Family Flaviviridae - Viruses And Human Disease

The rubella vaccine has now been in use for many years

genera are similar in size (11 kb for flaviviruses, 12.5 kb

and is generally safe and effective when given to children. The

for pestiviruses, 9.4 kb for hepaciviruses) and organization.

present vaccine has a high incidence of side effects in adults,

These viruses, like the picornaviruses, have a genome that

however, especially arthralgia and arthritis. Nonetheless,

contains only a single ORF. This ORF is translated into a

vaccination is recommended for women of childbearing age,

long polyprotein that is processed by cleavage into 10 or

as well as for certain health care personnel, who have never

more polypeptides. Processing of the precursor polyprotein

been vaccinated and who are seronegative. The need exists

is complicated. Cleavage is effected by a combination of

to improve the vaccine, and current efforts to understand the

one or two or three (depending on the virus) virus encoded

molecular biology of the virus in more detail will hopefully

proteases and two or more cellular proteases. The struc-

tural proteins are encoded in the 5′-terminal region of the

lead to the development of a better vaccine.

genome (like picornaviruses). However, all members of the

Flaviviridae are enveloped, unlike the picornaviruses, and

FAMILY FLAVIVIRIDAE

the structural proteins consist of a nucleocapsid protein and

two or three envelope glycoproteins. Cellular proteases make

The Flaviviridae are named after the prototype virus,

the cleavages that separate the glycoproteins, but the cleav-

yellow fever virus, flavus being the Latin word for yellow.

ages in the nonstructural region of the polyprotein, which is

The Flaviviridae are divided into three genera, the genus

required for RNA replication, are made by one or two virus-

Flavivirus, the genus Pestivirus, and the genus Hepacivirus.

encoded proteases. Even so, cellular signalase makes at least

A partial listing of viruses in the three genera is given in

one of the cleavages in the nonstructural domain of flavivi-

Table 3.12. In the following discussion, the term flavivirus

ruses. The cleavage pathways in this genus are described in

refers only to members of the genus Flavivirus unless

detail next.

otherwise specified.

All members of the Flaviviridae encode a serine

The genome organizations of members of the three

protease with a catalytic triad consisting of serine, histidine,

genera are shown in Fig. 3.27. The genomes of the three

and aspartic acid. The protease resides in the nonstructural

TABLE 3.12 Flaviviridae

Virus name

World

Genus/species

abbreviation

Usual host(s)

Transmission

Disease

distribution

Flavivirus

Dengue (Types 14)

DENV

Humans

Mosquito-borne

Dengue fever, shock,

Worldwide

hemorrhage,

Primatesa

Yellow fever

YFV

Mosquito-borne

Hemorrhage,

Africa,

liver destruction

Americas

Mammals,a

Japanese encephalitis

JEV

Mosquito-borne

Encephalitis

Widespread in

especially swine

Asia

Mammals,a birds

St. Louis encephalitis

SLEV

Mosquito-borne

Encephalitis

North America

Mammals,a birds

Murray Valley

MVEV

Mosquito-borne

Encephalitis

Australia

encephalitis

Mammalsa

Tick-borne encephalitis

TBEV

Tick-borne

Encephalitis

Europe, Asia

Mammals,a birds

West Nile

WNV

Mosquito-borne

Encephalitis

Europe, Africa,

North America

Hepacivirus

Hepatitis C

HCV

Humans

Parenteral,

Hepatitis,

Worldwide

transfusion

liver cancer

Pestivirus

Classical swine fever

CSFV

Swine

Contact

Fever, acute

Europe, Americas

gastroenteritis

Usually noneb

Bovine viral diarrhea

BVDV

Cattle

Contact

Worldwide

Including humans.

Calves infected in utero develop persistent infections that can lead to mucosal disease.

Flavivirus (Dengue)

CAP

NS1

NS2A

NS3

NS4A

NS5

NS2B

NS4B

Pestivirus (BVDV)

NS2-3

NS5A-B

Npro Erns

NS4A

NS4B

NS2

NS3

NS5A

NS5B

Hepacivirus (HCV)

NS2

NS3

NS4b

NS5

NS4a

C/F

(p58+p68)

Protease Cleavages

Enzyme Motifs

Coding Domains

NS3 proteinase

Polymerase (GDD)

Nonstructural proteins

NS2-3 autoproteinase

Helicase

Nucleocapsid protein

Npro autoproteinase

Serine proteinase

Virion glycoproteins

Signalase

Papain proteinase

Cellular protease

Ribosomal Frameshift

(furin?)

IRES

FIGURE 3.27 Genome organization of representatives of the three genera within the Flaviviridae. The cleavage sites

indicated with dashed lines have not been precisely localized. Adapted from Figures 2, 4, 5 of the Flaviviridae in Fauquet

et al. (2005) pp. 981998. The data on the F protein of hepatitis C virus, which is produced from the core protein sequence

by ribosomal frameshifting, came from Xu et al. (2004).

region called NS3, just upstream of a helicase. The crystal

translation, as described in more detail later. No nucleotide

structure of the dengue virus protease and of the hepatitis C

or amino acid sequence identity can be detected between

virus (HCV) protease has been solved to atomic resolution

members of different genera except for isolated motifs that

and they possess a fold similar to chymotrypsin, as is the case

are signatures of various enzymatic functions.

for other viral serine proteases whose structures have been

Viruses in the family are enveloped. As described later

solved. The enzyme is interesting in that a second polypeptide

and in Chapter 2, members of the genus Flavivirus have a

is required for activity, NS2B in flaviviruses and NS4A in

structure that is related to that of alphaviruses. Details of

HCV. From the atomic structure of the HCV protease com-

the structures of pestiviruses and hepaciviruses are lacking.

plexed with the region of NS4A required for activity, it is

Flaviviruses mature at intracytoplasmic membranes rather

clear that NS4A forms an integral part of the folded protease.

than at the plasma membrane.

Thus, it is puzzling that the protease consists of two cleaved

products rather than one continuous polypeptide chain.

Genus Flavivirus

Flaviviruses encode only the NS3 protease. Hepaciviruses

and pestiviruses encode a second protease in the NS2 region that

There are about 53 species of flaviviruses currently rec-

cleaves between NS2 and NS3. Pestiviruses also encode a third

ognized, and many species have important subtypes that are

protease at the N terminus of the polyprotein whose only known

also named. A representative sample is given in Tables 3.11

function is to cleave itself from the polyprotein precursor.

and 3.12. The relationships of the viruses to one another is

Flaviviruses have capped genomes whose translation is

illustrated by the dendrogram in Fig. 3.28. All members of

cap dependent. In contrast, the hepacivirus and pestivirus

the genus are closely related and share significant amino acid

genomes are not capped and have an IRES in the 5′ non-

sequence identity in their proteins, which results in serologi-

translated region. Members of family Flaviviridae do not

cal cross-reactivity. Historically, members of this genus were

have a poly(A) tail at the 3′ end of the RNA. Instead, a stable

assigned to it on the basis of these cross-reactions. Most are

stem-loop structure is present at the 3′ end of the genome that

arthropod-borne, and they were once referred to as Group

is required for replication of the genomic RNA and for its

B arboviruses. They can be divided into three major groups

Vector or Host

Serogroup

Louping Ill

TBE (Hypr)

Tick-borne

Tick

TBE (Neu)

Encephalitis

Langat Bat

Powassan

Rodent

APOI

Rio Bravo

Bat

Murray Valley

Japanese

Kunjin

Encephalitis

Culex

West Nile

SLE

Mosquito

Dengue 4

Dengue 2

Dengue

Dengue 1

Aedes

Dengue 3

Yellow Fever

Cell Fusing Agent

Aedes

CFA

Scale Bar equals distance of 0.13

FIGURE 3.28 Phylogenetic tree of the flaviviruses based on NS3 polyprotein region using the neighbor-joining method.

Data are from Billoir et al. (2000).

based on the vector utilized: the mosquito-borne group (which

introduced into the United States, is vectored by a very wide

includes yellow fever, the dengue complex, and the Japanese

variety of mosquitoes, and this has been in part responsible

encephalitis complex), the tick-borne encephalitis group (the

for the rapid spread of the virus across the United States.

TBE complex), and a group that lacks an arthropod vector.

The last are of limited medical importance. Notice that in

Expression of the Viral Genome

the phylogenetic tree in Fig. 3.28, the tick-borne viruses

and the mosquito-borne viruses belong to different lineages.

The genome organization of a typical flavivirus is illus-

These viruses are adapted to a tick vector or to a mosquito

trated in Fig. 3.27. As for all plus-strand RNA viruses, the

vector, and interchange of vectors does not occur. Further,

genomic RNA is a messenger and in the case of flaviviruses

the tree indicates that the mosquito-borne viruses separate

serves as the messenger for all of the virus encoded proteins.

The RNA is capped but lacks 3′ poly(A). There is a stem-

out into a lineage vectored primarily by mosquitoes belong-

loop structure at the 3′ end which serves the same function

ing to the genus Culex and lineages vectored by mosquitoes

belonging to the genus Aedes. However, in these lineages the

as poly(A) in other messengers. This structure increases the

restriction on the mosquito vector is not firm and mosquitoes

efficiency of translation of the RNA by about 10-fold and

belonging to other genera may vector many of these viruses.

will substitute for poly(A) in model systems. Viral proteins

As two examples, the ancestral yellow fever virus in Africa

are not required for this effect and therefore cellular pro-

is vectored by Aedes mosquitoes in both sylvan and urban

teins must interact with this structure in order to increase the

cycles, but in the Americas it is vectored by Hemagogous

efficiency of translation. It is known that during translation

mosquitoes in a sylvan cycle and Aedes mosquitoes in an

of mRNAs that are capped and polyadenylated, there is an

urban cycle, as described later. West Nile virus, recently

initiation complex formed that contains both cap-binding

protein and poly(A)-binding protein. Thus, the complex

nal signal sequences is responsible for the multiple insertion

interacts with both ends of the mRNA to initiate translation.

events required to insert prM, E, and the following protein,

It is assumed that a cellular protein binds the 3′ stem-loop of

NS1, into the endoplasmic reticulum. After separation of

flaviviruses and interacts with the initiation complex so as to

these three proteins by signalase, prM and E form a het-

perform the same function as the poly(A)-binding protein.

erodimer. prM is cleaved to M by furin during transport of

Formation of this complex in the case of flavivirus RNAs

the heterodimer or during virus assembly. Assembly of viri-

could be enhanced by cyclization of the viral RNA described

ons is described in more detail later.

later, although the primary function of cyclization appears to

Following the E protein is NS1 (NS for nonstructural).

be in the replication of the viral RNA.

NS1 is a glycoprotein and has multiple functions that are

The processing of the long polyprotein produced from

only poorly understood. It is found as dimers and higher

the genome is complicated and is illustrated in Fig. 3.29 as

multimers in three locations in mammalian cells: intrac-

an example of complex processing events that can occur in

ellular; anchored in the plasma membrane by a GPI (gly-

viral polyproteins associated with lipid bilayers. The nucleo-

cosyl-phosphotidylinositol) anchor; and as a soluble protein

capsid protein is 5′ terminal in the genome and is removed

secreted from the infected cell. It is required for RNA rep-

from the precursor polyprotein by the viral NS2BNS3 pro-

lication, presumably a function of the intracellular form of

tease. Two envelope proteins, prM (precursor to M) and E

the protein. For this function, it interacts with NS4A. The

(envelope), follow. Both are anchored in the endoplasmic

cell surface-anchored form is capable of antibody-induced

reticulum by C-terminal membrane-spanning domains and

signal transduction that may play a role in cell activation.

are usually, but not always, glycoproteins. A series of inter-

The function of the secreted form is unknown but it has

NS1

prM

NS4B

NS2B

Lumen

NS2A

NS4A

ER membrane

Cytoplasm

NH3+

NS5

NS3

COOH

Intramembrane

Proteolytic Cleavages

Structural proteins

domain

Golgi protease (Furin?)

NS2B-3

unknown protease

Stop transfer

Nonstructural proteins

NS2B-3 (alternative)

signal

Signalase

Signal

sequence

FIGURE 3.29 Processing of the flavivirus polyprotein into the structural and nonstructural proteins of the virus. The

structural proteins (blue) at the N terminus of the polyprotein are processed primarily by signalase, with one late cleavage in

prM due to furin. The nonstructural proteins (green) are mostly processed by the viral NS2BNS3 protease. As indicated in

the figure, the central 40 amino acids of NS2B interact with NS3, tying NS3 to the membrane, and this interaction is essential

for proteolytic function. The striped arrow shows the alternative site of cleavage within NS2A that may lead to an anchored

form of NS1. Adapted from Figures 3, 4, and 6 in Strauss and Strauss (1996).

been speculated that it has a role in counteracting immune

cyclize the RNA are illustrated in Fig. 3.30, where two possi-

responses to the virus.

ble structures are shown. Experimental data have shown that

Next in the polyprotein precursor are two hydrophobic

the RNA sequence in the capsid protein downstream of the

polypeptides called NS2A and NS2B. These proteins are

AUG start codon is involved in cyclization (region marked

cleaved by the viral NS2BNS3 protease. They are associ-

CS1). Sequences upstream of the start codon are also known

ated with membranes and may serve to anchor parts of the

to be required for cyclization, and the two structures show

replication machinery to internal membranes in the cell.

different ways that these might be used for cyclization. This

figure also illustrates the long stem-loop structure at the 3′

NS2A has multiple functions. It inhibits the production of

interferon-α /β by infected cells. As described in more detail

end of the RNA, discussed earlier. A stem-loop structure in

the 5′ region just upstream of the CS1 region has also been

in Chapter 10, the interferons are potent inhibitors of virus

replication and most, perhaps all, viruses encode products

shown to be important in translation of the RNA, in this case

to block interferon action. NS2A also has a role in the pro-

for recognition of the AUG start codon, which is found in a

duction of infectious particles from the infected cell, since

poor context for a start codon.

certain mutations in this protein block virus assembly but do

The sequences surrounding CS1 are illustrated for a

not affect other aspects of the virus life cycle. These mutants

number of mosquito-borne flaviviruses in Fig. 3.31. This

can be suppressed by changes in the NS3 helicase domain,

eight nucleotide sequence is invariant among the mosquito-

suggesting an interaction between NS2A and NS3. NS2B

borne flaviviruses, and experimental studies have shown

also interacts with NS3, but with the protease domain. It

that this sequence is important for cyclization and replica-

tion of the RNA. The 3′ sequences complementary to this

is a cofactor required for the NS3 protease activity and the

region are found in the 3′ nontranslated region (see also Fig.

central domain of NS2B forms a complex with NS3, which

follows NS2B in the polyprotein precursor. The NS2BNS3

3.30). Changes in these sequences that eliminate cyclization

serine protease cleaves many bonds in the polyprotein. NS3

prevent the RNA from replicating, even in model systems

also has at least two other activities--the middle domain of

in which translation of the RNA is not required for expres-

NS3 is a helicase, required for RNA replication, and the C-

sion of the replicase. Compensating mutations in the partner

terminal domain has RNA triphosphatase activity, an activ-

sequence that restore cyclization restore the ability of the

ity required for the capping of the viral genome.

RNA to replicate. Thus, cyclization is required for RNA rep-

NS4A and NS4B are hydrophobic polypeptides that are

lication.

associated with membranes. They may function in assembly

The identities of the promoters recognized by the RNA

of the viral replicase on intracellular membranes. Both the

replication machinery are as yet unknown, but the require-

viral NS2BNS3 protease and cellular signalase are required

ment for cyclization suggests that sequences at both ends of

to produce the final cleaved products.

the RNA are required. The conservation of the 8-nucleotide

NS5 is the viral RNA polymerase. It appears to be a

core sequence suggests that these sequences might be part of

soluble cytoplasmic protein that associates with membranes

the promoter recognized by the RNA replicase.

through association with other viral peptides. It also has

methyltransferase activity and thus is the capping enzyme

Formation of the Virion

that caps the viral genome. Thus, capping requires two fla-

viviral proteins, NS3 (RNA triphosphatase) and NS5 (cap-

Most flaviviruses mature at intracellular membranes.

ping enzyme). Note the similiarity to alphaviruses where the

Budding figures have been described only rarely and assem-

RNA triphosphatase activity is also on the helicase-protease

bly may be associated with the complex processing of the

protein (nsP2, the analogue of flaviviral NS3), and the meth-

polyprotein. West Nile virus is an exception to this general

yltransferase or capping activity is a different protein (nsP1).

rule. It grows to higher titers in cultured cells than other fla-

However, in alphaviruses the capping enzyme and the RNA

viviruses and budding of preassembled nucleocapsids at the

polymerase (nsP4) are distinct proteins, whereas in flavivi-

plama membrane is readily seen. Even in this case, however,

ruses they are present in the same polypeptide.

intracellular assembly of virions is also seen.

The processing of the structural proteins from the precur-

sor polyprotein was described earlier. prM and E form a het-

Replication of the Viral RNA

erodimer shortly after synthesis. The assembly of flaviviruses

RNA replication is associated with the nuclear mem-

has clear parallels with that of alphaviruses, as described in

brane. The composition of the replicase complex is not

Chapter 2. E of flaviviruses and E1 of alphaviruses are

understood but is assumed to consist of many (most? all?)

homologous proteins, having the same structure and func-

of the viral nonstructural proteins with associated cellular

tion (see Fig. 2.17). A heterodimer is first formed, between

proteins. Cyclization of the RNA is required for replication.

prM and E in flaviviruses and PE2 and E1 in alphaviruses.

Sequences from the 5′ and 3′ regions of dengue virus RNA

Immature virus particles can be isolated that have uncleaved

that form a number of stem-loop structures and that also

PE2 or prM whose infectivity is very low. The trimeric spikes

C C

G G

C G

C A

U A

C C

U C

G C

C U

A C

G U

A C

G U

A A

C C G A

U C

G G

C A

U G

C A

C G

U A

C C

G U

A C

U G

C A

G C

G U

A G

A GU

AA U

UG A

U G

C C

G A

U U

G C

G A

U U

A U

A C

G C

G A

G AC G

C CA

CS1

147) C C C UGGGC G UC AAUAUG GUAC GAC GAG (173)

(126) CC CC GGGUCG UC AAUAUG C UAA AAC GC G (153)

MVE

(126) AA CC GGGC UA UC AAUAUG C UGAA AC GC G (153)

(127) AA CC GGGC UG UC AAUAUG C UAA AAC GC G (154)

(129) AA CC GGGUUG UC AAUAUG C UAA AAC GC G (156)

SLE

(127) GA CC AC CU U UC AAUAUG C UGAAAC GC G (153)

DEN4

(125) C AC GC CU U UC AAUAUG C UGAAAC GC G (151)

DEN2

FIGURE 3.31 Conserved nucleotide sequence elements in the 5′ region encoding the capsid protein in six different

mosquito-borne flaviviruses. The number of the first and last nucleotides shown is given in parentheses. The boxed nucleotides

in red are those postulated to be important for cyclization of the RNA. Residues shaded in green are complementary to

sequences at the 3′ end and those given in blue are conserved but probably not involved in cyclization. Adapted from Figure

7 of Hahn et al. (1987).

in these immature particles are quite similar in structure (see

by fever and rash. Several important viruses and their dis-

Fig. 2.15). Flaviviruses have a triangulation number of 3 and

eases are listed in Tables 3.11 and 3.12. We begin the discus-

therefore 60 trimeric spikes each consisting of 3 heterodimers

sion of these viruses with yellow fever virus, the prototype

of prM and E. Alphaviruses have a triangulation number of 4

flavivirus and a virus whose history was important in the

and therefore there are 80 trimeric spikes each consisting of

development of the science of virology and of vaccinology.

3 heterodimers of PE2 and E1. After cleavage of prM or of

Yellow fever virus (YFV) was once greatly feared and is

PE2, however, the structures are quite different. Alphaviruses

still capable of causing large epidemics. The virus is viscero-

retain 80 trimeric spikes with E2-E1 heterodimers. In flavivi-

tropic in primates, the only natural hosts for it. The growth

ruses, however, the M-E heterodimer dissociates and there is

of the virus in the liver, a major target organ, causes the

a dramatic rearrangement whereby 90 E-E homodimers are

major symptoms of disease and the symptoms from which

formed and the particle shrinks from 60 nm in diameter to

the name of the virus derives, jaundice following destruc-

50 nm. Upon infection of a cell and exposure of the mature

tion of liver cells. The virus also replicates in other organs,

flavivirion to acidic pH there is another dramatic rearrange-

such as kidney and heart, and causes hemorrhaging. Illness

ment and 60 E-E-E homotrimers are formed that tilt up so

is accompanied by high fever. Death occurs in 2050% of

that the fusion peptide at the extremity of domain 2 is inserted

serious infections, usually on days 710 of illness and usu-

into the cellular target membrane and fusion results. It seems

ally as a result of extensive liver necrosis.

clear that the structures of alphaviruses and flaviviruses had a

YFV is present today in Africa and Latin America.

common origin, and that recombination during their evolution

It originated in Africa and spread to the Americas with

resulted in this common structure becoming associated with

European colonization and the introduction of slaves. The

different suites of RNA replication enzymes. It is possible that

virus is maintained in two different cycles. In an endemic or

this structure, which allows enveloped viruses to have a regu-

sylvan cycle, it is maintained in Aedes africanus and other

lar icosahedral structure, evolved only once. It is also of note

Aedes mosquitoes in Africa and in Haemogogus mosquitoes

that both groups of these viruses are primarily arboviruses and

in the Americas. Monkeys form the vertebrate reservoir. In

perhaps this common structure is important for this.

this cycle, forest workers and other humans who enter deep

The flavivirus nucleocapsid is thought to be icosahedral

forests are at risk. Infection of humans can lead to the estab-

in symmetry, perhaps having a triangulation number of 3.

lishment of an epidemic or urban cycle in which the virus

There appears to be no interaction between the envelope

is transmitted by the mosquito Aedes aegypti and humans

proteins and the capsid proteins in flaviviruses, however,

are the vertebrate reservoir. In this cycle, all urban dwellers

unlike the situation for alphaviruses, so that the icosahedral

are at risk. Aedes aegypti is a commensal of man, breed-

structure of the nucleocapsid, if it exists, is not coordinated

ing around human habitation. It is widespread in the warmer

with the icosahedral arrangement of the glycoproteins form-

regions of the world, including the southern United States,

ing the outer surface of the virion.

Central America and the Caribbean, large regions of South

America, sub-Saharan Africa, the Indian subcontinent,

Southeast Asia, Indonesia, and northern Australia.

Yellow Fever Virus

History of Yellow Fever

Many flaviviruses are important pathogens of humans.

Different viruses may cause encephalitis, hemorrhagic fever

In the 1800s, YFV was continuously epidemic in the

with shock, fulminant liver failure, or disease characterized

Caribbean region, where it had a pronounced influence on

the development and settlement of the Americas by the

there was no disease aboard, the ship was allowed to dock.

Europeans. Caucasians and Native Americans are very sen-

Yellow fever soon appeared in Norfolk. A number of early

sitive to yellow fever, usually suffering a serious illness

cases among the citizens of the town were ascribed to the

with a high death rate. Black Africans, who were brought

ship passing within a half mile of their homes, and it is pos-

as slaves to the New World to replace Native American

sible that infected mosquitoes were blown ashore, although

slaves who had died in large numbers from European dis-

it is also possible that workmen visiting the ship while laid

eases, in general suffer less severe disease following yellow

up for repairs may have brought the disease into the town.

fever infection, presumably having been selected for partial

The disease then spread in all directions at a uniform rate of

resistence by millennia of coexistence with the virus. Their

about 40 yards per day until it encompassed the whole city.

relative resistance to yellow fever resulted in the importa-

The epidemic peaked at the end of August and died out after

tion of even more black slaves into yellow fever zones. The

a frost in October. During the epidemic, an estimated 10,000

high death rate among French soldiers sent to the Caribbean

cases of yellow fever occurred in a population of 16,000, and

region to control black slaves was probably responsible for

2000 died of the disease. The report established two other

the decision by Napoleon to abandon the Louisiana territory

facts about the disease: Persons who had had yellow fever

by selling it to the United States, by which the United States

previously were immune, and the epidemic was not spread

underwent a huge territorial expansion. The high death rate

by person-to-person contact.

among French engineers and workers in the 1880s under de

The Walter Reed Investigation

Lesseps, who had previously supervised the construction of

the Suez Canal, led to the abandonment of the attempt by the

At the turn of the twentieth century, there was much

French to build a canal through Panama. The Panama Canal

debate as to the mechanism by which yellow fever spread.

through Panama was built by the United States only after

The Department of the Army sent an expedition, under the

yellow fever was controlled.

command of Walter Reed, to Cuba, recently acquired by the

From its focus in the Caribbean, yellow fever regularly

United States from Spain, to study the disease. The commis-

spread to port cities in the southern and southeastern United

sion undertook to test the thesis that the virus was transmit-

States and as far north as Philadelphia, New York, and

ted by mosquitoes, using themselves as human volunteers.

Boston. Epidemic yellow fever even reached London. The

Mosquitoes were allowed to feed on yellow fever patients

virus also spread up the Mississippi River from New Orleans.

and then on members of the commission. At first there was

The virus was transported from its focus in the Caribbean by

a lack of understanding about the fact that mosquitoes are

ships, which carried freshwater in which mosquitoes could

infected only by feeding on patients early in their disease,

breed. If there was yellow fever on the ship, the disease was

before an effective immune response arises, and about the

maintained and could be transmitted by the mosquitoes or

necessity for an extrinsic incubation period in the mosquito,

by infected individuals to ports at which the ships called.

during which the virus establishes an infection in the salivary

Yellow fever epidemics could afflict most of the population

glands, before it can transmit the virus. Ultimately, however,

of a city and result in death rates of 20% or more of the city's

the investigation team did succeed in infecting themselves

original population.

by mosquito transmission and one member of the commis-

One telling account of an epidemic in Norfolk, Virginia,

sion, Dr. Jesse Lazear, died of it. Fortunately, his was the

in 1855 is described in the report of a committee of phy-

only death recorded in these experiments. It is of note that in

sicians established to examine the causes of this epidemic.

the days before the introduction of a vaccine, most research-

Quarantine procedures to prevent the introduction of yellow

ers who studied yellow fever in the field or in the laboratory

fever were often thwarted by captains who concealed the

ultimately contracted the disease and many of them died.

presence of the disease to avoid a lengthy quarantine, even

With the discovery that the virus was mosquito borne, the

going to the extreme of secretly burying crew members who

U.S. Army began a campaign in Havana to eliminate mos-

died while in quarantine. On June 6, 1855, the steamer Ben

quito breeding places by eliminating sources of water around

Franklin arrived from St. Thomas and anchored at the quar-

human habitation. It was (and still is) common for drinking

antine ground. There had been three cases of yellow fever on

water to be stored around houses throughout Latin America

the ship during the voyage, of whom two died and were bur-

in large pots that served as excellent breeding places for

ied, one on land and one at sea. There was yet another case

Ae. aegypti. The campaign, which included smashing such

on board during quarantine who died and was buried ashore.

water containers, succeeded in breaking the mosquito trans-

Yet when the health officer, Dr. Gordon, visited the ship, he

mission cycle and yellow fever as an epidemic agent dis-

was told by the captain that there was no disease on the ship.

appeared from Havana within months. This approach was

The captain did admit that there had been two deaths dur-

later exported to other areas with great success, including

ing the voyage but ascribed them to other causes. After 13

Panama. These successes led to the belief that yellow fever

days in quarantine and continued inspection by Dr. Gordon,

could be eradicated, but the discovery of the endemic cycle

who finding nothing amiss believed the captain's report that

of yellow fever dispelled this idea. Forest workers who cut

down trees and brought the mosquitoes down from the upper

spread of epidemic yellow fever in Latin America and, with

canopy, where they transmit the disease to monkeys, were

less success, in Africa. The success of this vaccine has served

particularly at risk. Once infected, a person is able to bring

as a model for the development of other live virus vaccines,

the disease back to town where it can get into the Ae. aegypti

namely, passing the virus in cultured cells from a nonnative

population and start an urban epidemic.

host. Recent sequencing studies have found that the 17D vac-

cine differs from the parental Asibi strain at 48 nucleotides

The Yellow Fever Vaccine

that result in 22 amino acid substitutions. The substitutions

In the late 1920s, yellow fever virus was successfully prop-

responsible for the attenuation of the virus are not known, but

agated in rhesus monkeys, in which it causes a lethal disease

it is suggestive that 8 of the amino acid substitutions are found

and in which it can be experimentally passed from monkey to

in the E protein, where they might alter host range.

monkey. One such strain was derived from an infected human

Yellow Fever Today

named Asibi. Theiler and Smith passed the Asibi strain of

yellow fever in chicken cells, and after approximately 100

Although not as wide ranging as previously, yellow fever

passages, it was found that the resulting virus was no longer

continues to cause epidemics in Africa and South America as

virulent for rhesus monkeys. After additional passages, this

illustrated in Fig. 3.32. On an annual basis, 50300 cases are

virus, called 17D, was ultimately used as a live virus vac-

officially reported in South America and up to 5000 cases in

cine in humans and has proved to be one of the best and most

Africa, but these figures are significantly underreported

efficacious vaccines ever developed. The vaccine virus has

and the World Health Organization estimates that there are

been given to about 350 million people. It causes very few

200,000 cases of yellow fever each year with 30,000 deaths.

side reactions, although three recent vacinees developed full-

Between 1986 and 1991, annual outbreaks of yellow fever

blown yellow fever and died. The vaccine is essentially 100%

occurred in Nigeria that probably resulted in hundreds of

effective in providing long-lasting protection against yellow

thousands of cases. An intense campaign beginning in 1992

fever. This vaccine is routinely given to travelers to regions

to vaccinate the population of Nigeria has resulted in the vir-

where yellow fever is endemic and is used to control the

tual disappearance of yellow fever in Nigeria, but epidemics

182

150

309

1540

824

815 251 382 262

1246

1239

340

260

325

Cases of Yellow

Fever 1992-2004

1-10

11-100

101-1000

1000-10,000

FIGURE 3.32 Cumulative number of cases of yellow fever reported to the World Health Organization for the years 1992

through 2004, by country. It is suspected that cases in Africa may be underreported by a factor of 10 or more. Immunization

coverage in Africa has remained low and the disease has continued to spread. Major epidemics (>250 cases) have occurred

in Liberia, Burundi, and Peru in 1995, Guinea in 2000, Burkina Faso in 2002, and the Democratic Republic of Congo in

2004. Note that while Nigeria had 19,891 cases between 1980 and 1991, since 1994 there have been only 12 cases. Data

from: http://www.who.int/immunization_monitoring/data/data_subject/en/index.html.

continue to occur in other African countries. Epidemics of

of infected cells results in increased cytokine production and

yellow fever also continue to occur in Peru, Bolivia, Brazil,

can result in capillary leakage and shock. Although second

Ecuador, Columbia, and Venezuela, perhaps in part due to the

infections are common, infection by a third serotype is rare.

reemergence of Ae. aegypti in South America as described

Evidently the boost to the immune system from the second

in more detail later. There was one imported case of yellow

infection results in an increase in the amount and avidity of

fever in the United States in 1996, in which an American who

cross-reactive antibodies.

visited the jungles of Brazil along the Amazon River without

Although infection by a second serotype is important for

being immunized returned to the United States with yellow

the development of DHF, it is also known that the probability

fever and died of the disease. Because of the endemic cycle

of contracting DHF is in part a function of the virulence of

in which monkeys are the reservoir, it is probably impossible

the virus that is responsible for the second infection. Some

to eradicate the virus as has been done with smallpox and as

dengue strains grow better than others and are more likely to

is planned for poliovirus and measles virus.

cause DHF upon a second infection than other strains of the

same virus. As one example, there was very little DHF in Sri

Lanka before 1989 despite the continuing circulation of all

Dengue Viruses

four dengue serotypes. In 1989, however, a new strain of

The four dengue viruses, now considered by the ICTV to

DEN-3 appeared in Sri Lanka that caused a large number of

be serotypes of a single viral species, have recently undergone

cases of DHF. A second example, DEN-2 in the Americas,

a dramatic expansion in range. The incidence of dengue fever

is described in Chapter 8.

is estimated to have increased 30-fold over the last 40 years

Because second infections by a different serotype are

and dengue viruses now infect an estimated 50100 million

much more likely to lead to DHF than primary infections,

humans each year. Infection may be subclinical or may result

the development of vaccines against dengue has progressed

in dengue fever, which is usually uncomplicated but which

slowly. The possibility is real that immunizing against one

can progress to dengue hemorrhagic fever (DHF) or den-

serotype might put a person at risk for a more serious ill-

gue shock syndrome (DSS). Uncomplicated dengue fever is

ness. Current efforts in Thailand are directed toward devel-

characterized by headache, fever, rash, myalgia (muscle pain,

oping a quadrivalent attenuated virus vaccine that would

from myo = muscle and algia = pain), bone pain, and prostra-

immunize against all four serotypes simultaneously. U.S.

tion. The disease may be mild or it may be extremely pain-

scientists are independently attempting to develop vaccines

ful (an old name for the disease is break-bone fever which

for the viruses, based either on attenuated dengue viruses

dramatically describes the joint pain that can occur), but it is

or on the development of chimeric flaviviruses that express

almost never fatal. However, progression to DHF or DSS is

dengue envelope antigens in a yellow fever vaccine back-

associated with a significant mortality rate. In the absence of

ground (see Chapter 11). These various vaccine candidates

medical care, mortality can be as high as 20%, but with good

are in clinical trials as of this writing. A major problem has

medical care the mortality rate is a few percent. Up to 250,000

been the tendency of vaccinated humans to respond strongly

cases of DHF and DSS are recorded each year, most of them

to one of the four serotypes in live virus vaccines, often to

in children, and in Southeast Asia DHF and DSS are a lead-

DEN-3, while responding only weakly or not at all to other

ing cause of mortality in children. DHF and DSS have also

serotypes. Changing the ratios of the four viruses in the mix

become important in Latin America. It is thought that DHF

and use of multiple inoculations are being tested as possible

and DSS are caused by immune enhancement in which infec-

ways to overcome this problem.

tion by one serotype of dengue virus expands the population

Dengue viruses are maintained in Ae. aegypti in urban set-

of cells that can be infected by a second serotype. Many anti-

tings in most of the world, but also in Ae. albopictus in Asia,

bodies induced by the four dengue viruses are cross-reactive,

and humans are the vertebrate reservoir. Part of the difficulty

reacting not only with the infecting virus but with the other

in developing vaccines is that there is no animal model for

dengue viruses as well. Immediately following infection,

the disease. The virus will infect monkeys but does not cause

in fact, a person is immune to all four serotypes. With time

disease in them. Small animal models of infection exist but

this cross protection fades, and in less than a year the person

the infection process is artificial and the resulting disease is

remains immune only to the infecting virus, probably for life.

not dengue fever (DF) or DHF. DF and DHF are exclusively

After this time, infection with another serotype can occur and

human diseases and the dengue viruses that infect humans

cause disease. Still present, however, are cross-reactive anti-

are exclusively human viruses.

bodies that can react with the newly infecting virus but which

The Spread of Dengue Viruses

cannot neutralize the virus to provide protection. The nonneu-

tralizing antibodies are thought to enable the virus to infect a

The four serotypes of the dengue viruses arose in Old

larger number of lymphocytes by means of Fc receptors (see

World monkeys and jumped to humans an estimated 200

Chapter 10) than would otherwise result from infection only

1000 years ago. As noted before, the human viruses are now

of cells expressing the dengue receptor. This expanded pool

strictly human viruses. Although they will infect monkeys

under laboratory conditions (without causing disease), the res-

ease is greatly underreported. Only one JE virus infection

ervoir in nature is exclusively humans. However, the monkey

in 200 or 300 results in encephalitis, with children and the

viruses still exist as monkey viruses in a sylvatic cycle in Asia

elderly being at higher risk. The fatality rate following JE

and Africa. The human viruses have been continuously active

encephalitis is 240% in different outbreaks, but 4570%

over large areas of Asia and the Pacific region for many years.

of survivors have neurological sequelae. In endemic areas,

In areas of Thailand where the viruses are endemic, for exam-

virtually all people have been infected by the time they reach

ple, most people are infected by multiple serotypes in child-

adulthood. Birdmosquitobird transmission is the normal

hood and DHF is a leading cause of mortality in children.

transmission cycle, but domestic pigs are particularly impor-

The viruses have recently dramatically expanded their

tant amplifying hosts for transmission to humans because

range in the Americas. Before 1970 there was very little

they are found in proximity to their human owners. Various

dengue activity in the Americas, probably because of mos-

species of Culex mosquitoes transmit the virus. During

quito control efforts that were abandoned about that time.

peak transmission seasons, up to 1% of Culex mosquitoes

Following this, dengue activity increased dramatically, espe-

around human habitations may be virus infected. Travelers

to endemic regions have a probability of about 10-4/week

cially upon the introduction of new strains of the virus from

Asia. By the 1990s there occurred widespread epidemics

of contracting JE, and 24 cases of JE encephalitis in travel-

that affected many millions of people every year. Epidemics

ers were reported between 1978 and 1992. Inactivated virus

have resulted in an estimated 100 million cases of dengue

vaccines are in use in different regions of Asia. The Japanese

infection in Brazil alone. The introductions of Asian viruses

have long used such a vaccine to eliminate JE encephalitis

included more virulent strains of dengue that together with

from their population, and the Chinese have recently devel-

the circulation of multiple serotypes led to epidemics of

oped a vaccine that is being used in China and Thailand.

DHF. Dengue virus is now a major health problem in Latin

The Japanese vaccine is available in the United States for

America. Dengue has also become more active in the Pacific

travelers to endemic regions. Of considerable interest is the

region. Recent epidemics in Hawaii, the first in 50 years,

finding that JE virus infection may reactivate in mice after

have resulted in more than a hundred documented cases of

the immune system first damps it out. Reactivation in other

dengue fever. This topic of the origin and spread of dengue

animals may also occur and could be important for persis-

to the Americas is discussed in more detail in Chapter 8.

tence of the virus in nature.

For the dengue viruses, immune enhancement is impor-

tant for the disease caused in humans. There is no evidence

Japanese Encephalitis Virus

that immune enhancement plays a role in the disease caused

The Japanese encephalitis (JE) complex of flaviviruses

by JE virus or other flaviviruses such as MVE virus. It is

includes a large number of related viruses, many of which

interesting, however, that in a mouse model system, prior

cause encephalitis in humans. For these viruses, the majority

treatment with subneutralizing concentrations of anti-JE

of human infections are inapparent and, for most, fewer than

serum resulted in an increase in virus growth and in mortal-

1% of infections result in neurological disease. However,

ity in the mice following infection by MVE virus. This sug-

when encephalitis develops it is often serious with case

gests that the potential for immune enhancement exists for

fatality rates as high as 50% and neurological sequelae are

other flaviviruses but does not occur in humans other than

frequent among survivors. In addition to JE, these include

the dengue viruses because the immune reaction to flavivi-

St. Louis encephalitis (SLE), Murray Valley encephali-

ruses is normally strong and not cross-reactive and subneu-

tis (MVE), and West Nile viruses. The close relationships

tralizing concentrations of antiviral antibodies do not exist.

of these viruses are illustrated in Fig. 3.28. Some of these

It does raise a warning flag for vaccines, however, and vac-

viruses are widespread whereas others are much more local

cines that increase the seriousness of disease caused by sub-

in their distribution. West Nile virus, for example, is now vir-

sequent infection have occurred in the case of measles virus

tually worldwide (the Australian strain is often called Kunjin

and respiratory syncytial virus (see Chapters 4 and 10).

virus), whereas MVE virus is only found in Australia. Thus,

As indicated before, most human infections by JE virus

circulation of at least some of these viruses has occurred

do not result in invasion of the nervous system and encepha-

over widespread areas, as is the case for the dengue viruses

litis does not occur. Two laboratory experiments are of inter-

described in the preceding section. For most of the viruses

est in this regard. In one experiment, JE virus variants were

in this lineage, birds form the major vertebrate reservoir and

selected that failed to bind to mouse brain membrane receptor

culicine mosquitoes are the major vectors.

preparations. These mutants were attenuated for neuroinva-

JE virus is distributed throughout Asia, including Japan,

siveness and neurovirulence because their receptor-binding

India, Southeast Asia, Indonesia, the Philippines, and

preferences were altered. In a second experiment, passage

Borneo (Fig. 3.33). Reported cases of JE encephalitis aver-

of JE virus and of MVE virus in cultured cells selected for

age 30,00050,000 per year with 10,000 deaths, but the dis-

variants that bound to glycosaminoglycans (GAG). These

SouthKorea

(6)

Japan

(122)

Nepal

(1283)

China

(122,955)

Taiwan

India

(127)

(25,856)

Hong Kong

Bangladesh

(4)

Thailand

(5)

Philippines

(836)

Vietnam

(174)

(9745)

Sri Lanka

Malaysia

(1965)

(104)

Singapore

(10)

Reported Cases 19861990

None

Bali

120

(5)

21200

2012000

>2001

No case data, but endemism suspected

FIGURE 3.33 Range and reported cases of Japanese encephalitis, 19861990. Adapted from MMWR (1993) Vol. 42,

RR-1, p. 2. Since this detailed report, the first human cases were reported in Papua New Guinea in 1997, there were two fatal

cases on islands in the Torres Strait in 1995, and the virus was detected in mainland Australia (the Cape York Peninsula) in

1998.

variants were rapidly removed from the bloodstream when

But with the recent occurrence of outbreaks of encephalitis

inoculated into mice and were attenuated. It was suggested

in Europe, the Middle East, and North America the situa-

that GAGs present on cells and extracellular matrices result

tion changed. Not only has infection by the virus resulted in

in the removal of these variants from blood and tissues before

large numbers of cases of neurological disease in humans,

replication and neural invasion can take place. Evidence was

but domestic animals, especially horses, and wildlife, par-

presented that the attenuation of the live JE virus used as a

ticularly birds, have been severely impacted. The effect of

vaccine in China, called SA14-14-2, may have resulted, at

the virus in North America has been especially dramatic, as

least in part, from such an effect.

described in more detail in Chapter 8. Since its arrival in

1999, more than 20,000 Americans have become ill from

West Nile infection and more than 800 have died. The virus

West Nile Virus

has also had severe effects upon horses and many species

West Nile (WN) virus was first isolated in 1937 from the

of birds.

blood of an infected woman in the West Nile province of

Two lineages of WN virus are recognized. The lineage

Uganda. Until 1999 it was not considered an important path-

present in North America, Europe, the Middle East, India,

ogen, causing only sporadic cases of encephalitis in parts of

Australia (where the subtype present has been called Kunjin

Africa, Asia, and Europe and having little effect on wildlife.

virus), and parts of Africa, called lineage 1, contains both

virulent and attenuated strains and is responsible for WN

of contacts. Alligators were accidentally infected by feeding

disease. Lineage 2 is present only in sub-Saharan Africa and

them infected birds. In humans, transmission of the virus by

Madagascar and is mostly maintained in an enzootic cycle.

blood transfusion or via breast milk has occurred.

The association of the virus with significant outbreaks of dis-

Pathology of West Nile Disease

ease in humans, domestic animals, and birds, and the wide-

spread dispersion of the virus in Eurasia and the Americas,

About 80% of human infections appear to be asymptom-

appears to be the result of the emergence of a more virulent

atic. In the 20% of infections that result in clinical disease,

strain of the virus.

most result in a self-limited illness characterized by fever,

Various species of culicine mosquitoes are the princi-

headache, fatigue, malaise, muscle pain, and weakness. In

pal vectors of WN virus, although the virus has also been

fewer than 1% of infected humans does the virus cross the

isolated from species of Aedes, Coquillettidia, Culiseta,

bloodbrain barrier and cause neurological disease such

and Ochlerotatus, among others. In Europe and Africa the

as meningitis, encephalitis, or paralysis. In about 13% of

principal vectors are Culex pipiens, Cx. univittatus, and Cx

patients experiencing neurological disease, infection of

antennatus, in India Cx. vishnui, in Australia Cx. annuliros-

anterior horn cells of spinal motor neurons causes an acute

tris, and in North America Cx. pipiens, Cx. quinquefascia-

flaccid paralysis very similar to poliomyelitis resulting from

tus, and Cx. tarsalis, among others. During epidemics, from

poliovirus infection. The fatality rate for neuroinvasive dis-

0.1% to as many as 15% of Culex mosquitoes were found to

ease is about 10%, and many survivors, especially those with

be infected.

poliomyelitis type disease, never fully recover.

WN virus infects a wide spectrum of vertebrates. The res-

Infection with WN virus may be serious because the

ervoir of the virus is various species of birds. More than 150

virus interferes with the innate immune system (see Chapter

species of birds were shown to be infected by WN virus and

10). Several of the nonstructural proteins of the virus inter-

only in birds, with few exceptions, does the virus produce

fere with phosphorylation of the Janus kinases JAK1 and

high enough viremia titers to infect mosquitoes. Laboratory

Tyk2. This prevents the activation of the transcription fac-

studies showed that viremia titers of 105 to 107 are required to

tors STAT1 and STAT2 and their transport to the nucleus.

infect mosquitoes; at the lower titer fewer than 15% of feed-

Activation of these factors is required for the cell to respond

ing mosquitoes become infected, whereas at the higher titer

to the signaling of the interferons, normally a first line of

more than 70% become infected. Mammals in general do

defense against viral infection.

not generate such high viremia titers after infection by WN

Vaccines to protect horses have been developed. One is

virus. The maximum viremic titer in humans and horses, for

an inactivated virus vaccine. The other is a recombinant vac-

example, appears to be about 103. Among birds, grackles,

cine that uses canarypox virus to express WN virus antigens.

corvids (crows, ravens, jays, magpies), house finches, house

Two viruses for human use are also being developed. The

sparrows, shorebirds, hawks, and owls are most susceptible

first is an inactivated virus vaccine. The second is a chimeric

to the virus and show high mortality rates upon infection

YFWN live virus vaccine in which the prM and E proteins

(25100% in various studies). They develop sufficiently

of the 17D YF virus vaccine strain have been replaced with

high viremia to efficiently infect mosquitoes that feed upon

those of WN virus (see Chapter 11).

them. WN virus has also been isolated from amphibians and

reptiles, and the lake frog of Russia develops sufficiently

Other Flaviviruses of the JE Complex

high viremia that it might serve as a reservoir.

There are reports that WN virus can persist in infected

MVE virus is an Australian virus that is closely related to

animals for a considerable time. Virus could be isolated from

JE virus. It causes encephalitis in humans, but the number

experimentally infected ducks and pigeons for more than 3

of cases is small. Birds are the primary vertebrate reservoir,

months, for example. Such persistence could be important

and epidemics of MVE have been associated with wet years

in the persistence of the virus in nature. Also important is

when the mosquito population expands and nomadic water-

transovarial transmission, which occurs in many flavivi-

fowl invade regions that are normally too dry to support

ruses, and West Nile is no exception. During transovarial

them. Culex annulirostris is the primary vector for MVE.

transmission, the eggs laid by the mosquito are infected and

SLE virus is a North American virus belonging to the JE

the emergent mosquito is thus infected. This mechanism is

complex that causes regular epidemics of encephalitis in the

especially important in temperate climates where adult mos-

United States. The virus is widely distributed and cases of

quitoes die off during the winter and the species persists as

SLE encephalitis have been recorded in every state, with the

diapausing embryos or larvae.

majority of cases occurring in the Mississippi River valley,

WN virus can also be spread by means other than by mos-

Texas, California, and Florida. Data for the years 19642003

quitoes, although the importance of such spread in the main-

are shown in Fig. 3.34. In the epidemic year 1975 there were

tenance and spread of the virus is unknown. Birds excrete

1815 cases of SLE encephalitis officially reported in the

virus in their feces, which can serve as a source of infection

United States, but in nonepidemic years there may be fewer

131

540 368 440

118

125

141

Cumulative Cases of SLE

1964 to 2003

336 149 5

978

116

365

1-10

11-100

101-500

>500

FIGURE 3.34 Distribution of cases of St. Louis encephalitis occurring between 1964 and 2003 in the United States,

shown by state. The large number of cases in Florida includes the most recent U.S. epidemic, which occurred in 1990,

during which Florida reported 223 cases and 11 deaths. Data came from Fields et al. (1996) p. 981, and MMWR, Summary

of Notifiable Diseases, 1996, 1997, 1998, 1999, 2000, 2001, 2002, 2003. St. Louis encephalitis was not a notifiable disease

nationwide until 1998. Recent reported cases were 24 in 1998, 4 in Florida in 1999, 2 in Texas in 2000, 79 in 2001 of which

71 were in Louisiana, 28 in 2002, and 41 in 2003. These have been incorporated into the state totals shown.

that 50 cases. The most recent epidemic occurred in 1990

other TBE viruses, can also be contracted by drinking raw

in Florida with 223 cases and 11 deaths. The case fatality

goat's milk and possibly other forms of raw milk. The virus

rate is about 7% overall, but is higher in the elderly. Most

has a tendency to set up persistent infection in experimental

infections by SLE are inapparent, as is the case for many

animals and possibly in humans as well. Although Ixodes

encephalitis viruses. The ratio of inapparent to clinical infec-

ticks are the primary vector, Dermacentor ticks and ticks of

tion is age dependent and varies from 800 to 1 in children

other genera are also capable of transmitting the virus. The

to 85 to 1 in the elderly. The virus is transmitted by Culex

distributions of two species of Ixodes ticks that are important

mosquitoes, and the primary vertebrate reservoirs are wild

vectors of TBE are shown in Fig. 3.35 together with the geo-

birds.

graphic range of endemic TBE disease.

Powassan virus is a member of the complex found in

North America and in Russia. In North America, 20 cases of

Tick-Borne Encephalitis Viruses

Powassan encephalitis have been reported since 1958.

The tick-borne encephalitis (TBE) viruses are impor-

All known TBE viruses cause encephalitis in humans with

tant pathogens in Europe and Asia, and there is also a rep-

the exception of Omsk hemorrhagic fever virus, which causes

resentative in North America. The viruses include Central

hemorrhagic fever in humans, as its name implies, in the

European encephalitis (CEE), louping ill, Russian spring-

absence of encephalitis. Two other members of this complex,

summer encephalitis (RSSE), Kyasanur Forest disease,

Kyasanur Forest disease virus and Alkhurma virus, which are

Omsk hemorrhagic fever, and Powassan viruses. Members

closely related and may represent isolates of the same virus,

of the TBE complex form a distinct group within the fla-

also cause hemorrhagic fever in humans but it is associated

viviruses (Fig. 3.28), but share 40% amino acid sequence

with encephalitis. Omsk hemorrhagic fever virus also dif-

identity with the mosquito-borne flaviviruses, showing their

fers from other TBE viruses in that its principal tick vector is

close relationship to other flaviviruses. Most TBE viruses are

Dermacentor reticulates rather than an Ixodes tick.

transmitted by Ixodes ticks and can cause a fatal encephali-

tis in humans. An inactivated virus vaccine is widely used

Cell Fusing Agent

in Central Europe to protect people exposed to ticks. Even

so, several thousand cases of TBE encephalitis occur each

A flavivirus called cell fusing agent was discovered in

year. The case fatality rate is 12%, with 1020% of survi-

laboratory cultures of Ae. aegypti cells in 1975. The relation-

vors having sequelae in the RSSE form. RSSE, and perhaps

ship of this virus to other flaviviruses is shown in Fig. 3.28.

Both ticks

Ixodes ricinus

Ixodes persulcatus

Endemic tick-borne encephalitis

FIGURE 3.35 Geographic distribution of two major tick vectors of tick-borne encephalitis. Also shown is the major

region in which TBE is endemic. Adapted from Porterfield (1995) p. 207.

This virus is an insect only virus and is not known to infect

is the presence of two proteases rather than one. The NS3

vertebrates. Very recently, new isolates of a strain of cell

protease is shared with the flaviviruses but requires NS4A

fusing agent have been made from wild-caught mosquitoes

as a cofactor rather than NS2B. The second protease, which

in Puerto Rico belonging to at least two genera, Aedes and

has a catalytic cysteine, is present in NS2 and its only known

Culex. Remarkably, DNA sequences related to cell fusing

cleavage function in viral replication is to cleave the NS2

agent have been identified in the genomes of wild-caught

NS3 bond.

A second difference is the lack of a 5′ cap and the pos-

mosquitoes, presumably having integrated into the mos-

quito genome at some time in the distant past. This and other

session instead of an IRES, so that initiation of translation

recent isolates of new flaviviruses has led to the suggestion

of the plus-strand genome is not cap-dependent but uses an

that there are as yet many flaviviruses in nature that remain

IRES as does poliovirus. A third difference is the production

to be identified.

of a small (17 kDa), short-lived protein called F (for frame

shift) or ARFP (for alternative reading frame protein) that

is encoded within the C protein gene in a different reading

Genus Hepacivirus

frame. Translation of this protein requires initiation at the 5′

Hepatitis C virus (HCV) forms a second genus in the

end of the polyprotein followed by a frameshift near residue

Flaviviridae. The virus was discovered in 1989 as a causa-

11 of the capsid protein. There is evidence that it is produced

tive agent of nonA-nonB hepatitis in humans. Despite the

in infected humans but it is not known if this protein plays a

inability to grow the virus in culture or in a small animal

role in virus replication. Of note is the fact that no other plus-

model, the complete genome sequence of the virus was estab-

strand RNA virus is known to produce two different proteins

lished using the methods of modern biotechnology and veri-

from two different reading frames in the same nucleotide

fied by injection of viral RNA produced from cDNA clones

sequence, although this phenomenon occurs in several other

into the liver of a chimpanzee, the only animal other than

classes of viruses.

humans that is infectible by the virus. The HCV genome,

HCV also differs in the way that RNA replication is

which is slightly smaller than those of the flaviviruses and

anchored to a membrane. RNA replication in plus-strand

pestiviruses, has an organization similar to those of the other

RNA viruses occurs in association with membranes. In fla-

members of the family (Fig. 3.27). It has a number of impor-

viviruses, the RNA polymerase is thought to associate with

tant differences from the genome of members of the genus

membranes by means of its association with membrane

Flavivirus, some of which are illustrated in the figure. One

bound proteins such as NS4A or NS4B. In HCV, the RNA

polymerase NS5B is itself anchored in the membrane by a

allow a complete virus replication cycle with the release of

C-terminal transmembrane anchor. Interestingly, this anchor

infectious virus will allow more rapid progress in the future.

is required for RNA replication and the HCV sequence can-

A second advance has been the use of immunodeficient mice

not be substituted with that from the pestivirus bovine viral

(severe combined immunodeficiency or SCID mice) into

diarrhea virus. Thus, this anchor plays a role in RNA rep-

which have been grafted human liver cells. These can be

lication other than simply anchoring the polymerase in the

infected by HCV and although the numbers of such animals

membrane.

is limiting, they possess obvious advantages over the use of

Another interesting difference is the cleavage of the N-

chimps.

terminal capsid protein from the polyprotein precursor. The

capsid protein is anchored in the membrane by a C-terminal

Natural History of HCV

transmembrane anchor, as described earlier for flaviviruses.

In flaviviruses the capsid protein is cleaved from this sig-

HCV is a causative agent of blood-borne hepatitis in man.

nal sequence anchor by the NS3 protease, but in HCV it is

In the United States, HCV was once spread primarily through

cleaved by a cellular protein, signal peptide peptidase.

transfusion of contaminated blood, but the development of

Because of its importance as a human disease agent,

a diagnostic screen for the virus has virtually eliminated

HCV has been the subject of intensive study. Progress has

this source of infection in the developed world. However,

been relatively slow because the only animal model for the

the virus continues to be transmitted through the sharing

disease is the chimpanzee, which are rare and expensive,

of needles by drug users. The virus can also be transmitted

limiting the number of experiments that can be performed,

sexually or from mother to child but these mechanisms are

and because until very recently there was no cell culture sys-

inefficient. There are additional mechanisms of transmission

tem in which the virus would undergo a complete replication

that are not well understood. In some developing countries,

cycle and release infectious virus. Studies in cultured cells

circumcision or scarification practices may be important in

have relied upon the expression of parts of the genome in

the spread of the virus.

expression vectors, and more recently upon the replication of

HCV is worldwide in distribution, as illustrated in

Fig. 3.36. It has been estimated that ∼3% of the world's

truncated versions of the genome called replicons. Replicons

encode all of the genes required for RNA replication but lack

population, 170 million people, are infected by the virus.

the genes encoding structural proteins. Thus, only part of

The highest infection rate found was among Egyptian blood

the virus life cycle can be studied using these reagents. The

donors, where up to 19% were seropositive for HCV, which

very recent development of systems using cultured cells that

may have resulted in part from past treatment for bilharziasis

Equator

Prevalence

(% of population infected)

1 to 2.4

2.5 to 10

>10

No data

FIGURE 3.36 Worldwide prevalence of hepatitis C as of April 2003 based upon published data. Map was found at:

http://www.reliefweb.int/rw/RWB.NSF/db900LargeMaps/SKAR-64GDV4?OpenDocument.

using inadequately sterilized needles. There are six differ-

RNA polymerase NS5B and inhibit its activity, two inhibi-

ent clades or genotypes of the virus, which differ by more

tors of the viral NS34A protease, and three compounds that

than 30% in nucleotide sequence and are numbered from 1

modulate the immune system. Other drugs are also being

to 6. In turn, each clade has many isolates that may differ by

studied as possible antivirals.

up to 25% in nucleotide sequence, so that the clades can be

subdivided into subclades called a, b, etc. These different

HCV Suppression of the Immune Response

viruses all cause the same disease but differ in the severity

of the disease caused and in their ease of cure. Genotype 1

In order to establish a chronic infection, HCV interferes

is the clade commonly found in the United States and prob-

with many aspects of both the innate and adaptive immune

ably became widespread only with the introduction of blood

responses of the host. The importance of such interference

transfusion in the 1940s. In Africa and Asia, the virus has

for chronicity and the persistence of the virus in nature is

been endemic for a long time, and the different clades have

illustrated by the fact that the virus interferes in so many dif-

different geographic distributions. Thus, for example, clade

ferent ways. The immune system is described in some detail

5 is commonly found only in South Africa, clade 4 is widely

in Chapter 10. Here we note that the first line of defense

distributed in the Middle East, clade 6 in eastern Asia, and

against viral infection is the production of type 1 interferons

(IFN) α and β, components of the innate immune system.

clade 2b in the Mediterranean and the Far East.

The NS34A protease of HCV interferes with the induction

of IFN by cleaving two intermediates, called MAV5 and

HCV Disease and Its Treatment

TRIF, in two different but overlapping activation pathways.

Infection with HCV can be extremely serious. The ini-

MAV5 is required in the pathway that starts from an intra-

tial infection may cause no disease or may result in hepatitis

cellular sensor of double-strand RNA called RIG-1, whereas

accompanied by jaundice, but fulminant liver failure is rare.

TRIF is required in the pathway that starts from a membrane

However, in 7080% of infections, the infection becomes

bound sensor of double-strand RNA called Toll-like recep-

chronic. During chronic infection, up to 1012 viruses are

tor 3 (see Chapter 10). The result is that both pathways are

produced each day and turn over with a half-life of about 3

disabled.

hours, and the more or less constant viral load in the blood

The HCV core protein interferes with the activity of any

is 103107 per ml. This chronic infection is well tolerated

IFN that might be produced. It induces the expression of cel-

by some and in a minority of cases spontaneous remission

lular proteins called SOCS1 and SOCS3. These block the

may occur in the absence of medical intervention. However,

JAKSTAT pathway by which IFN induces the production

in many persons chronic hepatitis results. Most seriously,

of hundreds of proteins required for defense against viral

in about 20% of chronic infections liver cirrhosis develops

infection (Chapter 10). Protein NS5A independently inter-

after a long lag, usually more than 20 years, and hepato-

feres with the IFN system in at least two ways. It induces

cellular carcinoma develops in up to 2.5%. Liver failure due

the production of IL-8, which attenuates the expression of

to HCV infection is the leading cause of liver transplantation

genes induced by the activity of IFN. It also binds to a pro-

in the United States.

tein called PKR that is induced by IFN, thereby inhibiting its

The current treatment for chronic HCV infection is injec-

activity. Protein E2 also inhibits PKR. Other HCV proteins

tion of interferon-α conjugated to polyethylene glycol,

are also known to interfere with the activity of IFN.

which increases its stability, together with the inhibitor riba-

HCV also interferes with the adaptive immune system.

virin. This treatment results in curing the infection in about

Interestingly, instead of a general interference with the adap-

half the cases but the cure rate depends upon the genotype of

tive system, as happens with HIV, for example, that cripples

the virus. In one trial, 42% of patients chronically infected

immune responses against all pathogens, the modulation

with genotype 1 HCV were cured whereas patients chroni-

by HCV is limited to HCV-specific responses, leaving the

cally infected with genotype 2 or 3 virus exhibited a cure

immune system free to control other viral infections. The

rate of 80%. This treatment is not only expensive but rela-

mechanisms by which this occurs are incompletely under-

tively toxic and many patients tolerate it poorly. This con-

stood. What is known is that successful clearance of HCV

sideration, as well as the fact that half the patients show no

infection in humans is associated with a strong T-cell

response, both CD8+ and CD4+, and that immunologic mem-

effect or only transient relief from this treatment, has led

to intense efforts to develop new treatments. These include

ory results such that although reinfection by HCV can occur,

efforts to develop vaccines as well as efforts to develop anti-

it does not lead to chronic infection. In humans in which the

infection becomes chronic, CD8+ cytotoxic T cells are rela-

viral agents that will interrupt virus replication or prevent

the virus from interfering with the host defenses against the

tively few and these T cells recognize fewer epitopes. In one

study, CD4+ helper T cells from persistent infections recog-

virus. Antivirals currently in clinical trials include nucleo-

side analogues that when incorporated into viral RNA result

nized very few epitopes whereas those from humans who

in chain termination, two compounds that bind to the viral

cleared the infection recognized up to 14 different epitopes.

HCV-Related Viruses

sheep (BDV). These three viruses share more than 70%

amino acid sequence identity and exhibit extensive serologi-

Viruses related to HCV, called GB viruses (from the

cal cross-reactivity. Their genome organization is similar to

initials of a surgeon with hepatitis from which they were

those of other viruses in the family (Fig. 3.37). Pestiviruses

first isolated), are known. GBV-A and GBV-B viruses have

have also been isolated from a number of other mammals

a genome organization very similar to that of HCV, but

including giraffe, deer, bison, bongo, alpaca, and reindeer.

share little amino acid sequence identity with HCV or with

The taxonomic status of these isolates is still unclear. Some

each other. They may eventually be classified as new gen-

are classified as strains of one of the three viruses just listed

era within the Flaviviridae, more closely related to genus

but some, at least, may represent other species of pestivirus

Hepacivirus than to genus Flavivirus or genus Pestivirus.

BVDV exhibits an important and interesting disease

A third virus, GBV-C, also called hepatitis G virus or HGV,

syndrome in cattle. Animals infected as adults by the virus

is related to GBV-A. These three viruses appear to be widely

may exhibit no disease or may have symptoms that include

distributed and establish chronic infections in humans, but

diarrhea, but they recover uneventfully. However, when a

there is no evidence that they cause disease.

pregnant cow is infected by the virus, infection of the fetus

may cause the fetus to become immunologically tolerant to

the virus, resulting in a chronic infection that lasts for the life

Genus Pestivirus

of the animal. Such in utero infection may lead to develop-

Three closely related viruses belonging to the genus

mental abnormalities or runting in the calf, and may render

Pestivirus are important pathogens of domestic animals

the calf sensitive to infection by other microorganisms, all

and have been well characterized. These are bovine viral

of which have serious economic effects. A more interesting

diarrhea virus (BVDV), classical swine fever virus (CSFV)

effect of the chronic infection, however, is the development

(also called hog cholera virus), and border disease virus of

in some animals of fatal mucosal disease at the age of 12

Structural Proteins

Nonstructural proteins

noncytopathic (ncp) BVDV

NS3

NS2

(BVDV SD1)

Translation, processing

little cleavage, no NS3

cytopathic (cp) BVDV

NS2

NS3

(BVDV Osloss)

partial Ub cleavage produces NS3

cp BVDV

NS2

NS3

(BVDV CP1)

Ub cleavage produces NS3 from a

duplicated gene

Npro

cp BVDV

NS2

NS3

(BVDV 515CP)

Cleavage by Npro autoprotease produces

pro

NS3 from a duplicated gene

Cleavage Motif

Npro autoprotease (papain-like)

Nucleocapsid protein

Ub Ubiquitin

Virion glycoproteins

FIGURE 3.37 Genome organization of cytopathic and noncytopathic strains of the pestivirus BVDV. (A) In noncytopathic

(wild-type) strains little cleavage occurs between NS2 and NS3. In cytopathic strains, NS3 is produced either by an upstream

insertion of ubiquitin (see B), insertion of multiple ubiquitin sequences plus duplication of NS3 sequences (see C), or

duplication of the Npro proteinase and its insertion immediately upstream of a duplicated NS3 (see D). Data for this figure

came from Meyers and Thiel (1996).

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