Family Orthomyxoviridae - Viruses And Human Disease

Borna disease virus is neurotropic and establishes a

behavioral abnormalities. Because of these effects on other

chronic or persistent infection despite an immune response

animals, several recent studies have tried to determine if the

to viral infection. The chronic infection results at least in part

virus is associated with neurological disease in man, in par-

because the virus downregulates its replication, resulting in

ticular with schizophrenia. Serological surveys have found

very low production of infectious virus. Downregulation to

that psychiatric patients are more likely to have antibodies

establish a persistent infection uses a mechanism different

to bornavirus than normal controls. Surveys which assay for

from those described in Chapter 3 for alphaviruses (shut-

the presence of viral RNA in peripheral blood mononuclear

off of minus-strand RNA) and for pestiviruses (titration of

cells (PMBCs) are even more suggestive: in some surverys

a cellular component required for RNA replication). Borna

up to 66% of psychiatric patients, including schizophrenics,

disease virus, like other (-)RNA viruses, has an inverted ter-

are positive for bornaviral RNA, compared to <5% of normal

minal repeat at the ends of the genomic RNA that contains

controls. Furthermore, very small amounts of virus-specific

promoters for RNA replication. During replication, the four

RNA have been isolated from postmortem brain samples

terminal nucleotides at the 5′ ends of both the genomic and

from patients suffering from schizophrenia and bipolar dis-

antigenomic RNA are often trimmed so that the majority

order, but not from normal individuals or patients suffering

of RNAs are missing these four nucleotides. The truncated

from other neurological disorders. Interestingly, a recent

RNA can be transcribed to produce mRNA but cannot repli-

study found that two patients hospitalized for severe depres-

cate, thus resulting in downregulation of RNA replication.

sion exhibited a rise in bornavirus antigen in PMBCs during

Borna disease virus appears to have a very wide host

the course of the disease, which fell to very low levels on

range. It was originally described as a pathogen of sheep and

recovery. Whether these different associations are indicative

horses in Germany, but is now known to infect a wide vari-

of causality remains to be determined, but it is conceivable

ety of warm-blooded vertebrates, birds as well as mammals.

that the virus causes recurrent episodes of depression on

The reservoir host in Switzerland has recently been reported

reactivation of a latent infection.

to be a shrew (Crocidura leucodon). These rodents are

exclusively terrestrial and it is thought that horses become

FAMILY OR THOMYXOVIRIDAE

infected by grazing on forage contaminated by excretions

from infected shrews.

As described, the virus establishes a chronic infection

The family Orthomyxoviridae (ortho = true or correct)

characterized by neurotropism and low production of virus.

contains three genera of influenza viruses: Influenzavirus A,

Infection may be asymptomatic or may result in disease

which contains influenza virus A; Influenzavirus B, which

characterized by movement and behavioral abnormalities.

contains influenza virus B; and Influenzavirus C, which con-

Naturally infected horses exhibiting such abnormalities usu-

tains influenza virus C (Table 4.7). Thogoto virus, a tick-

ally recover, but the disease may progress to paralysis and

borne virus of mammals, forms a fourth genus, Thogotovirus

death. Experimentally infected rats and primates also exhibit

and infectious salmon anemia virus belongs to a fifth genus,

TABLE 4.7 Orthomyxoviridae

Virus name

World

Genus/members

abbreviation

Usual host(s)

Transmission

Disease

distribution

Influenzavirus A

Influenza A

FLUAV

Humans, birds, swine

Airborne

Respiratory disease

Worldwide

Influenzavirus B

Influenza B

FLUBV

Humans

Airborne

Respiratory disease

Worldwide

Influenzavirus C

Influenza C

FLUCV

Humans

Airborne

Respiratory disease

Worldwide

Thogotovirus

Thogoto virus

THOV

Mammals

Tick-borne

Respiratory disease

Southern Europe,

Africa

Isavirus

Infectious salmon anemia

ISAV

Fish

Waterborne

Anemia, hemorrhagic

North Atlantic,

liver necrosis

North America

those of M (when present) and N of other (-)RNA viruses,

Isavirus. Influenza viruses A and B are closely related, but

respectively. The three proteins encoded in the three largest

influenza A infects a wide spectrum of birds and mammals

segments of influenza, called PB2, PB1, and PA (B or A

including humans, with birds being the reservoir, whereas

refers to a basic or acidic pK), possess the RNA polymerase

influenza B infects primarily humans and humans are the

activities encoded in the L protein and the P protein of other

reservoir. Influenza C is more divergent. Eight segments

(-)RNA viruses. Influenza A and B have two surface glyco-

of (-)RNA, totaling about 14 kb, comprise the genomes of

proteins, called HA and NA, but influenza C has only one,

influenza A and B viruses (Fig. 4.1) whereas influenza C

called HEF. These glycoproteins have the receptor-binding,

has only seven segments. Influenza viruses use sialic acid as

fusion, and receptor-destroying activities present in surface

a receptor, but the form used by influenza A and B viruses

glycoproteins of (-)RNA viruses.

differs from that used by influenza C virus, and the enzymes

Two proteins, called NS1 and NS2 (NS for nonstructural),

encoded by the viruses to destroy receptors are correspond-

are produced from RNA segment 8. NS1 is produced from the

ingly different. All three influenza viruses infect humans

unspliced mRNA (replication occurs in the nucleus). It binds

and cause disease, but influenza A represents the most seri-

to RNAs in the nucleus, including cellular pre-mRNAs, cel-

ous human pathogen because it causes very large, recurrent

lular snRNAs which are involved in splicing, and dsRNA.

epidemics with significant mortality. Influenza A has there-

Its activities inhibit the transport of cellular mRNAs from

fore been the most intensively studied and has been the focus

the nucleus and promote the synthesis of influenza mRNA.

of efforts to control influenza in humans.

NS1 also regulates splicing of influenza mRNAs and their

transport from the nucleus to the cytosol. Another function

Proteins Encoded by the Influenza Viruses

of NS1 is to interfere with the interferon pathway (Chapter

10), in part by binding dsRNA, which is a major inducer of

The proteins encoded in the different gene segments of

interferon and a cofactor for some proteins in the interferon

influenza A and influenza C viruses are described in Table

response to viruses, and in part by interacting with cellular

4.8. Influenza A produces 10 proteins from its eight genome

proteins involved in the interferon response. Influenza virus

segments, and most of these proteins have analogues in other

lacking NS1 is very sensitive to interferon and is replication

(-)RNA viruses (Fig. 4.1). The matrix protein, M1, and

defective in cells or hosts capable of synthesizing interferon,

the nucleocapsid protein, NP, perform functions similar to

TABLE 4.8

Genome Segments of Influenza Viruses

Influenza A

Influenza C

RNA

Encoded Protein

RNA

Encoded Protein

Functiona

segment

Length (nt)

Name

(aa)

segment

Length (nt)

Name

(aa)

2341

PB2

759

Cap recognition,

2365

PB2

774

RNA synthesis

2341

PB1

757

RNA synthesis

2363

PB1

754

2233

716

RNA synthesis

2183

709

2073

566

Hemagglutinin, fusion,

2073

HEF

655

major surface

antigen, sialic acid

binding. HEF of

FLUCV also has

esterase activity

1565

498

Nucleocapsid protein

1809

565

1413

454

Neuraminidase

1027

252

Matrix protein

Spliced

242

1180

p42

374

ßSignalase

M1′(p31) + CM2

See footnoteb

Spliced

259 + 115

934

NS1

230

Nonstructural protein

934

NS1

286

See footnoteb

Spliced

NS2

121

Spliced

NS2

122

All functions other than those in footnote "b" apply to both influenza A and influenza C.

M2 of Flu A forms an ion channel, and NS2 of FluA is a nuclear export protein; the functions of the comparable moieties of Flu C are unknown.

Source: Adapted from Fields et al. (1996) Table 2 on p. 1355 and data in Fauquet et al. (2005) p 683.

this site. If the cleavage site consists of multiple basic residues

whereas the wild-type virus is resistant to the interferon

that can be recognized by the intracellular enzyme furin, the

pathway. NS2 is produced from a spliced mRNA. It inter-

virus can replicate systemically in at least some hosts. The SV-

acts with M1 attached to influenza RNP and promotes the

5 type 1 glycoprotein has only fusion activity and is called F.

transport of the RNP to the cytoplasm. It is present in small

As described before, it is produced as a precursor F0 which is

quantities in the virion and so is not truly nonstructural.

cleaved to F1 and F2, and the nature of the cleavage site affects

Protein M2 is produced from a spliced mRNA from seg-

the virulence of the virus (see Viral Glycoproteins under

ment 7. It forms ion channels in membranes, probably as a

tetramer, that allow passage of H+ ions. During transport of HA

Paramyxoviridae earlier in this chapter).

to the cell surface, the presence of M2 in the membrane of the

The receptor bound by both influenza A virus and by SV-

transport vesicle causes the pH within the vesicle to equilibrate

5 for entry into cells is sialic acid. The type 2 glycoprotein of

with that in the cytosol. This prevents low pH activation of

influenza has neuraminidase activity and is called the neu-

the fusion activity of HA during transport, because transport

raminidase or NA. It removes sialic acid from glycoproteins

vesicles are otherwise acidic. M2 is also present in virions and

for the same reasons as described for the paramyxoviruses

is required for the disassembly of the virus and for the activa-

that use sialic acid as a receptor. The type 2 glycoprotein of

tion of the RNA polymerase activity. To become active, the

SV-5 has both neuraminidase activity and receptor-binding

polymerase in the interior of the virus must be exposed to low

(hemagglutinating) activities and is called HN.

pH. Influenza virus enters the cell in endosomes, which are

Influenza HA is present as a trimer on the surface of the

progressively acidified. The acidic pH not only triggers a con-

virus (as is F of SV-5). The trimeric spike has a long stalk

formational change in HA that results in fusion of the viral

and a head containing the sialic acid binding sites. As shown

membrane with the endosomal membrane, but it also activates

in Fig. 1.6, exposure to acid pH in endosomes produces a

the RNA polymerase of the virion through the activity of M2.

dramatic rearrangement of the spike in which the fusion pep-

M2 is the target of the drug amantadine, one of the relatively

tide, which forms the N terminus of HA2, is moved over a

few drugs that are effective against a viral disease. Amantadine

distance of more than 10 nm to the tip of the spike. Here it

binds M2 of most influenza strains and prevents it from act-

inserts into the target membrane and promotes fusion of the

ing as an ion channel, which prevents the activation of the

viral membrane with the target membrane. NA is present as

polymerase. When taken early during infection, amantidine

a tetramer (as is HN of SV-5), and forms a spike that is dis-

ameliorates the symptoms of influenza. A worrisome trend is

tinguishable in the electron microscope from the HA spike.

the appearance of amantidine-resistant variants of influenza, in

There is only one surface glycoprotein in influenza C, the

particular the H5N1 strain referred to as "bird flu."

hemagglutinin-esterase-fusion protein (HEF). Influenza C

In most, but not all, influenza A viruses an 11th protein

virus has, therefore, one fewer gene segments than influenza

(PB1-F2) is made. This protein is translated from an alter-

A. HEF has receptor-binding (hemagglutination), fusion, and

native reading frame from the mRNA of PB1. It is present

receptor-destroying activities. The receptor is sialic acid, but

in mitochrondria in infected cells and may serve to regulate

the activity that destroys the receptor is an esterase activity.

apoptosis by the cell.

The esterase does not remove sialic acid from proteins as

does NA of influenza A. Instead it removes the 9-O-acetyl

group from 9-O-acetyl-N-acetylneuraminic acid, the recep-

Influenza Glycoproteins

tor used by influenza C, and the virus does not bind to the

Comparison of the glycoproteins of influenza A virus and

deacylated sialic acid.

the paramyxovirus SV-5 is of interest. In both influenza A

virus and SV-5, one of the glycoproteins is type 1 (N terminus

Replication of Influenza RNA and Synthesis

out) and one is type 2 (C terminus out). In both cases, the type

of mRNAs

1 glycoprotein is produced as a precursor that must be cleaved

to activate the fusion activity required for entry into cells. The

Synthesis of influenza virus RNAs occurs in the nucleus,

type 1 glycoprotein of influenza A has fusion and receptor-

rather than in the cytoplasm as for most RNA viruses. This

binding (hemagglutinating) activities and is called the hemag-

makes possible the differential splicing observed for two of

glutinin or HA. The precursor is called HA0 and the cleaved

the mRNAs. Following infection by the virus, the viral RNPs

products are called HA1 and HA2 (which remain covalently

are transported to the nucleus and mRNA synthesis begins.

linked by a disulfide bond after cleavage of the peptide bond)

During synthesis of mRNA, influenza engages in a process

(Fig. 1.6). Cleavage is required to activate the fusion activity

called "cap-snatching." Capped cellular pre-mRNAs present

in the nucleus are bound by NS1, and the 5¢-terminal 1013

of the virus and the nature of the cleavage site influences the

nucleotides, containing the 5¢ cap, are removed by PB2. This

virulence of the virus. If the cleavage site consists of a single

basic amino acid, cleavage is extracellular and influenza rep-

oligonucleotide is used to prime synthesis of mRNA from

lication is restricted to the respiratory tract, and in the case of

the influenza genome segments, as illustrated in Fig. 4.13.

birds the gut as well, where there are enzymes that can cleave

Once initiated, other aspects of mRNA synthesis resemble

vcRNA

ppp-AGC AAAGCAGG

CCUUGUUUCUACU

15-22 nt

Replication

UUUUUU

vRNA

HO- UCGCUUUCGUCC

GGAACAAAGAUGA

(genome)

mRNA synthesis

"Cap-snatching"

A G

mRNA

AAAAAAAAAAAAA(PolyA)

m GpppX Y

GC AAAGCAGG

G A

10-13 nt

FIGURE 4.13 Relationship between genome RNAs, mRNAs, and vcRNAs of influenza virus. Transcription of

mRNAs in the cell nucleus requires a primer of 1013 nucleotides derived from cellular pre-mRNAs by "cap-snatching,"

and mRNAs terminate with a poly(A) tail. Those portions of the mRNA which are not complementary to the genome

RNA are shown in red. In contrast, vcRNAs are exact complements of the genomic minus strands. Adapted from Strauss

and Strauss (1997).

those that occur in rhabdo- and paramyxoviruses. Synthesis

mRNAs are formed from each of two of the segments, and

continues to near the end of the genome segment, where an

in total, 10 mRNAs are formed and 10 proteins are produced

oligo(U) stretch is encountered. Here the enzyme stutters to

(11 in the case of viruses that also produce PB1F2 described

produce a poly(A) tail on the messenger and then releases

earlier). The formation of the two mRNAs from segment 7

it. In addition to its role as a primer, using a cap derived

and their translation into proteins is illustrated schematically

from cellular mRNA relieves the virus of the necessity of

in Fig. 4.14.

encoding enzymes required for capping and ensures that the

When sufficient amounts of viral proteins have been

virus mRNA has a cap suitable for the cell in which it is

synthesized and transported to the nucleus, viral RNA rep-

replicating. This mechanism also results in interference with

lication begins. Replication requires encapsidation of prog-

the synthesis and transport of host mRNAs. Furthermore,

eny genomic and antigenomic RNAs as described for other

because the mRNAs have a different 5¢ end and lack the 3¢

(-)RNA viruses, and the mechanisms that lead to a switch

end of the antigenomic RNA, they lack promoters required

between synthesis of mRNAs and replication are thought

for replication and packaging and are therefore dedicated

to be similar to those that occur in rhabdoviruses and para-

mRNAs.

myxoviruses. During replication, the viral genome is cop-

Each genome segment gives rise to one primary mRNA

ied into a faithful antigenomic RNA (vcRNA) (Fig. 4.13),

species. However, two of these can be spliced, and both the

which is a perfect complement of the genome and serves as

unspliced and spliced RNAs serve as messengers. Thus, two

a template for production of genomic RNA.

M1 protein

(252aa)

Translation

59 CAP

M1 mRNA

Poly(A)

Cap-snatching, mRNA synthesis

Genome

Segment 7

Cap-snatching, mRNA synthesis,

splicing

CAP

M2 mRNA

Poly(A)

Translation

M2 protein

(97aa)

FIGURE 4.14 Synthesis of two mRNAs for the M1 and M2 proteins from gene segment 7 of influenza A. M1 RNA

is translated from ORF 1 (open box). M2 RNA starts identically, but after the splice it is translated in ORF2 (checkered

box). Both proteins are found in infected cells. The AUG initation codon is shown as a triangle; termination codons are

shown as filled diamonds. Patterned boxes at the end of the genome RNA are self-complementary sequences that could

form panhandles.

Synthesis of viral RNA, whether plus strand or minus

Assembly of Progeny Virions

strand, requires that the synthetase interact with both ends

of the RNA, whether vRNA or vcRNA; that is, the promoter

Influenza virus matures by budding of nucleocapsids

for synthesis of RNA is composed of elements from both

through the cell plasma membrane. Virions are pleomorphic

ends of the RNA. This is analogous to what has been found

but clinical specimens are primarily filamentous and can be

for alphaviruses and flaviviruses, described in Chapter 3,

up to a micrometer or more in length. Upon passage in cell

and may be a general mechanism used by many or all RNA

culture, most strains eventually give rise to virions that are

viruses. Thirteen nucleotides at the 5¢ end of the vRNA and

primarily spherical, averaging 100 nm in diameter. The form

12 nucleotides at the 3¢ end are highly conserved in influenza

that the virions assume is genetically determined. Studies of a

A viruses and these seem to contain the entire promoter ele-

strain of influenza A that remained filamentous after passage

ment. These sequences form an inverted terminal repeat and

in cell culture could be induced to form spherical particles

are capable of forming a panhandle structure (Fig 4.15A),

by changes in the M1 protein. The significance of filamen-

bringing the two ends together where they might interact

tous versus spherical particles is unknown, but filamentous

with the RNA synthetic machinery. An alternative structure,

forms must have a selective advantage in the infected ani-

called the corkscrew structure, is thought to be the structure

mal, whereas spherical forms seem to be selected upon pas-

recognized by the synthetase for initiation of RNA synthesis

sage in cell culture.

(Fig. 4.15B). Cyclization is also hypothesized to play a role

During assembly, the eight genome segments are reas-

in addition of poly(A) to mRNAs, by causing the polymerase

sorted in progeny virions if the cell is infected with more

to stutter at the oligo(U) tract located just before the double-

than one strain of influenza. Reassortment to produce

strand stem of the circular structure. Figure 4.15C illustrates

viruses with mixed genomes is efficient--the segments

an experiment to examine the sequence requirements within

are almost randomly reassorted to give all possible com-

the panhandle or corkscrew structure.

binations of genome segments in the progeny virions. This

Panhandle Configuration

11 12 13

A G U A G A A A C

A G G

U C C

U C G U U U U C G

Region II

Region I

Corkscrew Configuration (vRNA promoter)

G A

U A

59 A G C

G U

U C

U U

Mutations in vRNA Promoter

Mutant D1

Mutant D2

Mutant D3

3 x 108 pfu

1 x 104 pfu

5 x 107 pfu

7 x 108 pfu

FIGURE 4.15 Models for the influenza A virus promoter. (A) The Panhandle model, with a partially double-stranded

structure for the 5′ and 3′ terminal sequences. (B) The Corkscrew model predicting base pairing within the ends. After

Neumann et al. (2004). (C) Alternative base pairs introduced into the vRNA promoter and the effect of these changes on

viral yield in MDBK cells. Adapted from Catchpole et al. (2003), Figure 1.

process is analogous to the reassortment of chromosomes

proteins do drift. This together with the fact that the viruses

that takes place during sexual reproduction in diploid

seldom cause disease in their avian reservoirs show that

organisms.

influenza in birds is ancient and the virus has adapted to its

Budding must result in the packaging of the 8 different

primary host.

genomic segments that constitute the viral genome into one

The gene segments of influenza A virus reassort read-

virus particle if it is to be infectious. Reoviruses (Chapter

ily during mixed infection, and viruses with new combina-

5) have an assembly mechanism whereby the 1012 differ-

tions of genes arise frequently. Newly arising reassortants

ent segments are recognized and assorted so that each virus

can cause major epidemics of influenza when introduced

particle has one each of the different segments. The case for

into humans, a process called antigenic shift. Not all com-

influenza virus is not completely clear. Evidence has been

binations of genes give rise to viruses that are capable of

presented that the virus appears to package more than 8 seg-

epidemic spread in humans. Only three subtypes of HA (H1,

ments, possibly about 10, that are randomly chosen from the

H2, and H3) and two or three subtypes of NA (N1, N2, and

intracellular pool. Random packaging of 10 segments would

possibly N8) have been found to date in epidemic strains of

result by chance in about 3% of the virions having at least

human influenza virus. The first influenza virus isolated, in

1 each of the 8 different genome segments. However, more

1933, was called H1N1. This virus first appeared as the cause

recent data argue that the virions package exactly 8 segments,

of the great influenza epidemic of 1918 (see later). The virus

one each of the 8 different segments. For this to occur, the

isolated in the epidemic of 1957 had a different subtype of

packaging machinery has to recognize internal sequences in

both HA and NA and was called H2N2. The H2N2 virus

each of the segments and not just a packaging signal in the

replaced the H1N1 virus as the cause of influenza epidemics

conserved ends of the viral RNAs.

(Fig. 4.17). The H2N2 virus was itself replaced by H3N2

virus beginning with the epidemic of 1968. Serological sur-

veys suggest that prior to 1918 the virus that circulated was

Influenza A Virus

an H3N8 virus that first appeared as the cause of an epi-

demic in 1890. The reason that only a subset of HAs appear

Natural History of Influenza Virus

to be capable of causing epidemics in humans is, at least in

part, the fact that the receptors for the virus are somewhat

Influenza A virus infects a wide variety of birds and mam-

different in birds and humans. Sialic acid is linked to galac-

mals. A phylogenic tree that shows the relationships of the

tose predominantly by α2,6 linkages in humans but by α2,3

NP genes of viruses isolated from humans, pigs, and birds is

linkages in birds.

shown in Fig. 4.16. The human isolates and the pig isolates

Similarly, only certain types of the other segments

are closely related; as described later the pig viruses prob-

are compatible with infection of and epidemic spread in

ably originated from a human virus. The humanpig clade is

humans. For example, the nucleocapsid gene has diverged

distinct from the avian clade, however.

into five lineages, but only one of these lineages is present

Influenza A viruses are characterized by their two major

in viruses isolated from humans (see, e.g., Fig. 4.16). NS1

surface antigens, HA and NA. There are 16 different HA

is also at least partially host specific and thus only certain

subtypes (numbered H1 to H16). HAs in different subtypes

NS1s are compatible with human infection. Other proteins

differ by 30% in sequence and are not immunologically

also differ somewhat for optimal replication in birds versus

cross protective. There are also 9 different NA subtypes

mammals. It is thought that reassortment can result in the

(numbered N1 to N9). The major reservoirs of influenza A

introduction of a new HA or NA gene into a human virus,

in nature are wild ducks and other waterfowl such as gulls,

that is, a virus whose other gene segments are optimized

terns, and shearwaters, and viruses containing all 16 sub-

for human infection. The HA and NA proteins are the most

types of HA and all 9 subtypes of NA have been isolated

important antigens of the virus, and change of one or both

from waterfowl. Influenza replicates in the lung and in the

of these antigens gives rise to a virus for which the major-

gut of birds and the infection is normally asymptomatic (but

ity of the human population has no immunity and which is

epidemics of fatal influenza have occurred in turkeys and

therefore capable of causing a global pandemic. One pos-

chickens, and the emerging H5N1 virus has caused fatal

sible scenario is that pigs serve as intermediates ("mixing

infection in a number of different bird species). Ducks can

vessels") in the recombination process, because pigs can

excrete virus in feces for weeks, infecting other ducks via

be infected by both avian and human viruses (they contain

contaminated water, and a significant fraction of ducks may

sialic acid in both α2,3 and α2,6 linkage) and reassortment

become infected by the virus in this process. Migratory

could occur in this host.

ducks then spread the virus around the world, normally in

Influenza A virus is an example of a zoonotic disease in

a northsouth direction. The viruses in birds are in stasis.

humans. The reservoir of the virus is ducks and other birds,

Almost no differences in amino acid sequences of the vari-

and human infection is irrelevant for the maintenance of the

ous proteins are present in viruses separated by many de-

virus in nature.

cades, although the nucleic acid sequences encoding these

HOST

CLADE

SH90 (H3N2)

Sw/DN83(H3N2)

TE78(H3N2)

Udorn72(H3N2)

Vic86(H2N2)

Sw/HK76(H3N2)

Bei68(H3N2)

HK68(H3N2)

Sin57(H2N2)

MI60(H2N2)

Loy57(H1N1)

England55(H1N1)

Brazil78(H1N1)

FtWa50(H1N1)

FtMo47(H1N1)

Hickox40(H1N1)

WS33(H1N1)

PR34(H1N1)

1918 Influenza

Sw/IO46(H1N1)

Sw/IO30(H1N1)

Sw/OH35(H1N1)

Sw/37(H1N1)

Sw/May54 (H1N1)

Sw/WI57(H1N1)

Sw/WI61(H1N1)

Ty/NC88(H1N1)

Sw/NE98(H3N2)

Sw/TN77(H1N1)

Sw/HK82(H3N2)

Eq/Prague56(H7N7)

Eq/FL63(H3N8)

Eq/KY88(H3N8)

Gull/MD77(H13N6)

Gull/USSR84(H13N6)

Duck/PA69(H6N1 )

Duck/NY78(H2N2 )

Duck/TN76(H3N8)

Duck/Man.53(H10N7)

Ty/Ont.66(H5N9)

Ch/PA83(H5N2)

Ty/MN80(H4N2)

Gs/GD96(H5N1)

FPV34(H7N1)

Ch/Ger49(H10N7 )

Sw/Neth85(H1N1)

Sw/Ger81(H1N1)

Duck/Bav77(H1N1)

Tern/SA61(H5N3)

Duck/HK75(H3N2)

FIGURE 4.16

Phylogenetic tree of the nucleotide sequences of the influenza A virus NP gene sequences, constructed

with a neighbor-joining algorithm. Each isolate name includes the location and year of isolation, preceded for non-

human viruses by a species designation and a diagonal slash. Species abbreviations: Sw, swine; Ty, turkey; Ch, chicken;

Gs, goose. Standard two letter abbreviations for states in the United States are used. Other location abbreviations: SH,

Shanghai; DA, Dandong; Vic, Victoria; HK, Hong Kong; Sin, Singapore; Loy, Loygang; Ft Wa, Fort Warren; Ft Mo,

Fort Monmouth; Man, Manitoba; Ont, Ontario; GD, Guangdong; Ger, Germany; Neth, Netherlands; Bav, Bavaria; SA,

South Africa. The boxed isolate is the probable source of the H5 hemagglutinin in the currently worrisome "bird flu"

spreading from China. Adapted from Reid et al. (2004), Figure 2.

Type H2N2 appears

Type H3N2 appears

1935 1940 1945 1950 1955 1960 1965 1970 1975 1980 1985 1990 1995 2000

Year

Influenza A type H1N1

Influenza B

Influenza A type H2N2

Cocirculating B and A

Influenza A type H3N2

Cocirculating A types H1N1 and H3N2

FIGURE 4.17 Excess mortality caused by influenza A and B virus in the United States between 1934 and 1998. "1935"

refers to the winter of 19341935. Excess mortality due to the three dominant subtypes of influenza A and influenza B

are indicated by the colors shown in the key. Green cross-hatched bars are excess mortality in years when both A and B

viruses circulated. In 1955 and 1965, type H2N2 circulated with B, in 1983 and 1988 type H1N1 circulated with B, and

in 1985, 1992, 1996, and 1998 H3N2 circulated with B. In 1995 H1N1 and H3N2 types of influenza A both circulated

(hatched purple bar). Redrawn from Fields et al. (1996) p. 1421, with additional data from Thompson et al. (2003).

Epidemics of Influenza

Lower respiratory tract infection can also occur follow-

ing influenza infection and result in primary viral pneumo-

Influenza A virus causes a serious human illness, influ-

nia. Invasion of the damaged lungs by pathogenic bacteria

enza. It is perhaps confusing and unfortunate that the term

may follow and result in secondary bacterial pneumonia.

flu is often used to describe any respiratory tract infection

Influenza can be fatal, usually because of pneumonia result-

(and at times even infections of the gastrointestinal tract),

ing from viral infection, whether the pneumonia is due to

even those that are fairly mild. The symptoms of true influ-

primary viral infection or, more commonly, due to secondary

enza are usually more severe than those resulting from

bacterial infection. Fatal infection is more common in the

other respiratory tract infections and include fever, head-

very young (whose immune system is not fully developed)

ache, prostration, and significant muscle aches and pains

and in the elderly (whose immune system may be waning).

(myalgias) that last for 36 days. Weakness and cough can

Before the advent of antibiotics, bacterial pneumonia killed

last 12 weeks more. The fever can be high (3940°C is

many following severe bouts of influenza, but even today

not uncommon in adults and can be higher, especially in

influenza remains a serious killer. It has been estimated that

children). The morbidity that accompanies the disease can

influenza virus infects 1020% of the world's population

cause the patient to remain bedridden for a week or longer.

every year causing five million cases of severe illness and

In young children, the high fever can result in Reye's syn-

250,000 to 500,000 deaths. In the United States alone the

drome, an encephalopathy that may be fatal. The probabil-

estimated death rate from influenza in an average year is

ity of contracting Reye's syndrome is higher if aspirin is

20,00030,000 and can be significantly higher in epidemic

administered to control the fever.

years. People over 65 are at particular risk from influenza.

derly in 1917, the normal pattern, is apparent. The dramatic

The annual death rate in the United States from influenza A

increase in the death rate in the 20- to 29-year-old group in

in people over 65 is 1 per 2200, and in an epidemic year the

1918, in which people of this age were more likely to die

death rate may be 1 in 300 (i.e., 1 of every 300 people over the

than the old and the young, is striking. Death rates in young

age of 65 die of influenza during the epidemic). The excess

adults 1534 years of age were more than 20-fold higher

mortality caused by influenza is illustrated in Fig. 4.17, in

in the 19181919 pandemic than in the preceding years,

which the different strains of influenza A or B responsible

and the death toll in young adults in the United States was

for the epidemics are indicated. Although influenza A is usu-

high enough that overall life expectancy dropped sharply, as

ally the most serious cause of mortality, in some years influ-

illustrated in Fig. 4.19.

enza B is more of a problem than influenza A.

The overall mortality was perhaps 2% of the world

population but in some regions of the world, for example,

regions of Central America and certain islands in the Pacific,

The 1918 Influenza Epidemic

1020% of the entire population died in the epidemic. In

A pandemic of influenza erupted in 1918 due to the emer-

some remote Alaskan villages, more than 70% of all adults

gence of a virulent H1N1 strain. This extremely virulent

died, usually as a result of the simultaneous incapacitation of

virus swept around the world over a period of about a year

the entire population so that supportive care was not avail-

and infected an estimated 30% of the world's population,

able. The final death toll can never be known with certainty

causing 20100 million deaths. Although the very young

and estimates vary widely, from 20 to 100 million. The death

and the elderly are normally at the most risk from influ-

toll exceeded that produced by World War I, which was

enza, this influenza pandemic of 19181919 was unusual

ongoing at the time. In fact, 80% of deaths in the U.S. Army

in that mortality was highest in healthy young adults. The

during World War I resulted from influenza, and it is thought

age distributions of people dying of influenza and the related

that the final collapse of the German army in 1918 may have

pneumonia are compared for the years 1917 and 1918 in Fig.

been precipitated by widespread influenza in the troops. The

4.18. The much higher death rates in the young and the el-

surgeon general of the United States had expressed the hope

Deaths due to:

Influenza and Pneumonia 1918

Pneumonia 1917

Influenza 1917

0-9

10-19

20-29

30-39

40-49

50-59

>60

Age Brackets (years)

FIGURE 4.18 Age distribution of deaths due to pneumonia and influenza in the United States in 1917 and 1918. Age at

death of patients has been divided into 7 intervals of 10 years each. The percent of deaths due to pneumonia in 1917, due to

influenza in 1917, and due to the combined effects of pneumonia and influenza during the great epidemic year 1918 which

fall into each age bracket are shown. The epidemic shows the atypical preponderance of deaths in the 2029 and 3039

year old brackets during the 1918 epidemic. Data from Crosby (1989). For comparison, from 1990 to 1998 only 3.8% of

deaths due to influenza and pneumonia occurred in persons <49, 4.75% in persons 5064, and 91% in persons over 65

years old (updated information from Thompson et al. 2003).

U. S. Life Expectancy

1918

1900

1910

1920

1930

1940

1950 1960

1970

1980

1990

2000

Year

FIGURE 4.19 Life expectancy in the United States, showing the precipitous drop in 1918 because of deaths due to

the "Spanish flu." This drop interrupted an otherwise fairly uniform increase in life expectancy that resulted from better

health care, sanitation, and living conditions. Note also the leveling off in the late 1980s and 1990s due to AIDS. Adapted

from ASM News, July 1999, and more recent data from the National Center for Health Statistics.

that WWI would be the first war in which more U.S. sol-

RNA in these tissue samples that could be used to reconstruct

diers died of war injuries than died of disease, but this hope

the complete sequences of genome segments. The sequences

was shattered by the influenza epidemic. Descriptions of

from these five victims are almost identical and showed that

the epidemic with a focus on its effects on U.S. society are

the virus belonged to strain H1N1. The HA genes from these

found in the books Flu, by G. Kolata, America's Forgotten

five humans differ by only one to three nucleotides despite

Pandemic, by A. W. Crosby, and, quite recently The Great

the fact that they came from five humans whose deaths were

Influenza, by John M. Barry.

separated by over 7500 miles and several months in time.

The reasons for the extreme virulence of the 1918 virus,

The sequence of this gene places it in the humanswine lin-

and why healthy young people were more likely to die, a topic

eage, not in the avian lineage, and at the root of the tree

made even more important by the appearance of H5N1 "bird

leading to later isolates of human or swine influenza (Fig.

flu" (see Chapter 8) have been addressed recently using the

4.20). Thus, the HA of the virus does not appear to have

power of modern molecular biology. The pandemic of 1918

come directly from an avian source.

occurred before influenza virus could be isolated. However,

It is now possible to use reverse genetics to take a cloned

the sequences of all eight gene segments of the 1918 influenza

DNA copy of an influenza gene and rescue a virus contain-

genes have been obtained starting from a number of tissue

ing this gene. To do this, cells are transfected with up to

isolates. Samples of preserved lung tissue taken at autopsy

17 plasmids that express the 8 genome segments of influ-

from two U.S. soldiers who died of influenza in September

enza as well as the RNA polymerase proteins PB1, PB2,

1918 in New York and South Carolina were found to con-

and PA, and the NP protein, and in some cases the other

tain detectable influenza RNA, albeit in fragmented condi-

influenza proteins as well. Infectious influenza virus is pro-

tion. A third source of influenza RNA came from an Alaskan

duced and buds from the cell. Using this system influenza

Inuit victim who died in November 1918 and was buried in

virus has been produced that contains various combinations

permafrost, and whose body was sufficiently well preserved

of the 1918 HA and NA genes with other cloned genes from

that lung samples containing (fragmented) viral RNA were

the 1918 virus or from recent isolates, including virus that

obtained. Two additional sources of influenza sequences

contains the complete complement of the 1918 genes and

come from two victims of influenza who died of pneumonia

thus is a complete reconstruction of the 1918 virus. Various

in November 1918 and February 1919 at the Royal London

constructs have been tested in mice. Whereas recent iso-

Hospital. Reverse transcriptasepolymerase chain reaction

lates of influenza virus cause only mild disease in mice, the

technology was used to obtain sequences from influenza

1918 virus causes severe, often fatal disease. In mice, a virus

Japan89

MA90

Stk90

Fiji88

USSR77

Len54

0.05

Brazil 78

Distance

PR34

Sw/Cambridge39

WS33

WSN33

1918 Influenza

Sw/IO30

Sw/29

Sw/IL63

Sw/NE92

Sw/Japan80

Sw/Quebec 91

Sw/IO88

Sw/NJ76

Sw/HK74

Sw/Italy81

Duck/Alb76

Duck/WI80b

Duck/WI80a

Duck/TN85

1917 Alaskan Brant

Gs/HK/76

Oy/Ger87

Duck/Aust/80

Ty/Ger91

Ty/Ger90

Duck/Bav77

Duck/HK76

Duck/HK77

H2 Japan57

FIGURE 4.20 Phylogeny of the H1 hemagglutinin genes (bases 494659 aligned to the comparable sequence of

PR34). Viral names include species of isolation followed by location and year of isolation. Species include: Sw, swine;

Gs, goose; Ty, turkey; Oy, oystercatcher. In the United States the standard two letter abbreviation for the state is used;

outside the United States the following abbreviations are used: Len, Leningrad; Ger, Germany; HK, Hong Kong; Bav,

Bavaria; Aust, Australia; Stk, Stockholm; Alb, Alberta. The sequence of the 1918 pandemic strain and the avian strain

most closely related chronologically are boxed. A distance bar, where a distance of 0.05 = 11.2 synonymous differences,

is shown above and beside the tree, and the H2 hemagglutinin of Japan 57 virus is used as an outgroup. Adapted from

Fanning et al. (2002).

containing only the H1 and N1 of the 1918 virus was found

immune system, and that healthy young people, who have

to be highly virulent and caused fatal infection in mice. Virus

the strongest immune systems, suffered from more extensive

grew to high titer in the lungs of the mice and was associ-

release of potent cytokines that resulted in more extensive

ated with an influx of neutrophils and macrophages into the

tissue destruction.

infected lung.

The devastation caused by the 1918 virus raises continu-

The complete 1918 virus has also been tested, under

ing concern that a strain of influenza of equal virulence might

BSL-4 high containment conditions, in monkeys as well as

appear and again cause immense suffering worldwide. New

mice. The virus caused severe, usually fatal, disease in mon-

pandemic strains of influenza appear three or four times a

keys that was marked by much higher replication rates and

century. If a pandemic strain emerged from a virus such as

more extensive spread in the lungs. It was also marked by an

the H5N1 strain of bird flu (see Chapter 10), which has a

abnormal innate immune response (see Chapter 10). Certain

very high mortality rate in humans, the resulting epidemic

elements of the innate response were attenuated, perhaps

could indeed be devastating.

because of the activity of the NS1 gene which is known

to interfere with the immune response. In contrast, other

Antigenic Shift and Drift

immune responses, in particular inflammatory cytokines,

were enhanced, resulting in a "cytokine storm." The results

Immunity to influenza A virus following infection is

are consistent with the hypothesis that in humans the 1918

long lived but may not be complete and is subtype specific

virus provoked an extreme but unbalanced reaction by the

and even strain specific. The continuing appearance of new

strains that arise from antigenic drift and of new subtypes

must be made by late spring in order to allow time for the

that arise from antigenic shift lead to continuing epidemics.

pharmaceutical companies to prepare the vaccines, and an

Normally, two or three strains of influenza A circulate in the

element of risk is involved that the right choices will not

human population at any one time. Spread from person to

be made. The World Health Organization publishes choices

person is by respiratory droplets, requiring close proximity,

and supplies seed virus based upon the recommendation of

but people travel extensively and new strains of the virus

an international group of scientists, but the final selections

speed around the globe as they arise. Antigenic drift is the

are made by individual health agencies and the choices are

process by which mutations accumulate in the virus genome,

usually, but not always, correct. The number of vaccine

usually because of immune selection, that result in the devel-

manufacturers has declined dramatically in the United States

opment of new strains of the virus. These new strains are

over the last 2 decades because of legal liability problems,

partially resistant to the immunity induced by infection with

and what limited capacity that exists for manufacturing flu

previous strains of virus. After several years of drift, the

vaccine is mostly present in Europe. Production problems

strain may be sufficiently distinct to cause disease in a per-

by one of the manufacturers has resulted in recent shortages

son previously infected, but the illness is usually less severe

of vaccine.

because of partial immunity to the new strain. However,

The necessity to grow the virus in fertilized eggs also

new strains capable of causing serious illness can arise by

limits the amount of vaccine that can be produced. There

antigenic shift whereby reassortment results in change of

are efforts to develop a cell culture system for virus pro-

the surface glycoproteins of the virus. The reassortants that

duction for vaccine use, which could then be produced in

cause the biggest problems are those belonging to a new

larger amounts. Efforts are also being made to develop bet-

subtype (as illustrated by Fig. 4.17). As described, such a

ter adjuvents for use with the vaccine, which could reduce

new subtype may cause a pandemic in the human population

the amount of antigen required per inoculation. In addition,

because there is little immunity to the virus carrying these

obtaining the reassortants required for vaccine production is

new surface antigens, as happened in 1918 (H1N1), 1957

a time-consuming endeavor using classical methods of coin-

(H2N2), and 1968 (H3N2).

fecting cells with two different viruses and searching through

H1N1 virus, which had disappeared with the appearance

the progeny for the wanted reassortants. If reverse genetics

of the H2N2 epidemic strain in 1957, suddenly reappeared

described earlier can be developed in a way that satisfies

in 1977. This H1N1 virus, which first appeared in northern

the regulatory agencies concerned with vaccine safety, the

China in May 1977 and was called the Russian flu, was vir-

desired reassortants could be obtained much more quickly,

tually identical to influenza virus isolated from an epidemic

allowing quicker responses to new strains of virus.

in humans in 1950. It circulated in young people who had

In addition to the inactivated virus vaccine that is very

not been exposed to H1N1 virus. Because it was virtually

widely used, a new live virus vaccine based on a cold attenu-

unchanged despite 27 years having elapsed, it seems unlikely

ated virus has been licensed recently. Reassortment is used to

that it arose again de novo. Presumably this virus had been

introduce the HA and NA of the predicted epidemic strains

preserved in a frozen state, probably in a laboratory freezer.

into this attenuated virus. Because the attenuation of the virus

In 1976, in response to reports that investigators outside

results from changes in other genome segments, the recom-

Western Europe planned to develop and test vaccines against

binant strain is also attenuated. The vaccine is administered

H1N1 influenza, a WHO meeting report urged extreme cau-

by nasal spray rather than by injection as is the inactivated

tion in developing live vaccines from epidemic H1N1 strains

virus vaccine. To date this vaccine is only licensed for use in

because of the possibility of spread of the virus. One year

people between the ages of 5 and 49, and thus it cannot be

later the virus reappeared.

used for the populations most at risk for serious illness, but

clinical trials are continuing. It remains to be seen how well

accepted this vaccine will be.

Vaccination against Influenza A Virus

The necessity of reformulating the vaccine every year

Because of the seriousness of influenza disease, especially

is inconvenient for a number of reasons including the fact

in the elderly, attempts are made each year to vaccinate the

that the vaccine cannot be stored for use in the follow-

population at risk. Because of drift and shift, the vaccine

ing years. In addition, the vaccine is not always effective

must be reformulated every year to reflect the viruses cur-

because wrong predictions were made about which strains

rently circulating in the human population. There are three

of virus would be the biggest problems. There is an effort

strains of virus included in the most common vaccine, an

being made to develop universal vaccines that would target

inactivated virus vaccine produced from viruses grown in

all strains of influenza A and B, and that would therefore

eggs. These are two influenza A viruses and one influenza B

provide protection against all influenza strains and that

virus. These viruses are chosen from those that are circulat-

could be used year after year. One possibility that is being

ing in late spring, because these viruses are usually those

pursued is to use influenza A M2 protein as an antigen.

that will cause epidemics the following winter. The choice

This protein is highly conserved among all A strains but is

not normally seen by the immune system for some reason.

pharmaceutical houses. The vaccine was never conclusively

Preliminary studies have shown that this protein linked to

shown to cause disease, although there seemed to be a slight

hepatitis B core protein is highly immunogenic in mice and

increase of Guillain-Barré syndrome following inoculation.

provides protection against influenza A infection in mice,

Litigation went on for years and substantial damages were

regardless of strain. For influenza B, a subunit vaccine

paid out. In retrospect it is easy to criticize the program as an

based upon the sequence surrounding the cleavage site of

overreaction, but what would have been the reaction if noth-

the HA precursor, which includes a highly immunogenic

ing had been done and an influenza epidemic developed that

part of the fusion peptide, shows promise in early animal

resulted in 50100 million Americans becoming seriously

trials.

ill with 12 million deaths? Given the state of knowledge

at the time, many leaders felt there was no choice. Further,

the decision to vaccinate was not so different from cur-

Swine Flu Virus

rent policy, where strains of influenza A circulating in the

Continuing surveillance of influenza strains in nature is

spring are incorporated into a vaccine to be given in the fall.

required in order to reformulate the vaccines each year. This

A quote from the U.S. Surgeon General at a meeting of the

surveillance also serves to watch for the possible appear-

Association of State and Territorial Health Officiers in 1957

ance of another killer strain of influenza. An episode that

is worth thinking about: "I am sure that what any of us do,

occurred during the Ford administration, however, illus-

we will be criticized either for doing too much or for doing

trates the potential difficulties of identifying such a strain

too little."

and reacting in time. In February of 1976, a young soldier

at Fort Dix died of influenza and others became seriously

Bird Flu

ill. Tests showed that most of the soldiers were suffering

from the A/Victoria strain of influenza that was epidemic in

A recent scare began when 18 people in Hong Kong

the United States at the time or from adenovirus infection.

became seriously ill from influenza in 1997 and 6 died. The

However, the soldier who died and three other soldiers who

culprit was an avian influenza (H5N1) that was epidemic

were ill were infected with an influenza strain that was epi-

in birds being sold in the markets for food. Avian viruses

demic in pigs, referred to as swine flu. Serology studies indi-

do not normally infect people, and there was fear that an

cated that 200 or more other soldiers had been infected by

avian virus had made the jump to humans and might cause

this virus as well, showing that the virus was being transmit-

an epidemic of lethal influenza. The Hong Kong authorities

ted from person to person. The swine flu virus was closely

destroyed 1.6 million domestic birds in order to eradicate the

related to the 1918 pandemic virus, and is thought to have

epidemic in birds. No human-to-human transmission took

been introduced into pigs in 1918 from humans and to have

place and the virus disappeared. In 2002, however, H5N1

continued to circulate in pigs after it had died out in humans.

virus reappeared and by 2006 it has spread throughout Asia

Could it be possible that the 1918 virus had reappeared as

and into Africa and Europe. This virus has a mortality rate of

an epidemic virus in humans? The decision was made by

about 50% in humans and more than 140 people have died

President Ford, in consulation with leading scientists, to

of H5N1 infection as of this date. There is no person-to-per-

begin a crash program to develop a vaccine against swine flu

son transmission to date, but there is concern that the virus

and to begin to immunize the American population. It was

might mutate and cause a wide and devastating pandemic

thought, with some justification, that to wait for an epidemic

of influenza. This subject is considered at more length in

to begin before an immunization program was undertaken

Chapter 8.

would mean that it would be too late to be effective, given

the speed with which influenza epidemics spread. Further,

Influenza B and C Viruses

influenza is usually epidemic in winter, and the early detec-

Humans are the reservoir of influenza B virus. It causes

tion of this virus made possible the preparation of a vac-

influenza in humans but there exists only one subtype and

cine before the (next) winter flu season set in. Forty million

antigenic shift does not occur. Antigenic drift does occur,

Americans were immunized against swine flu. No epidemic

and the virus can cause epidemics of serious illness that

of swine flu developed, however, and litigation began. The

result in increased mortality, particularly among the elderly,

pharmaceutical companies had been reluctant to participate

as shown in Fig. 4.17. For this reason, the current strain of

in the program, pointing out that at any one time a certain

circulating influenza B is included in the annual flu vaccine.

fraction of Americans would develop encephalitis or rheu-

However, wide-ranging pandemics do not occur and the

matoid arthritis or any one of hundreds of other diseases.

virus is therefore not as much of a problem as influenza A.

If disease developed in proximity to receiving a new and

Less attention has accordingly been given to the study of this

relatively untested vaccine, a lawsuit would certainly fol-

virus. Influenza C is not a serious human pathogen and has

low and the potential damages were enormous. The program

been even less well studied.

could only advance when Congress agreed to indemnify the

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