Chapter 5: Viruses That Contain Double-Stranded RNA: Family Reoviridae - Introduction - Overview of the Family Reoviridae

Viruses That Contain Double-Stranded

RNA: Family Reoviridae

INTRODUCTION

Many of the Reoviridae are transmitted by arthropod vec-

tors (Tables 5.1 and 5.2). The orbiviruses are transmitted

by phlebotomine flies, ticks, gnats, and midges of the genus

Most of the dsRNA-containing viruses that we know about

Culicoides, and members of this genus are true arboviruses,

belong to the family Reoviridae. All members of this large

as are the coltiviruses, transmitted by ticks, and the sea-

family have a genome consisting of 10, 11, or 12 segments

dornaviruses, transmitted by mosquitoes. The members of

of dsRNA totaling 1627 kb. The family is very successful.

the three genera of plant reoviruses are also transmitted by

Twelve genera are currently recognized and different viruses

arthropods and are effectively plant arboviruses (although

infect a wide spectrum of vertebrates, invertebrates, plants, and

the term arbovirus is reserved for vertebrate viruses that are

fungi. Several viruses are important pathogens of humans.

transmitted by arthropods). Thus, members of eight genera

Other dsRNA viruses in addition to the family Reoviridae

of Reoviridae possess the ability to replicate in arthropods,

include the birnaviruses, icosahedral viruses 60 nm in diam-

of which two genera contain viruses that replicate exclu-

eter containing two genome segments of dsRNA totaling

sively in insects and six genera contain viruses that have an

about 7 kb, which infect chickens, fish, and arthropods; the

alternate vertebrate or plant host. The relationships among

totiviruses of fungi and protozoa, which have only one RNA

these various genera are illustrated in the unrooted dendro-

segment; and the partiviruses and hypoviruses of fungi and

gram in Fig. 5.1, which is based on the sequence of the RNA

plants. Much less about these viruses is known and for this

polymerase. It is interesting that the various genera do not

reason as well as the fact that no human pathogens are known

group in any obvious fashion related to the host or the vector

among these viruses, they will not be considered further.

for the virus. As one example, note that the orthoreoviruses

are most closely related to the aquareoviruses, not to other

OVERVIEW OF THE FAMILY REOVIRIDAE

viruses of mammals. As a second example, the coltiviruses

are more closely related to the plant reoviruses than to the

other mammalian reoviruses.

Eleven genera of Reoviridae are listed in Tables 5.1 and

Members of the family Reoviridae replicate in the

5.2 together with a partial listing of viruses in each genus,

cytoplasm. The virion is icosahedral (T=13), 6080 nm in

their hosts and modes of transmission, the diseases they

diameter, and double or triple shelled (the shells consist

cause, and their distributions. A 12th genus, Mycoreovirus,

of protein). The structures of viruses belonging to three

contains viruses that infect fungi. As a taxon, Reoviridae have

genera of the Reoviridae were shown in Chapter 2: reovi-

a wide host range. Members of the genera Orthoreovirus,

rus (genus Orthoreovirus) in Figs. 2.1 and 2.5; rotavirus

Rotavirus, Orbivirus, Coltivirus, and Seadornavirus infect

(genus Rotavirus) in Fig. 2.5; and bluetongue virus (genus

humans as well as other vertebrates, aquareoviruses infect

Orbivirus) in Fig. 2.11 (in this case only the core of the

fish, cypoviruses and entomoreoviruses infect arthropods,

virion is shown). The members of the genus Orthoreovirus

and members of three genera infect plants. Unclassified

have been the best studied and have served as a model sys-

viruses that infect scorpions, crabs, and bed bugs are also

tem for the family, but because of the medical importance

known. Characteristics of the five genera that contain human

of the members of the genus Rotavirus and the veterinary

pathogens are compared in Table 5.3.

TABLE 5.1 Reoviridae of Vertebrates

Virus name

Transmission

World

Genus/members

abbreviation

Usual host(s)

or vector

Disease

distribution

Orthoreoviruses

Non-fusogenic--Subgroup 1

Mammalian reoviruses

MRV

Humans, cattle,

Oralfecal

Gastroenteritis,

Worldwide

types 1, 2, 3

sheep, swine

respiratory disease

Fusogenic--Subgroup 2

Nelson Bay orthovirus

NBV

Flying foxes (bats)

Avian orthoreoviruses

ARV

Birds

Oralfecal

Australia, Worldwide

Wide range of

8 serotypes

symptoms from

inapparent to lethal

Fusogenic--Subgroup 3

Baboon orthoreovirus

BRV

Monkeys

Oralfecal

Orbiviruses

Bluetongue

BTV

Sheep, cattle

Culicoides

Rhinitis, stomatitis

Asia, Americas

24 serotypes

Africa, Australia,

African horse sickness

AHSV

Equines

Culicoides

Cardiopulmonary

Africa

10 serotypes

disease

Changuinola

CGLV

Humans

Phlebotamines

Fever

Panama

Kemerovo serogroup:

Kemerovo

Humans

E. Europe,

Great Island

GIV

Ticks

Fever, encephalitis

Seabirds

United States

Chenuda

CNUV

Wad Medani

WMV

Domestic animals

Coltiviruses

Colorado tick fever

CTFV

Humans

Ticks

Fever, encephalitis

North America,

Europe

Rotaviruses

Group A

RV-A

Humans, animals

Oralfecal

Infant diarrhea

Worldwide

Group B

RV-B

Humans, animals

Oralfecal

Epidemic adult

Primarily China

diarrhea

Group C

RV-C

Humans, animals

Oralfecal

Clinical significance

Worldwide

unknown

Groups D, E, F, G

Birds, mammals

Oralfecal

Seadornaviruses

Kadipiro

KDV

Vertebrates

Culex and Anopheles

Fever, encephalitis

China, Indonesia

mosquitoes

Banna

BAV

Aquareoviruses

Six serogroups

Fish

Representative members of each genus are shown, and the first listed is the type species of the genus.

TABLE 5.2 Reoviridae of Plants and Insects

Type virus name

Transmission

World

Genus/members

abbreviation

Usual host(s)

or vector

Disease

distribution

Cypoviruses

Cytoplasmic polyhedrosis

BmCPV-1

Arthropods

Ingestion

Diarrhea,

Worldwide

viruses (16 species)

starvation due

to changes in

the gut

Idnoreovirus

Ten-segmented, insect-derived,

DpIRV-1

Arthropods

non-occluded

Fijivirus

Fiji disease virus; five other

FDV

Plants

Delphacid leafhoppers

Australia, Asia,

groups of viruses

South America,

Northern Europe

Phytoreovirus

Wound tumor virus

WTV

Plants

Cicadellid leafhoppers

Rice dwarf virus

RDV

Rice

Cicadellid leafhoppers

Stunting

Southeast Asia, China,

Japan, Korea

Rice gall dwarf virus

RGDV

Oryzavirus

Rice ragged stunt virus

RRSV

Plants (Gramineae)

Planthoppers

Mycoreovirus

3 species of 11 or 12

CpMYRV-1

Fungi

segmented viruses of fungi

Abbreviations: BmCPV-1, Bombyx mori cypovirus 1; DpIRV-1, Diadromus pulchellus idnoreovirus 1; CpMYRV-1, Mycoreovirus 1.

These three orthoreoviruses, now considered strains of a

importance of the members of the genus Orbivirus, these

single species, are virtually ubiquitous viruses of mammals.

viruses have recently come under increased scrutiny.

They have been isolated from many different mammals as

well as from sources such as river water and untreated sew-

Genus Orthoreovirus

age. There is little evidence for host range specificity among

these mammalian viruses. However, reovirus infection of

The genus Orthoreovirus (the "true" reoviruses, to dis-

lower mammals is sometimes associated with more serious

tinguish the genus from the family) contains three viruses

illness than infection of humans.

that infect many mammals, including humans, referred to as

Other branches of the Orthoreovirus genus contain

mammalian orthoreovirus (MRV) types 1, 2, and 3. They

fusogenic reoviruses that infect birds, flying foxes, baboons,

were originally named from the first initials of the words

or reptiles. In general, the avian viruses do not grow in mam-

respiratory enteric orphan virus--they grow in the respi-

malian cells or must be adapted to mammalian cells before

ratory tract and in the enteric tract but were orphans, not

they will grow in them, and thus have a host range distinct

known to cause human illness at the time of their discovery.

from that of the mammalian viruses. The relationships among

The viruses are widespread and the majority of humans have

these viruses are illustrated by the tree in Fig. 5.2. There are

antibodies against all three serotypes by the time they are

five distinct lineages. The three nonfusogenic viruses group

adults. Most infections do not result in symptomatic disease

closely together, consistent with their classification as a sin-

or result in only mild symptoms. Studies with human vol-

gle species, mammalian orthoreovirus (species I). There are

unteers have shown that some individuals develop a mild

several strains of avian orthoreovirus (species II) that form

disease characterized by headache, pharyngitis, sneezing,

a second lineage. Nelson Bay orthoreovirus of flying foxes

rhinorrhea, cough, and malaise.

Comparison of Orthoreovirus, Orbivirus, Rotavirus, Coltivirus, and Seadornavirus

TABLE 5.3

Characteristic

Orthoreovirus

Orbivirus

Rotavirus

Coltivirus

Seadornavirus

Segments

Size of genome

23.5 kb

19.2 kb

18.6 kb

28.5 kb

20.0 kb

Type virus

Reovirus type 3

Bluetongue-1

Simian rotavirus SA11

Colorado tick fever

Banna

Portal of entry

Oral

Skin

Oral

Skin

Tissue tropism

Intestinal tract, upper

Hemopoietic

Intestinal tract

Hemopoietic and muscle

CNS

respiratory tract

Vector

None

Culicoid flies, ticks,

None

Ticks

Culex and Anopheles

mosquitoes,

mosquitoes

phlebotomines

Human disease

Upper respiratory

See Table 5.5

Diarrhea, especially in

See Table 5.5

infections, infant

children <5 yrs old

enteritis

Consensus terminal nucleotide sequences

5¢GGCUAUUAAAa

5¢(G/C)ACAUUUUGU

5¢GUAU(A/U)(A/U)AA

5¢ Terminal

5¢-GC(U/A)(U/A)

5¢ GU(A/U)AAA

5¢GGC(A/U)NAAAUUb

GAUGUGACC-3¢a

3¢ Terminal

UCAUC-3¢

AC(U/A)UAC-3¢

UGCAGU(G/C)-3¢

(A/G)C(C/U)GAC-3¢

AUAAAAACCC-3¢b

Rotavirus A.

Rotavirus B.

ROTAVIRUS

Rota "A"

PoRV-A

SiRV

AvRV

BoRV

PoRV-C

Rota "C"

PHYTOREOVIRUS

Rota "B"

RDV-H RDV-A

HuRV

RDV-Ch

KDV

FIJIVIRUS

NLRV

BAV

SEADORNAVIRUS

RRSV

ORYZAVIRUS

CYPOVIRUS

BmCPV

BTV

CTFV

AHSV

COLTIVIRUS

CHUV

GSV

GCRV

MRV-2,3,4 MRV-1

ORBIVIRUS

SBRV CSV

AQUAREOVIRUS

ORTHOREOVIRUS

FIGURE 5.1 Neighbor-joining tree constructed from the amino acid sequences of the RdRp (polymerase) gene of

representatives of 10 different genera of Reoviridae. The size of the oval illustrates the amount of diversion within a

genus, and each oval is color coded for the major host of its members (vertebrates, blue; humans, pink; insects, yellow;

fish, blue-green; and plants, green). Abbreviations are as follows: PoRV, porcine rotavirus; AvRV, avian rotavirus;

SiRV, simian rotavirus; BoVR, bovine rotavirus; HuRV, human rotavirus; NLRV, Nilaparvata lugens reovirus; RRSV,

rice ragged stunt virus; BAV, Banna virus; KDV, Kadipiro virus; BTV, blue tongue virus; AHSV, African horse sickness

virus; CHUV, Palyam virus; MRV-1,2,3,4 mammalian reoviruses types 14; CSV, SBRV, group A aquareoviruses; GSV,

GCRV, group C aquareoviruses; CTFV, Colorado tick fever virus; BmCPV, Bombyx mori cypovirus; RDV-H, RDV-A,

RDV-Ch, isolates of rice dwarf virus. Adapted from Fauquet et al. (2005) Figure 3 on p. 453.

Serotype 1 (Lang)

Mammalian orthoreovirus (I)

Serotype 3 (Deering)

(non-fusogenic)

Serotype 2 (Jones)

Avian orthoreovirus (II)

Fusogenic

NBV Nelson Bay orthoreovirus (III)

reoviruses

BRV Baboon orthoreovirus (IV)

RRV Reptilian orthoreovirus (V)

100 aa changes

FIGURE 5.2 Phylogenetic tree of the orthoreoviruses, derived from amino acid sequences of the σ2 core proteins,

which are encoded in the top three species by the S2 RNA segment, but encoded on a polycistronic S1 segment in the case

of BRV and RRV. The orthoreoviruses are now considered to consist of five species (I to V). A virtually indistinguishable

tree is given by the σNS sequences, and one with only slightly longer arms, but the same topology, by the outer capsid

protein sequences. The scale bar indicates the length of the arm for 100 changes. Adapted from Duncan (1999), and

modified according to Fauquet et al. (2005).

segments of dsRNA (Fig. 5.3), which range in size from 3.9

forms a third lineage (species III). Baboon orthoreovirus

to 1.2 kb and sum to 23.5 kb for MRV-3. The 10 segments

(species IV) and reptilian orthoreovirus (species V) form

fall into three size classes called L for large (3 segments

the remaining two lineages.

called L1, L2, L3), M for medium (3 segments called M1,

The species identified by their alignment in a phyloge-

M2, M3), and S for small (4 segments called S1, S2, S3,

netic tree such as Fig. 5.2 are confirmed by other attributes

S4) (Fig. 5.3 and Table 5.4). Eleven or 12 distinct proteins

that they share, the most important of which is the ability to

are produced, described in more detail later. The proteins

undergo reassortment during mixed infection. Members of

are named by Greek letters corresponding to the L, M, or S

the same species can reassort their genomes during mixed

segment that encodes them, but the numbering of the pro-

infection which, as discussed in Chapter 4 for influenza

teins does not reflect the number of the segment encoding

virus, is important for the evolution of these viruses. A

it (Table 5.4). Of the 11 proteins, 8 are components of the

second feature, which is certainly related to the ability to

virion, 4 in the outer shell and 4 in the inner shell. One

undergo recombination, is conservation of sequences at the

of the viral structural proteins, µ1, is cleaved during virus

ends of the genomic RNAs. For MRV, for example, the 5′

assembly to produce two different products called µ1C and

end of all RNAs begins GCUA and the 3′ end is UCAUC,

µ1N.

whereas the 5′ sequence differs for other species of orthoreo-

viruses. These conserved sequences are probably important

for replication of the RNA and for packaging of the 10 RNA

Entry of Orthoreoviruses into the Cell

segments. These 5′ and 3′ sequences are compared for dif-

After attachment to receptors, normally molecules that

ferent genera of the Reoviridae in Table 5.3.

contain sialic acid, orthoreoviruses are internalized into

Mammalian orthoreovirus serotype 3 (MRV-3) has

endosomes. In endosomes or in lysosomes, proteolysis

been extensively studied as a model for the members of the

of two proteins in the outer shell, σ3 and µ1C, produces

Orthoreovirus genus. In the following discussion, in which

what has been termed an ISVP (infectious subviral particle

aspects of the genome organization, replication, and struc-

or intermediate subviral particle). In this process, µ1C is

ture of orthoreoviruses are described, specific details refer to

cleaved to produce two fragments, δ and a small C-terminal

MRV-3. These details are summarized in Table 5.4.

φ, whereas σ3 is degraded. These cleavages are illustrated

schematically in Fig. 5.4. They can be blocked by agents that

The Genome of Orthoreoviruses

prevent acidification of endosomes, which demonstrates the

importance of the endosomes in the process. ISVPs can also

The orthoreovirus virion is a double-shelled icosahe-

be produced by treating virions with proteases in vitro.

dral particle 85 nm in diameter. The genome consists of 10

TABLE 5.4

Characteristics of the Proteins Encoded by the Ten Genome Segments

of Mammalian Orthoreovirus Serotype 3

Genome

Protein

Segment

Location/molecules of

5¢ NT (nt)

3¢ NT (nt)

segment

product

(length in nt)

ORF (aa)

Function of protein

protein per virion

λ3

3854

1267

Poly(C) polymerase, catalytic subunit

Core/12

of polymerase/transcriptase

λ2

3916

1289

Guanylyltransferase

Turrets on core/60

λ1

3896

1233

184

NT binding motif, Zn finger

Core/ 120

2304

736

Putative transcriptase/polymerase

Minor core component/12

component

m1®m1C

2203

708

Major structural protein

Outer capsid shell/600

mNS(mNSC) 2235

719

Binds ss RNA

Nonstructural

σ1

1416

455

HA, neut Ag, cell attachment protein

Capsid/36

σ1NS

120

Unknown

Nonstructural

σ2

1331

418

Structural component

Core/240

σNS

1198

366

Binds ss RNA

Nonstructural

σ3

1196

365

Major structural protein

Outer capsid shell/600

Abbreviations: HA, hemagglutinin; neut Ag, contains epitopes recognized by neutralizing antibodies; 5¢ NT, nucleotides at the 5¢ terminus of the RNA

segment that are not translated into protein; 3¢ NT, nucleotides at the 3¢ terminus of the RNA that are not translated.

Source: Joklik and Roner (1996).

Lane

which confirms the importance of proteolytic processing for

the activation of domains required for penetration.

Large (L)

The general scheme of the replication of reoviruses is

Segments

shown in Fig. 5.5. The various steps are discussed in more

detail next.

Medium (M)

Synthesis of mRNAs

Segments

On penetration of ISVPs into the cytoplasm, they are

converted into cores by further loss of δ, φ, and the σ1

fiber, and the rearrangement of the protein λ2 (Fig. 5.4).

Core particles are transcriptionally active: Each of the

10 segments of dsRNA within them is used to synthesize

Small (S)

an mRNA molecule that is the same length as the plus

Segments

strand of the genome segment. These mRNAs are capped

but not polyadenylated. All enzymatic activities required

for the initiation of RNA synthesis, capping, and elon-

gation of the product are present in the core, and occur

FIGURE 5.3

Gel electrophoresis of the mammalian orthoreovirus RNA

in vitro if cores are supplied with appropriate substrates.

genome segments, showing the variation of the segment size with serotype.

Synthesis of mRNA is conservative: The newly synthe-

Lane 1: reovirus serotype 2 (Jones), Lane 2: reovirus serotype 1 (Lang), and

Lane 3: serotype 3 (Deering). The segments cluster into three groups: 3L

sized mRNAs are extruded from the core and both strands

(large), 3M (medium), and 4S (small). From Fields et al. (1996), p. 1559.

of the parental dsRNA remain within the core. Extrusion

is an active process. Electron microscopic studies have

suggested that the mRNAs are extruded from the 12 ver-

The ISVP is capable of breeching the endosomal mem-

tices of the icosahedral structure. The enzymatic activi-

brane and gaining entry into the cytoplasm. The process of

ties are organized about these 12 fivefold axes, and it has

penetration may involve µ1N, whose N terminus is myris-

been suggested that there is an independent transcription

toylated and lipophilic. When ISVPs are produced by treat-

unit for each genome segment, consistent with the fact

ment of virions with proteases in vitro, they are capable of

that no member of the family Reoviridae has more than

penetrating into the cell by way of the plasma membrane,

12 genome segments.

ISVP

CORE

VIRION

Outer Capsid

λ2, µ1C, σ1, σ3

λ2, δ, φ, σ1

λ2

Rearrangement of σ1

Rearrangement of λ2

Cleavage of µ1C to δ and φ

Core

RNA

λ3, µ2

Loss of σ3

λ3, µ2

Loss of

δ, φ, σ1

λ1, λ3, µ2, σ2

σ3

Inner Capsid

σ1

Inner Capsid

FIGURE 5.4 Structure of a reovirion and subviral particles derived from it. At left, a cross section through the particle

shows the two protein shells (in yellow and blue) surrounding the RNA (green). In the middle, the intermediate subviral

particle (ISVP) is shown, after the loss of σ3, the cleavage of µ1C to δ and φ, and the extension of σ1. The right illustration

shows the core after the loss of σ1, δ, and φ and the rearrangement of λ2. Redrawn from Niebert and Fields (1995).

outer shell of the virion. Protein σ1 is the cell attachment pro-

Translation of Proteins

tein on the surface of the virion. Trimers of this protein are

In MRV, the 10 mRNAs are translated into 12 proteins

located at the 12 fivefold axes. Interestingly, a complete com-

of which 11 are distinct (Table 5.4). For 8 mRNAs, only

plement of σ1 trimers is not present in all virions. Virions

one reading frame is used and only one protein is produced.

contain from 0 to 12 trimers, with the median number of trim-

For the mRNA produced from segment M3, only one read-

ers being 7. Virions devoid of σ1 are not infectious, but viri-

ing frame is used but two different in-frame AUGs are used

ons containing one or more trimers are infectious.

to initiate translation. Thus, two proteins (µNS and µNSC)

Four proteins of MRV, µNS, µNSC, σ1NS, and σNS,

are produced from this segment that differ only in that the

are nonstructural. µNS, µNSC, and σNS are RNA-binding

longer version has an N-terminal extension. The mRNA

proteins and probably participate in virion assembly. The

from segment S1 is translated using two different, out-of-

function of σ1NS is not known; it is found in the nucleus of

frame AUGs, however, so that two different proteins (σ1

infected cells.

and σ1NS) are produced. In all orthoreoviruses, the mRNA

from the segment corresponding to S1 is translated into two

or three different proteins. In MRV the ORF for σ1NS is

Assembly of Progeny Virions

completely contained within the ORF for σ1, whereas in

species other than MRV the reading frames for the two or

The mRNAs serve as intermediates in the replication of

three proteins overlap only slightly. The various mRNAs are

reoviruses. Following synthesis and release from the core,

mRNAs quickly become associated with proteins µNS, σNS,

translated with widely different efficiencies so that different

and σ3 to form complexes containing single-strand RNA. All

amounts of the 11 or 12 proteins are produced.

complexes contain µNS, but only half contain σ3 and one-

Of the proteins produced, eight are components of the

quarter contain σNS. Complexes containing dsRNA appear

virion and the rest are nonstructural, present only within the

infected cell. Proteins λ1 and σ2, present in 120 and 240

later, which contain the three proteins just named but also

copies, respectively, form the shell of the core. Proteins λ3

contain protein λ2 and the RNA polymerase. Significantly,

and µ2 are present in 12 copies within the core, at the 12

all 10 dsRNA genome segments are present in equimolar

fivefold axes. Protein λ3 is the catalytic subunit of the RNA

quantities in these complexes, suggesting that the selection

polymerase, and µ2 is believed to be a component of this

and assortment of the 10 genome segments into progeny viri-

enzyme complex.

ons is associated with the conversion of (+)RNA into double-

Pentamers of protein λ2, present in 60 copies, form "tur-

stranded RNA. Because the particle-to-infectious virus ratio

rets" at the 12 fivefold axes of the core through which the

is almost one, the assembly process is clearly precise.

During maturation, protein µ1 undergoes a cleavage to

mRNAs are extruded. This protein has guanyltransferase

produce two fragments called µ1N (the N-terminal fragment)

activity and is a component of the complex that caps the

mRNAs. Protein µ1 and its cleavage products, µ1N and µ1C,

and µ1C (the C-terminal fragment). Fragment µ1N is small

together with protein σ3, both present in 600 copies, form the

(4 kDa) and myristoylated, and this process bears a striking

Virion

ISVP

Virion

Proteolysis

Attachment

Endocytosis

Proteolysis

Capped

transcripts

Primary

transcription

Penetration

RNA

Assortment

Translation

Core

RNase-sensitive

Complex

Minus-strand

Synthesis

RNase-resistant

Complex

Capsid

Assembly

NUCLEUS

Progeny

Virion

Release

FIGURE 5.5 The reovirus replication cycle. The primary steps are labeled in blue, while the intermediate products are

labeled in black. This diagram was adapted from Figure 6 in Fields et al. (1996) on page 1706. The color coding of the

protein components is the same as in Fig. 5.4.

similarity to the cleavage event that occurs during the matu-

activities induced by interferon. Thus, the sequestering of the

ration of picornaviruses (see Chapters 2 and 3).

dsRNA within particles where it is not available for interaction

It is significant that the dsRNA is never free but always

with cellular sensors that detect the presence of dsRNA or with

present in particles. As described in Chapter 10, dsRNA is a key

enzymes that are activated by binding dsRNA is important for

molecule in both the induction of interferon and in the antiviral

avoiding this potent cellular defense against viral infection.

Reoviruses and Cancer

rotavirus particle resembles a wheel with spokes from which

it derives its name (rota = wheel in Latin). The assembly

One fascinating recent development is the possibility

of rotaviruses differs in one important detail from that of

that orthoreoviruses might be useful in controlling some

orthoreoviruses. Subviral particles are assembled in the

cancers. They selectively kill mammalian cells whose Ras

cytoplasm and bud through the endoplasmic reticulum,

signaling pathways have been activated. This comes about

acquiring an envelope that is subsequently lost. During this

because orthoreovirus replication is constrained by the inter-

maturation process, the outer capsid layer of the virion is

feron stimulated gene RNA-activated protein kinase (PKR)

acquired. Perhaps because of this transient association with

(Chapter 10). However, tumor cells whose Ras pathways

membranes, two of the rotaviral proteins are N-glycosylated

are activated lack PKR activity, allowing the virus to rep-

and one is O-glycosylated. In contrast, no orthoreovirus

licate vigorously. Since nearly two-thirds of human tumors

protein is known to be glycosylated.

may have an activated Ras pathway, and since orthoreovirus

Activation of infectivity of the virion requires cleavage

infection is fairly benign, it is possible that MRV could be

of one of the major outer capsid proteins, similar to the

used to control or eradicate some tumors.

case for the orthoreoviruses where cleavage of an external

protein also occurs. In rotaviruses this process normally

Genus Rotavirus

occurs in the enteric tract where the virus is exposed to

secreted cellular proteases. Uncleaved rotavirus will bind

Rotaviruses are viruses of higher vertebrates and are

to cells but cannot penetrate and virus produced in cultured

very widely distributed. They cause gastroenteritis in their

cells must be treated with proteases to render it infectious.

various hosts and many different serotypes are known.

The protein is cleaved at a hydrophobic sequence that is

They have been isolated from monkeys, cattle, dogs, cats,

postulated to possess fusion activity, analogous to the proc-

pigs, sheep, horses, chickens, and turkeys, as well as from

ess that occurs in enveloped viruses such as influenza virus

humans. Viruses isolated from different animals exhibit

where precursor glycoproteins must be cleaved to activate

extensive serological cross-reactivity but limited ability to

fusion activities.

replicate in other hosts. As one example, rotaviruses isolated

from monkeys and cows can infect humans but cause much

milder symptoms than human rotaviruses, and have been

The Human Rotaviruses

examined for use as vaccines.

The rotaviruses cause diarrhea, primarily in newborns

Rotaviruses have been assigned to five species to the cur-

and the young, and the human rotaviruses are the single

rent time, rotavirus A, B, C, D, and E. Two tentative spe-

most important cause of severe diarrheal diseases of infants

cies may also be recognized in the future, rotavirus F and

and young children. In one study in the United States, non-

G. Assignment to species is based on sequence identities

bacterial infectious gastroenteritis, of which rotaviruses

and, where known, on the ability of members of a species

account for about half (Fig. 5.6), was found to be the sec-

to undergo reassortment during mixed infection. Human

ond most common disease in humans, accounting for 16%

infection is caused by viruses in rotavirus A, B, and C.

of illnesses over a 10-year period and occurring on average

1.5 times per person per year. Severe diarrhea can result in

Rotavirus Structure and Replication

dehydration that can be fatal if fluids are not replaced. In

Rotaviruses contain 11 genome segments that sum to

the developing world where hospitalization is not readily

18.6 kb (Table 5.3) and which are simply numbered from

available, diarrheal disease, about half of which is caused

1 to 11 in order of decreasing size. As for orthoreoviruses,

by rotaviruses (Fig. 5.6), are a major cause of infant mor-

one segment encodes two proteins in overlapping reading

tality. Firm estimates of the extent of diarrheal disease in

frames and the mRNA transcribed from another segment is

developing countries are difficult to establish, but in Asia,

translated into two proteins using two in-frame start codons

Africa, and Latin America there may be more than 1 bil-

so that they differ only in that one is a truncated form of the

lion cases of diarrhea each year with 23 million deaths.

other. Of the 12 proteins that differ in primary sequence, 6

The majority of deaths occur in children less than 5 years

are structural and 6 are nonstructural. Proteins are numbered

of age, where 14% of diarrheal episodes are fatal. Overall

in order of decreasing size as either virion structural proteins

it has been estimated that rotaviral disease causes between

(VP1-8, where VP4 is cleaved to produce VP5 and VP8) or

300,000 and 800,000 deaths worldwide each year. The glo-

nonstructural proteins (NSP1-6).

bal distribution of deaths caused by rotaviruses is illustrated

The replication of the virus and the overall structure of

in Fig. 5.7.

the virion resemble those of the orthoreoviruses, but with

Replication of rotaviruses is normally confined to termi-

some exceptions. Virions are triple shelled rather than dou-

nally differentiated enterocytes that line the tips of micro-

ble shelled and the virion is distinguishable from that of

villi in the small intestine. How the deaths of these cells

orthoreoviruses in the electron microscope (Fig. 2.5). The

provokes diarrhea has not been definitively established,

Developed Countries

Third World

Parasites

Unknown

1.4%

18%

Rotavirus

Unknown

Other

Rotavirus

5.5%

46%

39%

Bacteria

46%

Toxigenic

E. coli

21%

Bacteria

6.9%

Astrovirus

0.7%

Astrovirus

Adenovirus

Calicivirus

0.7%

Calicivirus

6.7%

0.7%

FIGURE 5.6 An estimate of the role of various etiologic agents in severe diarrheal disease requiring hospitalization in

infants and young children in developed countries and in the Third World. Adapted from Kapikian (1993).

each dot = 1000 deaths

FIGURE 5.7 Deaths (estimated) from rotavirus in 2003 in children under 5 years old. Adapted from Glass (2006).

The 10 countries that suffered the greatest losses are Ethiopia (28,905); Nigeria (47,525); the Democratic Republic of

Congo (28,905); Tanzania (11,440); India (146,044); Bangladesh (18,986); Indonesia (14,064); China (41,076); Pakistan

(36,450); and Afghanistan (17,830). These countries together account for about two-thirds of the worldwide deaths.

although maladsorption and osmotic diarrhea are thought to

confined to replication in the enteric tract, recent studies

be involved. Rotaviral diarrhea is watery, suggesting either

have shown that rotavirus infection may result in systemic

a secretory process or an osmotic process. It is also known

infection, albeit rarely.

that one of the viral proteins, NSP4, is an enterotoxin that

In temperate climates, epidemics of rotaviral disease occur

will induce diarrhea when given to mice. Although normally

in the winter. This is illustrated in Fig. 5.8 for epidemics in

Melbourne

Drought

JSN

JM M

JSN

JM M

J SN

JMM

JSN

J MM

J SN

JMM

J S N JMM J S

JMM

1980

1981

1982

1983

1984

1985

1986

1987

Washington, D. C.

J MM

JSN

JM M

JSN

JMM

JSN

JMM J S N

JM M

S N JMM

J SN

JMM

JSN

1975

1976

1977

1978

1979

1980

1981

1982

FIGURE 5.8 Monthly rotavirus infections over a multiyear period. Seasonal fluctuations are clear, with the cooler

months shaded in blue. Upper panel shows hospitalizations for gastroenteritis in Melbourne, Australia. The lower panel

shows hospitalizations in a children's hospital in Washington, D.C. Note the anomalous pattern for Melbourne in 1983,

a year in which there was a severe drought. Data from Barnes et al. (1998) and Brandt et al. (1983).

Melbourne, Australia, and in Washington, D.C. In southern

Rotavirus Vaccines

Australia, epidemics peak in the JuneSeptember time frame

Because of the seriousness of rotaviral disease on a world-

(their winter), while epidemics peak in the JanuaryMarch

wide basis, there have been ongoing efforts to develop vac-

time frame in the eastern United States. In the United States,

cines against rotaviruses. Such vaccines have been directed

epidemics peak in November and December in the warmer

at newborns and infants, in whom the problem is most

southwestern states and move to progressively later months

acute. Vaccine development and interpretation of results

as they spread to cooler states north and east (Fig. 5.9). As

from clinical trials have been complicated by several fac-

is the case for seasonality in epidemics of disease caused

tors, including the possible presence of maternal antibodies

by other viruses, the reasons for the association of rotaviral

in newborns, the fact that rotaviral infections often do not

outbreaks with cool weather are poorly understood.

Nov

Dec

Jan

Feb

Mar

Apr

FIGURE 5.9 Average time of peak rotavirus activity in the contiguous 48 states, United States, using cumulative data

from July 1991 to June 1997. This contour plot was derived using the median value for time of peak activity reported by

each regional or state diagnostic laboratory. The surveillance system and analytic methods used to create this map are

described in greater detail in Török et al. (1997).

produce absolute and lasting immunity to subsequent rein-

severe rotaviral disease in newborns and the very young.

fection, and the fact that there are multiple serotypes of rota-

This vaccine (RotaShield) was licensed in 1998 for general

viruses that infect humans. There are four major serotypes

use in the United States. It was anticipated that this vaccine

of rotaviruses that cause widespread and serious infections

would be useful not only in the developing world where

of humans, and thus recent efforts have been directed toward

rotavirus infection causes many deaths in infants, but also in

developing a quadrivalent vaccine that would protect against

the developed world where rotaviral infections lead to many

all four serotypes. Furthermore, the objective of vaccination

cases of diarrhea each year that require hospitilization or vis-

has now been defined as the prevention of severe rotaviral

its to physicians. However, on widespread use in the United

disease in very young children rather than prevention of any

States, it was found that a small number of infants developed

rotaviral disease.

the bowel obstruction called intussusception after immuni-

With these objectives, a rotavirus vaccine was developed

zation. This obstruction sometimes clears spontaneously but

using a virus from another animal species, the rhesus rotavi-

can require a fairly benign treatment by medical personnel.

rus, as a human vaccine. The rhesus rotavirus replicates well

In a small minority of cases, surgery is required to correct

enough in humans to elicit an immunizing response but does

the defect. The vaccine was subsequently withdrawn.

not cause serious illness, and the use of this virus as a human

The withdrawal of the rotavirus vaccine raises interest-

vaccine has been compared to the use of cowpox virus by

ing legal and moral questions, and illustrates the difficul-

Jenner to immunize against smallpox (Chapter 7). The rhe-

ties associated with developing and introducing vaccines.

sus rotavirus will successfully immunize people against only

Roughly 1 out of 2000 infants develop intussusception in

one of the four major rotavirus serotypes, and not against

the first 2 years of life. It seems clear that vaccination with

the others. However, rotaviruses contain 11 genome seg-

the rotaviral vaccine triggers intussusception in a small

ments and the different segments readily undergo reassort-

fraction of vaccinees, since immunization with RotaShield

ment when tissue culture cells are infected by more than

increased the risk of intussecption 20- to 30-fold within the

one strain, that is, genome segments can be interchanged in

first 2 weeks after vaccination, and one in 10,000 vaccinees

progeny viruses. This property was used to isolate reassorted

developed the condition immediately after immunization.

rhesus rotaviruses in which all of the segments were derived

However, it is not known whether vaccinated infants are

from rhesus rotavirus except for the segments encoding the

more likely to develop the obstruction in the first 2 years of

surface proteins, which were derived from human viruses

life than are nonvaccinated infants. It is even possible that

of the other three major serotypes. Clinical trials showed

the vaccine is actually protective in that fewer vaccinated

that a quadrivalent vaccine based on rhesus rotavirus and

infants will ultimately develop intussusception than non-

three reassorted rotaviruses, so that all four serotypes impor-

vaccinated infants. Furthermore, in the United States, about

tant for humans were present, was successful at preventing

55,000 children are hospitalized every year for rotaviral

disease and 20 to 40 die. Use of the vaccine in the United

ticks. Members of five species, including African horse

States would drastically reduce hospitalizations and the

sickness virus, bluetongue virus, and epizootic hemorrhagic

death rate would probably decline since intussusception is

disease virus, cause disease in domestic animals, and mem-

almost never fatal with proper treatment. However, because

bers of four other species cause human disease. Human dis-

of the legal atmosphere in the United States and the ethical

ease caused by naturally acquired orbiviruses may require

dilemma of giving a problematical vaccine for a disease that

hospitalization but is normally not life threatening, and no

is seldom life threatening in developed countries, the vac-

orbivirus is considered to be an important human pathogen.

cine has not been used in the United States since its with-

However, the animal pathogens may cause serious illness in

drawal. Since the vaccine was not used in the United States

domestic or wild animals (Table 5.5).

or other developed countries because of concerns about its

safety, developing nations did not adopt it, despite the fact

Veterinary Pathogens

that the vaccine would undoubtedly have saved hundreds of

thousands of infants from fatal rotaviral infection if it were

African horse sickness virus (AHSV) has caused many

widely used in such countries, where as many as 0.5% of

epidemics of fatal illness in horses in sub-Saharan Africa.

infected children die. A riskbenefit study in Peru, for exam-

The distribution of this virus is illustrated in Fig. 5.10C.

ple, concluded that the vaccine would prevent 1440 deaths

The first recorded epidemic occurred in the Cape Colony

and 23,000 hospitalizations as compared to perhaps 78 cases

in 1719. Thereafter, disastrous epidemics occurred every

of intussusception that might be vaccine related.

20 years or so up until the twentieth century, when vaccines

Now, 8 years and perhaps 5 million deaths later, the

became available. The virus appears to have been endemic,

problem appears to have been solved with the licensing of

presumably in the zebra, but wherever horses were intro-

two new rotavirus vaccines, Rotateq from Merck, licensed

duced, epizootic AHSV was sure to follow. The mortality in

worldwide, and Rotarix from Glaxo Smith Kline, licensed

introduced horses is close to 90%, and AHSV had a major

in Latin America, Africa, Asia, and the European Union. No

impact on agriculture, exploration, and conquest in Africa.

association with intussusception has appeared to date with

The military had to operate without cavalry and the early

the use of these vaccines. These vaccines are also live virus

explorers often walked rather than rode. The virus represents

vaccines, which is important for use in developing coun-

an example of an endemic virus that appears to cause little

tries because they can be given orally. Furthermore, because

disease in its native host (zebra), but which causes very seri-

the viruses replicate in the enteric tract, such vaccines give

ous illness when transmitted to a nonnative host (horses).

rise to IgA that protects the mucosal surfaces infected by

Another important veterinary pathogen is bluetongue

rotaviruses.

virus, which causes a serious disease in sheep. Bluetongue

virus is very widely distributed (Fig. 5.10A) and causes wide-

spread disease, but, interestingly, the strains of bluetongue

Genus Orbivirus

virus in Australia are not pathogenic. Also widespread are

The genus Orbivirus is widely distributed. It contains 21

viruses that cause epizootic hemorrhagic disease in animals

recognized species, each of which contains multiple sero-

(Fig. 5.10B), including outbreaks in deer in North America.

types. As examples, African horse sickness virus contains

9 serotypes and bluetongue virus contains 24 serotypes.

Persistence of Orbiviruses on Erythrocytes

Altogether, more than 150 serotypes of orbiviruses are

currently recognized. Orbiviruses contain 10 segments of

At least some orbiviruses have evolved an unusual way

dsRNA totaling 19.2 kb (Table 5.3) in an icosahedral vir-

of persisting in nature as arboviruses. In tropical areas of the

ion 86 nm in diameter. Reminiscent of rotaviruses, orbivi-

world, arthropods may be continuously active and viruses

ruses may exit the cell by budding through the cell plasma

associated with such arthropods may persist by continuous

membrane to acquire an unstable envelope that is soon lost.

passage between the vertebrate and the invertebrate host.

Viruses can also be extruded from cells without acquiring

However, in many areas of the world, including some tropi-

an envelope.

cal areas, the vector activity may become low or nonexistent

Different orbiviruses infect a wide range of higher ver-

during some periods, such as the dry season or the winter.

tebrates including ruminants, horses and their relatives,

Arboviruses must be able to survive such periods of low vec-

rodents, bats, marsupials, sloths, primates including humans,

tor activity, and different arboviruses have solved this prob-

and birds. Unlike reoviruses and rotaviruses, orbiviruses are

lem in different ways. Most have evolved ways to persist

arboviruses, transmitted by biting flies, mosquitoes, or ticks,

in the invertebrate host during periods of inactivity. Many

and able to replicate in the vector as well as in the vertebrate

can be passed transovarily in the arthropod host, others can

host. Among the vectors known for various orbiviruses are

survive in diapausing insects. Orbiviruses, in contrast, have

insects of the genus Culicoides (midges and gnats), phlebot-

evolved a mechanism to persist in the vertebrate host for long

omine flies, culicine and anopheline mosquitoes, and Ixodes

periods. Viral infection of vertebrates is normally cleared

TABLE 5.5

Representative Orbiviruses, Coltiviruses, and Seadornaviruses Causing Disease

in Humans and Domestic Animals

Genus

Virus

Hosts

Vector

Disease syndromes

Distribution

Orbivirus

African horse sickness

Horse, dog, zebra

Culicoides

Cardiopulmonary disease,

Africa, Asia

(midges)

hemorrhagic fever

Africa, Asia, Australiaa,

Bluetongue

Sheep, cow, goat,

Culicoides

Fever, frothing at mouth,

deer

(midges)

shock, coronitis

Americas

Epizootic hemorrhagic

Deer

Culicoides

Similar to bluetongue

Americas, Australia, Africa

disease

(midges)

Palyam

Cow

Culicoides

Abortion

South Africa, Japan

(midges)

Orungo

Humans

Mosquitoes

Febrile illness

Africa

Changuinola

Humans

Phlebotomines

Febrile illness

Panama

Kemerovo

Humans

Ticks

Febrile illness, encephalitis

Russian, Eastern Europe

Coltivirus

Colorado

Humans

Ticks

Febrile illness, encephalitis,

North America

tick fever

hemorrhagic fever

Eyach

Humans

Ticks

Encephalitis ?

Europe

Seadornavirus

Banna,

Humans

Mosquito

Meningoencephalitis

Southeast Asia, Indonesia, China

Kadipiro

Australian isolates of bluetongue virus are not pathogenic.

rapidly by the immune system so that viremia of sufficient

shown in Fig. 5.11. The life cycle of the virus is illustrated in

titer to infect a new arthropod taking a blood meal normally

Fig. 5.12. Although CTF infects many mammals, including

lasts only a few days. However, bluetongue virus becomes

humans, the natural cycle of transmission involves primarily

associated with red blood cells by binding to glycophorins

small mammals and larval and nymphal ticks. Adult ticks may

on the surface of the cell, where it persists in indentations in

transmit the virus to humans and large mammals outside the

the membrane in a nonreplicating state protected from the

normal transmission cycle. Transovarial transmission does not

immune system. The virus evidently can remain attached

occur in ticks and larval ticks are normally infected by feeding

and viable for the life of the erythrocyte. When an arthropod

on small mammals that are viremic. In humans, CTF causes

takes a blood meal, the virus bound to erythrocytes is able

an illness characterized by fever, myalgia, chills, headache,

to initiate infection of the arthropod. Because erythrocytes

and malaise, and 20% of cases require hospitalization. The

have an average lifetime of 160 days, the virus can persist in

acute illness lasts 510 days. Recovery may be uneventful

a viable state in the vertebrate host for many months.

or convalescence may be prolonged for several weeks. CNS

infection or hemorrhagic fever may occur, almost always in

children. Fatalities are rare (<0.1%).

Genus Coltivirus

The second species in the genus is Eyach virus. This

The coltiviruses possess 12 genome segments summing to

virus is widely distributed in Europe where the major host

28.5 kb (Table 5.3). The virion is icosahedral and 80 nm in

is thought to be the European rabbit Oryctolagus cunniculis.

diameter. Like the orbiviruses, the coltiviruses are arboviruses,

It is transmitted by Ixodes ticks and has been associated

transmitted by ticks. Two species are recognized, one found in

with febrile illness and encephalitis in humans. It is hypoth-

North America and the second in Europe. The prototype virus

esized that the virus was introduced into Europe from North

is Colorado tick fever virus (CTF), from which the genus gets

America through migration of lagomorph ancestors from

its name (Colorado tick fever). CTF is present in the Rocky

America to Europe about 30 million years ago.

Mountain area of North America at elevations from 4000 to

10,000 feet, and two serotypes are known. It has been isolated

Persistence of CTF in Erythrocytes

from a number of mammals including humans and from ticks

and mosquitoes that serve as vectors. Transmission to humans

CTF has evolved a way to persist in the vertebrate host

is usually by Dermacentor andersoni ticks whose range is

that resembles that used by bluetongue virus. CTF infects

Bluetongue virus (BTV)

Equator

Pathogenic BTV

Nonpathogenic BTV

Hemorrhagic disease of deer (IHDD), Ibaraki virus, and the

Kemerovo group viruses

Equator

IHDD

Ibaraki

Kemerovo

African Horse Sickness (AHSV)

Equator

AHSV

FIGURE 5.10

Geographical distribution of orbiviruses that causes disease in animals and humans. Adapted from

Fields et al. (1996) p. 1736.

British

Columbia

Saskatchewan

Alberta

(1)

Washington

Montana

(130)

Oregon

South

Idaho

(14)

Dakota

Wyoming

(6)

(17)

(88)

Nevada

Utah

Colorado

(255)

(907)

California

(21)

New

Arizona

Mexico

Range of Tick Vector

Dermacentor andersoni

FIGURE 5.11

Distribution of the primary vector of Colorado tick fever, Dermacentor andersoni, shown in color,

and the number of diagnosed human cases of Colorado tick fever in various states between 1980 and 1988. From Tsai

(1991).

bone marrow cells early after infection, including erythro-

recognized, Banna virus, Kadipiro virus, and Liao ning

cyte precursors. The virus remains within the erythrocyte

virus, of which Banna virus was the first isolated and the best

after it matures, in a nonreplicating state, and appears to

characterized. Vectors include Anopheles, Culex, and Aedes

persist for the life of the erythrocyte. When a tick takes a

mosquitoes. Banna has been isolated from humans suffering

blood meal, it can be infected by the virus within the eryth-

from encephalitis or febrile illness, but its association with

rocyte. The differences in the mechanisms used by CTF

human illness has not been conclusively shown since there

and bluetongue virus to persist in the blood of vertebrates

are many viruses in this region that cause such diseases.

are thought to reflect the different vectors used. Culicoides

flies, the vector of bluetongue, take smaller blood meals

Comparison of the Reoviridae with Other

and digest the blood meal in a different way than ticks, the

RNA Viruses

vector of CTF.

Some aspects of the replication cycle of the Reoviridae

resemble those of single-strand (+)RNA viruses (Chapter 3)

Genus Seador navirus

and other aspects resemble those of single-strand (-)RNA

Seadornaviruses contain 12 genome segments and are

viruses (Chapter 4). Replication proceeds through a plus-

transmitted by mosquitoes. The viruses are found in tropi-

strand intermediate that is a messenger RNA and that

cal and subtropical regions of Southeast Asia, primarily

exists, at least transiently, free in the cytoplasm, a charac-

China and Indonesia. The name of the genus is derived from

teristic of (+)RNA viruses. Reoviruses could have origi-

Southeastern Asia dodeca RNA viruses. Three species are

nated from the plus-strand viruses through the acquisition

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