliovirus infections of sus-

ous viruses were sequenced, it became apparent that these

ceptible humans are inapparent or result in a mild febrile ill-

viruses fell into five lineages or clades whose members are

ness in which cells of the pharynx and the gut are infected

closely related to one another. As described in Chapter 1,

and recovery is uncomplicated. However, a transient viremia

the definition of a virus species is somewhat arbitrary, but

is established following infection (viremia = virus present

the purpose of classification is to recognize evolutionary

in the blood), and in a small percentage (<2%) of infections

relationships, and 63 of these 65 human enteroviruses have

the virus invades the central nervous system (CNS), where it

now been reclassified into five species, called poliovirus and

infects motor neurons in the spinal cord and, in severe cases,

human enterovirus A, B, C, D. These assignments are shown

other regions of the CNS. The mechanism by which the virus

in Table 3.3, and the various members of a species are now

enters the CNS is still controversial. Current information sup-

considered serotypes. This table contains information on

ports the hypothesis that viremia allows the virus to enter by

serotypes accepted as of the 2005 ICTV report. More than

penetrating through the bloodbrain barrier, but entry via ret-

80 serotypes are now known and as new serotypes continue

rograde transport in axons that serve the periphery may also

to be identified and characterized, it is to be expected that

be involved. In any event, infection of the CNS can result in

this number will continue to grow. The extensive sequence

paralysis, which can be severe enough to be fatal because

data have also uncovered examples of recombination that

of paralysis of respiratory muscles. The name poliomyelitis

have occurred during the evolution of these viruses, both

comes from the Greek words polio = gray and myelo = spinal

within species and between species.

cord, from the pathology caused by damage to the motor neu-

Sequence information has also been used to identify

rons in the spinal cord, which are located in the gray matter.

one species of bovine enterovirus and two species of pig

Polioviruses readily undergo recombination with other

enteroviruses (Porcine Enterovirus A and B) in the genus

polioviruses and with at least some other enteroviruses. The

Enterovirus (Table 3.3). There are 2 serotypes assigned to

distinguishing feature of a poliovirus, what makes it a polio-

Simian enterovirus A and 17 other known monkey enterovi-

virus, is the structural protein module and not the nonstruc-

ruses have as yet to be classified into species. The monkey

tural protein module associated with the structural proteins.

viruses form a distinct clade related to porcine enterovirus 8

This has importance implications for vaccines that protect

(Porcine Enterovirus A) and will probably be classified into

against poliomyelitis, as described later.

one or two species.

TABLE 3.3 Current Taxonomy of the Genus Enterovirus

Species

Strains, subtypes, and serotypes

Human enterovirus A

Human Coxsackie viruses A28, 10, 12, 14, 16, human enterovirus 71, 76

Human enterovirus B

Human Coxsackie virus A9

Human Coxsackie viruses B16 (including swine vesicular disease virus)

Human echoviruses 17

21 other human echoviruses

Human enterovirus 69, 7378

Human enterovirus C

Human Coxsackie viruses A1, 11, 13, 15, 17, 1922, 24

Human enterovirus D

Human enteroviruses 68, 70

Poliovirus

Human poliovirus types 1, 2, and 3

Bovine enterovirus

Bovine enteroviruses 1, 2

Porcine enterovirus A

Porcine enterovirus 8

Porcine enterovirus B

Porcine enterovirus 9, 10

Simian enterovirus A

Simian enterovirus A1, A2-plaque

Unassigned viruses

17 simian enteroviruses

higher standards of hygiene led eventually to epidemics of

Epidemic Poliomyelitis

poliomyelitis, these standards also led to a reduction in dis-

Polioviruses appear to have been important pathogens

eases caused by numerous other infectious agents, both viral

of humans for a very long time. The depiction of a lame

and bacterial (see Fig. 1.1).

priest on an Egyptian stele that dates from 3500 years ago

suggests that poliovirus was present in ancient Egypt, and

Control of Epidemic Poliomyelitis

references to clubfoot in ancient Greek and Roman writings

Before it was controlled with vaccines, epidemic polio-

probably signifies that polio was present at these early times.

myelitis was greatly feared, and it is hard now for people

However, although it is very likely that poliovirus has been

to realize the extent of fear that the disease induced. It was

widespread in humans for thousands of years, there is no

not simply that the disease could be fatal, but the specter of

firm evidence for poliomyelitis in human populations until

the iron lung and the wheelchair hanging over teenagers or

about 200 years ago, when the virus appears to have been

young adults who were the most likely to contract the dis-

(or to have become) widespread. Serosurveys in the United

ease. Furthermore, the epidemics struck during the summer,

States in the 1930s and 1940s, before the introduction of the

during the summer breaks of schools or universities. Many

Salk and Sabin vaccines, indicated that 80100% of adults

human pathogenic viruses are known to prefer a season for

had been infected by poliovirus at some time in their lives.

attack on humans: influenza during the winter, measles in

Studies in other areas of the world, including studies of lame-

early spring, enteroviruses during the summer. It is thought

ness in populations, also suggest that, at least in the 1900s,

that this phenomenon relates to air temperature and humid-

the majority of the world's population had been infected

ity. For example, poliovirus infections are correlated with

with poliovirus.

humidity in the Americas and in Europe.

Paradoxically, even though poliovirus was surely wide-

In the United States, there were huge poliovirus epidem-

spread earlier, poliomyelitis epidemics of large proportions

ics every summer in the 1950s in which more than 50,000

evolved only during the twentieth century and they were con-

people, mostly children or adolescents, became ill. Of these

centrated at first in countries practicing the highest standards

cases, about 20,000 were paralytic and 20003000 people

of hygiene. This startling phenomenon has been explained

died (Fig. 3.4). Death was often the result of the paralysis of

as resulting from changes in human behavior. Originally,

the muscles required for breathing, and iron lungs were intro-

the highly infectious virus was contracted by infants shortly

duced for mechanical ventilation of poliomyelitis patients

after birth when they were still protected by maternal anti-

until their muscles recovered sufficiently that they could

bodies (see Chapter 10 for a discussion of maternal antibod-

breathe on their own. Wards containing dozens of patients in

ies). This natural infection served to immunize the infant,

iron lungs became a common sight in the large epidemics of

protecting it from poliomyelitis for life. However, when the

the 1950s (Fig. 3.5), and there were fears that larger wards

chain of immunization was interrupted upon removal of the

containing still more iron lungs would be required as the

virus from the environment by the development of hygienic

epidemics became more virulent. Of the survivors of polio-

conditions, unprotected children grew up, giving rise to sus-

myelitis, many were permanently paralyzed and confined to

ceptible populations. If the virus invades such populations,

wheelchairs or required the use of crutches for walking. One

epidemics rapidly evolve.

of the best known poliomyelitis cases is that of Franklin D.

Notice that this scenario requires that infants be infected

Roosevelt, who contracted poliovirus in 1921 at the age of

very early, while still protected by maternal antibodies.

39 and was in a wheelchair for the rest of his life, although

After these antibodies wane, the infant is susceptible to

he continued to lead an active political life.

poliomyelitis, although it has been thought that infection of

Introduction of the Salk and Sabin vaccines in the 1950s

susceptible but very young children is less likely to cause

and 1960s led to the elimination of poliovirus in the United

poliomyelitis. Statistics of the fraction of young children

States over a period of about 2 decades (Fig. 3.4) and more

who contract poliomyelitis in societies in which the virus

recently has led to the elimination of poliovirus throughout

is endemic, rather than epidemic, are not well defined, in

the Americas. The Salk vaccine, which was the first to be

part because of the high death rate of children in such socie-

developed, is an inactivated virus vaccine that is given as a

ties due to many infectious diseases. However, surveys con-

series of injections. Introduction of this vaccine resulted in a

ducted in the twentieth century of lameness in populations,

rapid decrease in the number of poliovirus cases. However,

most of which is probably due to paralytic polio, found simi-

because the vaccine induces circulating antibodies but little

lar extents of lameness whether the virus was endemic or

in the way of mucosal immunity (see Chapter 10), it prevents

epidemic.

poliomyelitis, the disease, by preventing spread of the virus

In any event, it is clear that changes in human behavior

from the gastrointestinal (GI) tract to the CNS, but not infec-

can bring about serious complications relating to infectious

tion of the GI tract by the virus. The virus thus remained in

disease, and such scenarios have recurred many times during

circulation. The Sabin vaccine, introduced shortly thereafter,

the last century. However, it is important to note that although

Cases of Poliomyelitis in the United States from 1951-2004

Salk Vaccine

(IPV)

100,000

Sabin Vaccine

(OPV)

10,000

Number of cases/year

1000

Vaccine-related cases

100

Sabin Vaccine

Discontinued

1950

1960

1970

1980

1990

2000

Year

FIGURE 3.4 Total number of cases of poliomyelitis in the United States from 1951 to 2004 and the number of vaccine-

related cases after the introduction of the live virus Sabin vaccine. IPV is inactivated polio vaccine; OPV is oral polio

vaccine. Data from N. Nathanson et al. (1996) p. 556 and from Morbidity and Mortality Weekly Report (MMWR). Note that

the scale is logarithmic, with each division portraying 10 times as many cases as the one below.

is a live attenuated vaccine that is given orally. Attenuation

(oral administration of relatively small doses of live virus

was achieved by blind passage of the virus followed by test-

versus injection of large amounts of inactivated virulent

ing of the resulting virus in monkeys. The changes resulting

virus), making it suitable for widespread use in developing

from passage are now known and are shown in Table 3.4;

countries. Worldwide use of this vaccine has resulted in the

two changes are sufficient to make the virus avirulent in the

eradication of wild-type poliovirus in the United States and

case of types 2 and 3. The introduction of the Sabin vaccine

throughout the Americas (the last case of indigenous polio-

led to a further rapid decline in paralytic poliomyelitis. This

virus infection in the Americas occurred in Peru in 1991).

vaccine has the drawback that it induces a very small number

Poliovirus is in the process of being eradicated in other parts

of cases of paralytic disease, termed vaccine associated para-

of the world, although it is still endemic in areas of Africa

lytic poliomyelitis (VAPP), that result from reversion of the

and Asia. With the extirpation of polio in the United States,

attenuated virus to virulence. The incidence rate is about 1

the use of Sabin vaccine was discontinued in this country

per million persons inoculated, and there were about 10 such

and it has been replaced with the Salk vaccine, in order to

cases per year in the United States until use of this vaccine

eliminate VAPP.

was discontinued in the year 2000 (Fig. 3.4). The efficacy of

As described before, polioviruses undergo recombina-

the Sabin vaccine is very high, however, because it induces

tion with other enteroviruses. It is important, therefore, to

mucosal immunity as well as other forms of immunity. It

note that the mutations in the Sabin vaccines that render the

prevents subsequent infection by the wild-type virus, thus

virus attenuated are all found in the structural region of the

allowing eradication of the wild-type virus if coverage is

genome (Fig. 3.6). Thus, recombination with other entero-

sufficiently broad. In addition, it is much cheaper and sim-

viruses cannot restore the virulence of the virus. Reversion

pler to manufacture and administer than the Salk vaccine

to virulence requires the back mutation of the attenuating

FIGURE 3.5 Ward of iron lungs and rocking beds at the poliomyelitis rehabilitation center in Rancho Los Amigos,

California. From Halstead (1998) with the permission of the author and the publisher.

mutations in the vaccine viruses or, conceivably, recombina-

and the current difficulties in complete eradication of polio

tion between two attenuated poliovirus strains that eliminates

have important lessons for us. The original Salk vaccine was

the attenuating mutations. In the latter case, however, it is

incompletely inactivated because the science of virology

unlikely that most recombinants would be virulent because

was insufficiently developed to assay for minute amounts of

of the incompatibility of the various nonstructural proteins

residual live virus in solutions containing very high concen-

with one another.

trations of virus. The result was that this vaccine caused a

small number of cases of poliomyelitis, but in retrospect the

Development of the Polio Vaccine

riskreward ratio was favorable because of the significant

The polio vaccine has been enormously successful in con-

decline in natural infections (Fig. 3.4). As soon as the prob-

trolling this virus scourge, but the history of its development

lem was recognized, more stringent methods of inactivation

TABLE 3.4

Characteristics of Poliovirus Vaccines

Salk vaccine

Inactivated wild-type poliovirus (three types)

Sabin vaccine

Live poliovirus, attenuated by mutations in:

Type 1

Type 2

Type 3

5′NTR

A480G

G481A

C472U

VP1

G2795A

A106T

C2909U

T143I

U2 493C

I6T

C2879U

L134F

VP3

U2438A

L225M

C2034U

S91F

VP4

G935U

A65S

5 NTR

AAAn

VP2

VP3

VP1

A106T

SABIN 1 A65S

L225M

L134F

U2438A C2879U

A480G G935U

G2795A

T143I

SABIN 2

G481A

C2909U

I6T

SABIN 3

S91F

C472U

C2034U

U2493C

FIGURE 3.6 Diagrams of the genomes of the Sabin vaccine strains of poliovirus types 1, 2, and 3 showing the locations

of the attenuating mutations.

were quickly developed that resulted in complete inac-

Health Organization (WHO) initiated a campaign, the

tivation of the infectivity of the virus, solving this prob-

Global Polio Eradication Initiative, to eradicate poliovi-

lem and serving as an example for development of other

rus worldwide by the year 2000. Although falling short of

vaccines. The introduction of the Salk vaccine, although

this goal, significant progress has been made. The number

enormously successful in controlling polio, also suffered

of polio cases worldwide fell from an estimated 300,000+

from early problems when it was found that early lots were

cases in the mid 1980s to fewer than 3000 by 2000 and sub-

contaminated with the monkey virus SV40 (described in

sequently to still lower levels (Fig. 3.7). In 2000 the Centers

Chapter 7). Infection of humans by this virus appears to be

for Disease Control and Prevention (CDC) said that 2971

benign, although there is some evidence that, very rarely,

cases were reported of which 719 were confirmed by labo-

brain tumors may be associated with infection. In any event,

ratory analysis. In 2001, 537 cases were reported of which

this episode brought to light the issue of adventitious con-

473 were confirmed by laboratory analysis, and these cases

tamination of cell cultures with viruses that infect the host

occurred in just 10 countries. It appeared that eradication

supplying tissues for culture. A third problem that arose

would be achieved soon. The eradication campaign hit a

during development of polio vaccines was the infection of

snag recently, however, when Muslim clerics in Nigeria

a number of laboratory workers in Germany with Marburg

claimed that the vaccine could cause AIDS or infertility.

virus. These workers were employed in the isolation of

In 2003, officials in some parts of Nigeria suspended local

cells from the kidneys of wild-caught monkeys that were

vaccination programs, and an epidemic of poliomyelitis in

to be used in propagating polioviruses, and some of the

Nigeria resulted that then spread to neighboring countries

monkeys were infected with Marburg virus, at that time an

that had been free of polio (Fig. 3.8). By 2005 polio had

unknown virus. Several people died in the ensuing epidemic

spread to a total of 16 countries that had previously been

(described in Chapter 4).

polio free. Further setbacks in the polio vaccination ini-

tiative have resulted from civil unrest in Sudan and other

Eradication of Polioviruses

countries that resulted in interference with vaccine cam-

Introduction of the Sabin vaccine led to the eradication

paigns and the reestablishment of poliovirus transmission.

of poliovirus from the Americas, and in 1988 the World

Health ministers from Africa are stepping up vaccination

Tunisia

Morocco

Algeria

Western Sahara

Libya

Egypt

Mauritania

Mali

Niger

Chad

Sudan

Senegal

Gambia

Burkina Faso

Guinea Bissau

Guinea

Nigeria

Ethiopia

Sierra Leone

Ivory Coast

Ghana

Liberia

Cameroon

Somalia

Benin

Togo

Equatorial

Uganda

Guinea

Kenya

Congo

Gabon

Rwanda

Zaire

Burundi

Cabinda

Tanzania

Countries with cases of poliomyelitis

Malawi

Angola

2002

Zambia

Mozambique

2003

Zimbabwe

Caprivi Strip

2004

Namibia

Botswana

Swaziland

South Africa

Lesotho

FIGURE 3.8 Reemergence of poliomyelitis in Africa 20022004. Data from MMWR.

programs to reestablish control of poliovirus transmission,

in nonimmunized or incompletely immunized contacts

but these situations illustrate problems that result from the

(e.g., 21 cases in Hispaniola in 20002001), illustrating the

continuing conflicts among societies.

potential for continued outbreaks arising from vaccination.

The Salk vaccine could still be used in developed countries,

Should Routine Poliovirus Immunization Be Eliminated?

but it seems unlikely that routine administration of Salk

If poliovirus is finally eradicated worldwide, should

vaccine would be used in developing countries. It should

vaccination against the virus be scaled back? Limiting vac-

be possible to design new attenuated viruses for vaccine

cination would be important because of the residual viru-

purposes that would be safer than the Sabin vaccine, but

lence of the vaccine virus. Virulent revertants of vaccine

in the absence of poliovirus epidemics no drug company

virus not only cause a small number of individual cases

would want to undertake the very expensive development

(Fig. 3.4), but have led to small epidemics of poliomyeli-

of a new vaccine, especially in view of legal problems that

tis when the virulent virus, derived by reversion, circulates

would be sure to arise.

Associated problems are the difficulties in being sure that

infection of mice, it has been found that the virus can

poliovirus is truly eradicated and the possibility that virulent

persist in neurons in a latent state for at least 12 years.

poliovirus could reemerge. It is known that immunodeficient

There is no evidence that poliovirus might similarly per-

children who have received the vaccine virus can continue

sist in humans for 40 years, however, and such persis-

to secrete virus for long periods of time, during which the

tence seems unlikely. Other possible explanations for the

virus may revert to virulence. Even wild-type poliovirus can

failure of motor neurons in post-polio syndrome have also

circulate silently because most infections are inapparent. In

been suggested. Fortunately, paralytic poliomyelitis and

regions where poliomyelitis has reemerged following inter-

its sequelae may soon be a thing of the past.

ruption of vaccination campaigns, it has been found in some

cases that the virus had been circulating for at least 2 years

before reemergence. In addition, poliomyelitis can be caused

Other Enteroviral Diseases of Humans

by enteroviruses other than poliovirus (see later), a possible

As described, 62 human enteroviruses other than polio-

source of confusion in diagnosis.

viruses are currently recognized in the ICTV catalog and

If vaccination were terminated and poliovirus were to

classified as serotypes of 4 different species. Although most

reemerge in a naïve population, it could lead to a widespread

of these have been known for 50 years, it is only recently

epidemic. Possible sources of reemerging virus include new

that the association of many of these viruses with signifi-

strains that might arise from other enteroviruses, circulating

cant human illness has been shown. In fact, it has now been

strains of wild poliovirus or vaccine-derived virus that have

established that most enteroviruses do cause disease, and

escaped detection, or the inadvertent or deliberate release of

many of them cause significant episodes of serious disease

wild poliovirus by escape from a laboratory or introduction

(Table 3.5). Study of disease caused by these viruses has

by terrorists. It would be necessary to maintain stockpiles

been complicated by the fact that there are so many entero-

of polio vaccine to counter such threats, and in the case of

viruses, of which at least some have multiple strains that

the Salk vaccine this stockpiling itself could serve as a pos-

may differ in disease-causing potential, and by the fact that

sible source of accidental escape of the wild virus. Since

serious disease is an uncommon complication of infection

the events of 9/11, it is also obvious that terrorists would

by most enteroviruses (even for poliovirus most infections

have no compunctions about releasing a virulent virus into

do not result in significant disease). This has made it dif-

the U.S. population if such a virus could be obtained. Thus,

ficult to ascribe any particular disease to infection by any

even if (when?) poliovirus is eradicated, the policies with

particular virus. However, even though serious disease is

respect to vaccination will require careful consideration.

an uncommon complication, enteroviral infections are very

Post-Polio Syndrome

common, and the total number of cases of disease caused by

these viruses is large. These illnesses include very infrequent

Although poliovirus has been eradicated from devel-

paralytic disease essentially indistinguishable clinically

oped countries, there is a large cohort of people infected

from that caused by poliovirus; myocarditis and pericarditis

in the 1950s who are or were paralyzed. Many paralyzed

(caused especially by the Coxsackie B viruses) that is usu-

poliomyelitis patients were ultimately able to resume

ally subclinical but can be acute and result in significant car-

almost normal activities. Through a process of axonal

diac compromise; aseptic meningitis; encephalitis; hepatitis;

sprouting and reenervation of muscles by the motor neu-

the common cold (perhaps a quarter of summer colds are due

rons that survived the infection, many learned to walk and

to enteroviruses); diarrheal disease; febrile illnesses; rash;

use their previously paralyzed limbs. In many, recovery

hand-foot-and-mouth disease (a common childhood illness

was effectively complete. However, a syndrome called

caused by several serotypes in human enterovirus A); and

post-polio syndrome has emerged to plague a significant

epidemic acute hemorrhagic conjunctivitis (an epidemic

fraction, perhaps 40%, of the survivors of paralytic polio-

disease caused by enterovirus 70 that appeared recently and

myelitis. This syndrome appears 3040 years after polio

spread around the world). The Coxsackie B viruses are also

infection and is characterized by fatigue, pain, and weak-

associated epidemiologically with juvenile onset diabetes in

ness. The weakness may be severe enough to require the

humans but how (or even whether) they cause diabetes is still

use of a wheelchair. The syndrome results from the degen-

unresolved. There are no vaccines for any of these viruses.

eration of motor neurons, but the reasons for the degenera-

tion are not clear. The favored hypothesis is that it is the

result of overuse of the surviving motor neurons, which

are forced to do the work of many. A second possibility is

Genus Rhinovirus

that the surviving neurons were damaged by the original

poliovirus infection and fail prematurely. A third, albeit

The human rhinoviruses are the causative agents of about

unlikely, possibility is that poliovirus persists in neu-

half of human colds, the most characteristic symptom of

rons and is somehow reactivated, even in the presence of

which is rhinitis (inflammation of the nasal mucous mem-

anti-polio antibody. In model studies using Sindbis virus

brane and characterized by a runny nose). Other viruses that

TABLE 3.5

Clinical Syndromes Associated with Human Enteroviruses

Clinical syndrome

Poliovirus

Enterovirus A

Enterovirus B

Enterovirus C

Enterovirus D

Paralysis

Types 1, 2, 3

Coxsackie A7, A9

Coxsackie B2B5

Enterovirus 70

Enterovirus 71

Echoviruses 4, 6, 9, 11, 30

Aseptic meningitis

Coxsackie A2, A4, A7,

Coxsackie B1B6

A9, A10

All echoviruses except

12, 24, 26, 29, 32, 33

Pericarditis, myocarditis

Coxsackie B1B5

Echoviruses 1, 6, 9, 19

Encephalitis

Enterovirus 71

Coxsackie B1B5

Enterovirus 70

Echoviruses 2, 6, 9, 19

Hepatitis

Coxsackie A4

Coxsackie A9, B5

Echovirus 4, 9

Upper respiratory

Coxsackie B4, B5

Coxsackie A21, A24

Enterovirus 68

disease, pneumonia

Hand, foot, and

Enterovirus 71

mouth disease

Coxsackie A5, A10, A16

Acute hemorrhagic

Coxsackie A24

Enterovirus 70

conjunctivitis

Undifferentiated

Types 1, 2, 3

Coxsackie B1B6

febrile illness

serve as major causes of the common cold include some

years have not been done to establish whether immunity

of the enteroviruses, just described, and the coronavi-

to a particular rhinovirus following infection is long lived.

ruses, described later. One hundred serotypes of human

For the same reasons, there are no vaccines for any of these

rhinoviruses are currently recognized. Eighteen of these

viruses.

have been assigned to the species human rhinovirus A

Rhinoviruses replicate in the upper respiratory tract and

and three of them to human rhinovirus B. The remain-

are transmitted by direct person-to-person contact. Coughing

ing 79 serotypes have not yet been assigned to a spe-

and sneezing, common syndromes of rhinovirus infection,

cies. There are also three serotypes of bovine rhinovirus

help spread the virus to nearby contacts. It is not clear how

known to exist, and there are rhinoviruses for other ani-

much of the spread is due to aerosolization of the virus on

mals that have as yet to be well characterized. In general,

coughing or sneezing followed by inhalation of the aero-

rhinoviruses are specific for a particular species or for

solized virus by a susceptible contact, and how much is due

a limited range of species, and this restriction appears

to contact with mucus that contains virus, such as by hand-

to work at the level of receptors required for virus entry

shake or contact with contaminated doorknobs, followed by

(see Chapter 1).

transmission of the virus to mucosal membranes in the nose

The 100 serotypes of human rhinoviruses are not cross

or the mouth.

protective and the result is that we are subject to many

It is an interesting and informative historical fact that

rhinovirus colds during our lifetimes. Young children, not

early attempts to isolate rhinoviruses using standard cell cul-

having been exposed to rhinoviruses and other viruses

ture techniques were unsuccessful. Most cells in the body

that cause colds, contract many colds a year. Adults, hav-

are maintained at 37°C at a pH of 7.4, and cells in culture are

ing become immune to many of these viruses through

normally maintained under these conditions. However, cells

hard experience, have fewer colds per year, usually only

in the upper respiratory tract are maintained at a lower tem-

about one. However, the extent and duration of immunity

perature, about 33°C, because the inhalation of outside air

to a particular rhinovirus induced by infection are not well

through the upper respiratory tract keeps this area cool, and

established. There are so many rhinoviruses (and although

at a pH significantly less than 7.4 because of the high con-

rhinoviral disease may be miserable it is not life threaten-

centration of CO2 in expired air. Rhinoviruses replicate well

ing) that detailed studies on cohorts of people over many

in cultured cells under these altered conditions and appear

to require the lower temperature and lower pH for efficient

the viruses are unrelated. For reference, Table 3.6 presents

growth. In part because of this, rhinovirus infection is limited

a description of the currently known viruses whose name

to the upper respiratory tract, and rhinoviruses almost never

includes hepatitis. Figure 3.9 shows the incidence of hepati-

cause lower respiratory tract infections.

tis in the United States in 1997 caused by hepatitis viruses A,

It is also of interest that rhinoviruses are sensitive to very

B, and C, which are the most important causes of viral hepa-

low pH, and infectivity is destroyed by exposure to pH 3.

titis in the United States. Identification of which hepatitis

The related polioviruses, however, survive exposure to pH

virus is responsible for any specific case of hepatitis requires

2, which is necessary because, being enteroviruses, they

immunologic tests or virus isolation, because symptoms are

must survive passage through the stomach in order to infect

similar.

an animal.

Hepatitis A virus is a picornavirus and will be considered

here. The other viruses will be considered when their respec-

tive families are introduced.

Genus Cardiovirus

The Cardiovirus genus consists of several viruses of mice

Hepatitis A Virus

of which encephalomyocarditis virus (EMC) has been exten-

Hepatitis A virus (HAV) is a causative agent of infectious

sively studied as a model picornavirus. It is closely related to

hepatitis in humans. The virus is worldwide in distribution.

other picornaviruses (Fig. 3.1) although differing in certain

Only one serotype is known, but isolates from different areas

important characteristics. The EMC IRES has proved more

or different times can be grouped into different genotypes or

useful than the poliovirus IRES in experiments that require

strains. The most distantly related HAV isolates share about

polycistronic mRNAs or that express proteins in a cap-inde-

75% nucleotide sequence identity, but most isolates are

pendent fashion in vertebrate expression systems. Theiler's

much more closely related. HAV is a typical picornavirus

virus, another member of this genus, causes demyelinating

but is an outlier in the family (Fig. 3.1). It shares only 28%

disease in mice and has been extensively studied as a model

amino acid identity in its structural proteins with any other

for multiple sclerosis in humans.

picornavirus, whereas most picornaviruses are more closely

related to one another.

Genus Hepatovirus

The number of cases of hepatitis A in the world has been

estimated to be more than 1.4 million each year. In 1998

Hepatitis in Humans

in the United States, for example, ∼37,000 cases of hepa-

Many different viruses, belonging to several virus fami-

titis were reported, of which two-thirds were diagnosed as

lies, are known to cause hepatitis (inflammation of the liver)

caused by HAV. HAV is spread through contaminated food

in humans. These different viruses have different modes

and water. Filter-feeding shellfish like oysters are known to

of transmission and cause illness of different degrees of

concentrate the virus, and consumption of raw shellfish has

severity (although all hepatitis is serious) that results from

been the cause of many epidemics of hepatitis A. A 2003

destruction of liver cells caused by growth of these viruses

epidemic of more that 500 cases in Pennsylvania was caused

in the liver as a target organ. Hepatitis is characterized by

by consumption of green onions that are believed to have

fatigue and other symptoms that result from inadequate liver

been contaminated during harvest. Infection by HAV usually

function, and it may be fatal if sufficient destruction of the

results in a self-limited illness in which the patient recovers

liver takes place. A characteristic feature of acute hepatitis is

with relatively few sequelae. The illness can be quite serious,

the presence of elevated levels of liver enzymes circulating

even fatal, however, because 90% of the liver tissue can be

in the blood that results from the destruction of liver cells.

destroyed by virus infection, and liver function is severely

Many cases of hepatitis are accompanied by jaundice (turn-

impaired until the liver recovers. The seriousness of disease

ing yellow) because of the destruction of the liver, which is

is age dependent. Very young children suffer little disease

responsible for clearing bilirubin from the blood.

but with advancing age infection by the virus becomes more

The viruses whose primary disease syndrome in humans

serious. The mortality rate in children younger than 14 is

is hepatitis, and which therefore target the liver as the princi-

only 0.1%, but HAV infection in people older than 40 results

pal or only organ infected, or viruses that are closely related

in a fatality rate of 2.1%.

to viruses that cause such hepatitis, have historically been

Before the introduction of a vaccine, the only prophylaxis

named hepatitis virus followed by a letter, in the order of

for HAV was injection of immune gamma globulin, which

isolation. Thus we have hepatitis A virus, the first to be iso-

provided protection from the virus for a few weeks. Two

lated, hepatitis B virus, the second, and so forth. Because

inactivated virus vaccines against HAV were licensed in the

these viruses belong to a number of different families, con-

mid 1990s that have been found to give long-lived protec-

fusion can arise because of the similar names even though

tion. Results from a clinical trial in Thailand that showed the

TABLE 3.6 Causative Agents of Viral Hepatitis in Humans

Annuala U.S. acute

Chronic

Family/

Genome type/

Long-term

cases/deaths

hepatitis (millions

Virus

genus

size in kb

Transmission

Chronicity?

effects

in 2004

of cases) U.S. / World

Hepatitis A

Picornaviridae/

ss (+) RNA/

Fecaloral

Very little

Few if any

5683/76

0/0

Hepatovirus

7.5 kb

ds DNA (RT)/b

HCCc, cirrhosis

Hepatitis B

Hepadnaviridae/

Parenteral, sexual,

10% of adults,

6212/659

1.2/300400

Orthohepadno-

3.2 kb

vertical

90% of

virus

neonates

HCCc, cirrhosis

Hepatitis C

Flaviviridae/

ss (+) RNA/

Parenteral, sexual,

>50%

720/4321

3/50100

Hepacivirus

9.4 kb

vertical

Hepatitis D

Deltavirus

ss, circular

Parenteral, (sexual,

Yes

Exacerbates

7500/

0.07/?

RNA/1.7 kb

vertical?)

symptoms

1000

of Hep B

Hepatitis E

Hepeviridae/

ss (+) RNA/

Fecaloral

Few if any

Very rare

0/0

Hepevirus

7.5 kb

Hepatitis Fd

Hepatitis G

Flaviviridae/

ss (+) RNA/

Parenteral, other?

Yes

??/none

Hepacivirus

9.4 kb

Data from MMWR Summary of Notifiable Diseases--2004. It is noteworthy that acute cases of both hepatitis A and hepatitis B have declined significantly

since 1990 with the introduction of vaccines that are now in widespread use; see Figure 3.9 below.

RT is reverse transcriptase. Nucleic acid in virion is partially ds DNA, consisting of a full-length minus-strand DNA of 3.2 kb, and an incomplete

plus-strand DNA that is variable in length.

HCC, Hepatocellular carcinoma.

Isolate from a fulminant case of hepatitis, not further characterized.

Hepatitis A

Hepatitis B

Hepatitis C

Hep A vaccine

Hep B vaccine

ACIP guidelines

1973

1978

1983

1988

1993

1998

2003

Year

FIGURE 3.9 Incidence (cases per 100,000 population) of viral hepatitis in the United States between 1973 and 2003.

Hepatitis A incidence was the lowest ever in 2004, but there has been a trend for cyclic increases every decade, and future

increases could occur. However, with the expansion of recommended vaccination to include children in all communities

where the incidence was consistently above the national average (1999 ACIP guidelines), the incidence of HAV has

continued to plummet. No vaccine for hepatitis C exists, but an antibody test for hepatitis C was first introduced in May

1990. This graph is adapted from MMWR Vol. 52, #54, Summary of Notifiable Diseases United States 2003.

Dose

300

Start

250

Surveillance

Booster

Surveillance

200

Control Group

150

Controlled Trial Ends

100

Hep A Vaccine Group

200

400

600

800

Time after dose 1 in days

FIGURE 3.10 Controlled trial of a hepatitis A vaccine in Thailand. Children were divided into two groups, and at the

times indicated by the blue arrows the vaccine group was given hepatitis A vaccine and the control group was given hepatitis

B vaccine. There were two periods of surveillance for cases of hepatitis A, indicated by the pink overlays. The controlled

trial ended at 540 days when the control group was given hepatitis A vaccine at the times indicated by the magenta arrows

and the vaccine group was given hepatitis B vaccine. Adapted from Figure 3 in Innis et al. (1994).

efficacy of this vaccine are shown in Fig. 3.10, as an exam-

interesting differences in the replication of this virus from

ple of the type of data that can be obtained in clinical trials.

those of other picornaviruses, such as the presence of VP0

The introduction of these vaccines and the recommendation

in virus particles rather than the cleaved products VP2 and

in 1996 of routine childhood vaccination has resulted in a

VP4 (see Fig. 3.2). The worldwide burden of human gas-

steady decline in the rate of hepatitis A in the United States.

troenteritis caused by Aichi virus or closely related viruses

In 2003 there were 7653 cases reported (2.7 per 100,000

is unknown at present. As described later, many viruses

population), and childhood vaccination appears to have been

belonging to a number of virus families cause epidemic gas-

important in the control of this disease. Of interest is the

troenteritis in humans, and sorting out the causative agents is

finding that hepatitis A rates were much higher in the west-

difficult and requires time.

ern United States before the introduction of the vaccines, but

A virus related to Aichi virus appears to be widespread

the rates are now similar across the United States, and an

in cattle in the Aichi area of Japan. This virus, classified for

increasing proportion of cases occur in adults.

now as a member of the Kobuvirus genus, apparently causes

inapparent infection in cattle and has been called bovine

kobuvirus. Thus, it is possible that kobuviruses are widely

Genus Kobuvirus

distributed in the world and infect a number of species.

In Aichi, Japan in 1989, a stool specimen from a patient

suffering from gastroenteritis associated with consumption

Genus Aphthovirus

of raw oysters was found to contain a new picornavirus, sub-

sequently called Aichi virus. Since then the virus has been

Foot-and-mouth disease viruses (FMDV) belong to

found not only in Japan but also in Pakistani children with gas-

seven currently recognized serotypes. They cause a debili-

troenteritis and in Japanese travelers returning from Southeast

tating disease, foot-and-mouth disease, in cattle and other

Asia with gastroenteritis, and the virus thus appears to be

animals, and are economically important pathogens. FMDV

widespread and to be one of the many viruses that are causa-

was eliminated from the United States many years ago by

tive agents of human gastroenteritis. The virus is sufficiently

the simple expedient of killing all infected animals until

distinct in its sequence from other picornaviruses to be clas-

such time as the virus was extirpated. The last epidemic

sified in a separate genus, Kobuvirus (Fig. 3.1). The name

in the United States occurred in 1929. The virus still cir-

is derived from the Japanese word for bump, because the

culates in Europe, South America, and other parts of the

virion appears bumpy in the electron microscope. There are

world, and the U.S. Department of Agriculture maintains

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