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Orthomyxoviridae

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Orthomyxovirus (Influenza) Family
The name myxovirus was originally applied to influenza viruses. It
meant virus with an affinity for mucins. Now there are 2 main groups
– the orthomyxoviruses and the paramyxoviruses. Their differences
in simple terms are shown in Table 1.
All orthomyxoviruses are influenza viruses. Isolated strains are
named after the vims type (A, B, C), the host and location of initial
isolation, the year of isolation, and the aniigenic designation of the
hemagglutinin and neuraminidase. Eleven hemagglutinin antigenic
subtypes and 8 neuraminidase antigenic subtypes are designated.
Both of these are glycoproteins under separate genetic control, and
they vary independently. Examples of influenza designations follow:
A/swine/New Jersey/8/76 (H1N1), previously (HswINI)
A/Brazil/78 (H1N1)
B/Singapore/79
A/Bangkok/79 (H3N2)
Table 1. Differences between orthomyxoviruses and
paramyxoviruses
0rthomyxoviruses
Paramyxoviruses
Viruses and Influenza A,B,C
diseases
Mumps, measles,
respiratory syncytial,
parainfluenza
Genome
Single-stranded RNA
in 8 pieces, MW2-4X
106
Single-stranded RNA in
single piece, MW5-8X
106
Inner
ribonucleoprotein helix
9-nm diameter
18-nm diameter
INFLUENZA
Influenza is an acute respiratory tract infection that usually
occurs in epidemics. Three immunologic types of influenza
virus are known: A, B, and C.
Antigenic changes continually take place within the A group of
influenza viruses and to a lesser degree in the B group,
whereas influenza C appears to be antigenically stable.
Influenza A strains are also known for pigs, horses, ducks,
and chickens (fowl plague). Some animal isolates are
antigenically similar to the strains circulating in the human
population.
Influenza virus type C differs from the type A and type B
viruses; its receptor-destroying enzyme does not appear to be
a neuraminidase, and its virion structure is not fully
understood. The following descriptions are based on influenza
virus type A.
Properties of the Virus
A. Structure: Influenza virus consists of pleomorphic,
approximately spherical particles having an external diameter of
about 110 nm and an inner electron-dense core of 70 nm.
The surface of the virus particles is covered with 2 types of
projections, or spikes, approximately 10 nm long possessing
either the hemagglutinin or the neuraminidase activity of the
virus. A model of the influenza virion is shown in Fig.
Influenza Virus Model Cutaway
The RNA genome consists of 8 distinct pieces with an aggregate
molecular weight of 2-4 x 106.
Viral RNA has not proved to be infectious. But this structure
contains the virion-associated RNA-dependent RNA polymerase as
well as the genome. Evidently, all messenger RNA is
complementary to the virion RNA.
Because of a divided genome, viruses of this group exhibit several
biologic phenomena such as high recombination frequency,
multiplicity reactivation, and ability to synthesize hemagglutinin and
neuraminidase after chemical inactivation of viral infectivity.
The results of hybridization studies on RNA have supported the
immunologic grouping of the hemagglutinins of the influenza A
viruses. Similar studies of the neuraminidase genes have been in
agreement with N antigen subtype designations based on the
results of serologic tests.
The nucleocapsid is further organized by the coiling of the
whole RNP strand into a double helix 50-60 nm in diameter The
protein component of this structure has a molecular weight of
60,000 and is associated with the group-specific CF antigen A
protein (M) shell surrounds the nucleoprotein and forms the
inner part of the virus envelope It is composed of a small protein
(MW 26,000) and constitutes about 40% of the virus protein
About 20% of the virus particle is composed of lipid, apparently
derived from the host cell The lipid is formed into a bilayer
structure
The hemagglutinin spike is responsible for the agglutination of
erythrocytes by this virus It is composed of 2 molecules of a
glycoprotein (MW 75,000) that may or may not be cleaved to form
2 disulfide-linked glycopeptides of molecular weight 27,000 and
53,000 The smaller of these is present at the end of the molecule
which is attached to the lipid.
The neuraminidase spike is responsible for the receptor
destroying activity of the virus, this activity results in elution of the
virus from host cells or erythrocytes The role of its activity in virus
replication is unknown It is composed of 4 polypeptide molecules
with a molecular weight of about 60,000 The arrangement of
these molecules is still a matter of debate Both the hemagglutinin
and the neuraminidase spikes have been purified, and a study of
the purified protein has helped to explain antigenic changes of the
virus
B. Reactions to Physical and Chemical Agents:
Influenza viruses are relatively stable and may be stored
at 0-4 °C for weeks. The virus is less stable at -20 °C than
at +4 °C. Ether and protein denaturants destroy infectivity.
The hemagglutinin and CF antigens are more stable than
the infective virus. Ultraviolet irradiation destroys
infectivity, hemagglutinating activity, neuraminidase
activity, and CF antigen, in that order. Infectivity and
hemagglutination are more stable at alkaline pH than at
acid pH.
Animal Susceptibility and Growth of Virus: Human strains
of the virus can infect different animals; ferrets are most
susceptible. Serial passage in mice increases its virulence,
producing extensive pulmonary consolidation and death. The
developing chick embryo readily supports the growth of virus,
but there are no gross lesions.
Wild influenza viruses do not grow well in tissue cultures. In
most instances, only an abortive growth cycle occurs, ie, viral
subunits are synthesized but little or no new infectious progeny
is formed. From most influenza strains mutants can be
selected that will grow in cell culture. Because of the poor
growth of many strains in cell culture, initial isolation attempts
should employ inoculation both of the amniotic cavity of the
embryonated egg and of monkey cell cultures.
The process of infection begins by adsorption of the virus onto
its
receptor
sites
(neuraminic
acidcontaining glycoproteins). The hemagglutinin protein is
involved in this reaction. The other spike protein,
neuraminidase, can destroy the site. The virus particle is taken
into the cell, where it is disrupted, causing a decrease in
detectable virus shortly after infection. Intracellular synthesis of
the viral RNA and protein then occurs. Viral RNA pieces are
synthesized individually in the nucleus within 2-3 hours. All
viral proteins are synthesized in the cytoplasm. Structural
proteins bind to the cell membrane and are joined by the
ribonucleoprotein. At 8 hours, new virus particles bud through
the membrane. Neuraminidase may be important in release of
the completed virion.
Transcription of Influenza
Virus Genes
Minus-sense RNAs (genome segments)
Viral RNA-dependent
RNA polymerase
Positive-sense mRNAs
Influenza Viruses: Replication
(-) genomic RNA
(+)RNA
(-) genomic RNA
In most influenza virus systems, noninfectious particles capable of
hemagglutination are produced (von Magnus phenomenon).
These particles, called "incomplete," increase in number upon
serial, high-multiplicity passage of the virus. The incomplete
paticles are smaller and more pleomorphic than standard virus,
and they interfere with replication of standard virus. They are
known as defective interfering, or DI, particles. The largest virus
RNA piece is missing from such particles.
Biologic Properties:
1. Hemagglutination. All strains of influenza virus agglutinate
erythrocytes from chickens, guinea pigs, and humans and—
unlike paramyxoviruses – agglutinate erythrocytes from many
other species as well. Agglutination of red blood cells occurs
when the hemagglutinin interacts with a specific receptor on the
red blood cell membrane. This receptor is a glycoprotein (MW 3 x
104) that contains sialic acid. This glycoprotein serves both as the
receptor site for the hemagglutinin and as the substrate for the
viral neuraminidase. Cleavage of the glycoprotein by the enzyme
dissociates the virion from the red cell, resulting
in spontaneous elution. After elution, the cell receptors are
destroyed and hence can no longer be agglutinated with fresh
virus; however, the eluted virus can reattach and agglutinate
additional cells.
Group antigen. All influenza A virus strains share a common
antigen, distinct from those of influenza B and C. This soluble
(S) antigen is found in the medium from infected cell cultures
and is a component of the ribonucleoprotein of the virus. It can
be identified by CF. Antibody to this nucleoprotein antigen
does not induce resistance to the virus in humans. The other
internal proteins and the RNA polymerase also have groupspecific antigenic activity.
Specific antigens. The infectious virus particles induce in
animals the development of virus-neutralizing and other
antibodies, and the inoculated animals become resistant to
infection. Influenza virus administered in large amounts is toxic.
The effect is apparently associated directly with the virus
particles and can be prevented by specific antibody.
Virions contain 2 subtype or strain-specific antigens – the
hemagglutinin (H1-H16) and the neuraminidase (N1-N9). The
hemagglutinin is the principal specific envelope antigen, and
differences in this antigen among strains of virus can be shown
by HI tests. Antibody to the hemagglutinin neutralizes virus and
is a protective mechanism.
Man – H1-H3 and N1-N2
Neuraminidase is antigenically distinct from the hemagglutinin
and is governed by a separate gene (RNA fragment); hence, it
can vary independently of the hemagglutinin. The antigens of
the hemagglutinin and the neuraminidase of the virus are the
basis for classifying new strains. Antibody against the
neuraminidase does not neutralize the virus, but it modifies the
infection, probably by its effect on the release of virus from the
cells. The antibody against the neuraminidase occurs in sera of
humans who experience infection. The presence of
antineuraminidase antibody results in marked protection against
disease.
Influenza A
• In Birds
– 16 HA A variants
– 9 NA A variants
• In Humans
– 3 HA A variants
• (H1, H2, and H3)
– 2 NA A variants
• (N1 and N2)
Influenza Type A Viruses:
Subtypes
 Subtypes
are distinguished by the antigenic
properties of HA and NA
 Three HA subtypes found in influenza type
A viruses that exhibit human-human spread
(H1, H2, H3)
 Two NA subtypes found in influenza type
A viruses isolated from infect humans (N1,
N2)
Influenza Type A Viruses:
Antigenic Shift 1889-1977
Year
1889
1900
1918
1957
1968
1977
Subtype
H2N2
H3N8
H1N1
H2N2
H3N2
H1N1
Common Name
Spanish flu
Asian flu
Hong Kong flu
Russian flu
A Pandemic Strain Emergence:
Reassortment of Influenza Viruses
Antigenic Shift
Avian
Reservoir
Human
virus
Avian
virus
Other
mammals?
Swine
New
Reassorted
virus
Influenza Viruses:
Antigenic Drift
 Gradual
accumulation of mutations that
allow the hemagglutinin to escape
neutralizing antibodies (Point mutation in
HA gene)
 Epidemic
strains thought to have changes
in three or more antigenic sites
ANTIGENIC DRIFT
GRADUAL ANTIGENIC CHANGE
WITHOUT A CHANGE IN SUBTYPE
H3N2
H3N2
H3N2
H3N2
1968
1975
1993
2004
HONG
KONG
VICTORIA
BEJING
FUJIAN
Two possible mechanisms for the antigenic variation
of influenza virus have been suggested:
(1) All possible configurations may be present in a pool of
antigens that exist throughout the globe; from these, highly
infectious strains arise and initiate epidemics. High
antibody levels to recent strains in the human population
will inhibit strains with major antigens that were dominant in
recently prevalent strains and will select strains of different
antigenic composition.
Serial passage of virus in mice vaccinated with the
homologous strain yields a virus with an apparent
rearrangement of antigens or the appearance of new
antigens. The change in antigenic character evolves slowly
on passage (antigenic drift).
(2) Antigenically different strains may be selected by
means of genetic recombination induced by selection
factors such as passage in a partially immune host.
When 2 strains of influenza virus are simultaneously
injected into mice or eggs, a new strain sharing the
properties of each parent strain may be recovered; this
has been attributed to genetic recombination (antigenic
shift).
Antibodies are important in immunity against influenza,
but they must be present at the site of virus invasion.
Resistance to initiation of infection is related to antibody
against the hemagglutinin. Decreased extent of viral
invasion and decreased ability to transmit virus to
contacts are related to antibody directed against the
neuraminidase.
Filamentous forms. In addition to the spherical
particles, elongated forms possessing the same surface
projections exist. The filamentous forms also agglutinate red
cells and elute from them. In its early passages in chick
embryos, the virus is usually in filamentous form, but with serial
passage it takes on a spherical appearance.
Recombination. The multisegment nature of the
influenza vims genome allows recombination to occur with high
frequency by reassortment between orthomyxoviruses of the
same group. The RNA fragments of different influenza A viкгses
migrate at different rates in polyacrylamide gets. Similarly, the
polypeptides of different influenza A viгuses can be
differentiated. Thus, using 2 different parental viruses and
obtaining recombinants between them, it is possible to tell
which parent donated which RNA fragment to the recombinant.
These techniques enable rapid and more complete analysis of
recombinants that emerge in nature
What is an Epidemic?
• The occurrence of more cases of disease than
expected in a given area or among a specific
group of people over a particular period of
time*.
Epidemic
What is a Pandemic?
• An epidemic occurring over a very wide
area (several countries or continents) and
usually affecting a large proportion of the
population.
• Examples:
– Cholera
– AIDS
– Pandemic Influenza
Pandemic
Pandemic Influenza
• Normal influenza season
about 36,000 deaths in US
• Pandemic influenza
1918 -- 20,000,000 deaths worldwide
-- 500,000 US deaths
1957 and 1968 pandemics
-- 104,000 US deaths
Epidemiology
Influenza occurs in successive waves of infection, with peak
incidences during the winter. Influenza A infections may vary
from a few isolated cases to extensive outbreaks that within a
few weeks involve 10% or more of the population, with rates of
50-75% in children of school age. The period between
epidemic waves of influenza A is 2-3 years. All known
pandemics were caused by influenza A strains. During the
pandemic of 1918-1919 more than 20 million persons died,
mainly from complicating bacterial pneumonias. Recent
pandemics occurred in 1957-1958 owing to A influenza (H2N2)
and in 1968 owing to A influenza (H3N2). In 1976 in New
Jersey, a new type of influenza arose that resembled swine
influenza (HswIN1), hut it failed to spread in spite of a lack of
immunity in most people under age 50 years. An enormous
government-sponsored vaccination campaign was stopped
because Guillain-Barre syndrome appeared in some
vaccinated individuals. The predominant influenza A in the
USA in 1978-1979 was an H1N1 variant of the strains
prevalent in the 1950s.
Influenza B tends not to spread through communities as
quickly as influenza A. Its interepidemic period is from 3 to
6 years. Small outbreaks of influenza B were frequent in
the USA in 1979-1980.
The main reason for the periodic occurrence of epidemic
influenza is the accumulation of a sufficient number of
susceptibles in a population that harbors the virus in a few
subclinical or minor infections throughout the year.
Epidemics may be started when the virus mutates to a new
antigenic type that has survival advantages and when
antibodies in the population are low to this new type. A
much more drastic change in the segmented RNA genome
occurs when antigenic shift occurs. This involves the
recombination of different segments of the RNA, each of
which functions as an individual gene.
In early life, the range of the influenza antibody spectrum is
narrow, but it becomes progressively broader in later years.
The antibodies (and immunity) acquired from the initial
infections of childhood are of limited range and reflect the
dominant antigens of the prevailing strains. Later exposures
to viruses of related but differing antigenic composition result
in an antibody spectrum broadening toward a larger number
of the common antigens of influenza viruses. Exposures later
in life to antigenically related strains result in a progressive
reinforcement of the primary antibody. The highest antibody
levels in a particular age group therefore reflect the dominant
antigens of the virus responsible for the childhood infections
of the group. Thus, a serologic recapitulation of past infection
with influenza viruses of different antigenic makeup can be
obtained by studying the age distribution of influenza
antibodies in normal populations.
Antibodies against swine influenza (perhaps related to the
pandemic influenza strain of 1918) have not been found in
persons born after 1923. Persons born during 1923-1933 had
their first influenza experience with a type A virus closely related
to the 1933 WS strain. Those born between 1934 and 1943 do
not possess swine or WS antibodies but have antibodies against
another type A virus, PR-8 (H0N1).
Another antigenic change occurred among the A viruses in 1946.
Strains occurring between 1946 and 1957 have been called Al,
or H1N1. strains. The influenza antibodies in persons born
between 1946 and 1957 are chiefly against the H1N1 strains.
With the widespread appearance of the type A2 Asian strain in
1957, the H1N1 subtypes were replaced by H2N2 viruses.
Type A2 virus seemed to be related to previous influenza
viruses, in that sera in 1957 from people who were age
70 years or older often contained antibody against A2
isolates. Furthermore, anti-A2 antibody increases were
found in sera from this age group after injections of type
A vaccine that did not contain the A2 virus (anamnestic
response). This suggests that viruses prevalent during
the 1889 pandemic contained H2N2 antigens shared with
the 1957 Asian strains.
Influenza B appears to be changing antigenically, since
almost all strains isolated in 1965-1966 were closely
related to B/Singapore/3/64, which differed significantly
from the formerly prevalent variant represented by
B/Maryland/1/59. In 1972, a new variant was isolated in
Hong Kong (B/HK/5/72) and then became the
predominant type B virus around the world. In 1979-1980,
for the first time in 6 years, type B viruses caused most of
the reported cases in the USA. Most of the type B strains
were closely related to B/Singapore/79.
Surveillance for influenza outbreaks is more extensive
than for any other disease in order to identify the early
appearance of new strains, with the aim of preparing
vaccines against them before an epidemic occurs.
Surveillance also extends into animal populations,
especially birds, pigs, and horses. Some believe that
pandemic strains arise from recombinants of human and
animal strains.
Since the virus causing fowl plague was identified as
human influenza A type in 1955, many influenza viruses
have been isolated from a wide variety of domestic and
wild bird species. Some of these include the major H and
N antigens related to human strains.
Avian influenza ranges from highly lethal infections in chickens
and turkeys to inapparent infections in these and other avian
species that harbor the same strains. Domestic ducks and quail
often manifest influenza infection by coughing, sneezing, and
swelling around the beak, with variable mortality rates. Wildlife
species and most domestic fowl show little or no signs of
disease.
The possibility that influenza viruses are transmitted between
birds and mammals, including humans, may seem unlikely,
particularly if the transfer were to be only by the respiratory
route. However, influenza viruses of ducks multiply in the cells
lining the intestinal tract and are shed in high concentrations into
water. These viruses remain viable for days or weeks in water. It
is possible that influenza among birds is a waterborne infection,
moving from wild to domestic birds and even to humans.
Where does influenza come from?
Type A constantly circulates in natural
reservoirs
Birds are the natural reservoir of all
subtypes of Influenza A viruses
Migratory waterfowl
Chickens, turkeys, ducks, geese
Humans
Pigs
Horses
Other
Pathogenesis and Pathology
The virus enters the respiratory tract in airborne droplets.
Viremia is rare. Virus is present in the nasopharynx from 1-2
days before to 1-2 days after onset of symptoms. The
neuraminidase lowers the viscosity of the mucous film in the
respiratory tract, laying bare the cellular surface receptors and
promoting the spread of virus-containing fluid to lower portions
of the tract. Even when neutralizing antibodies are in the blood
they may not protect against infection.
Antibodies must be present in sufficient concentration at the
superficial cells of the respiratory tract. This can be achieved
only if the antibody level in the blood is high or if antibody is
secreted locally.
Inflammation of the upper respiratory tract causes necrosis of
the ciliated and goblet cells of the tracheal and bronchial mucosa
but does not affect the basal layer of epithelium. Interstitial
pneumonia may occur with necrosis of bronchiolar epithelium
and may be fatal. The pneumonia is often associated with
secondary bacterial invaders: staphylococci, pneumococci,
streptococci, and Haemophilus influenzae.
Clinical Findings
The incubation period is 1 or 2 days. Chills, malaise, fever,
muscular aches, prostration, and respiratory symptoms may
occur. The fever persists for about 3 days; complications are not
common, but pneumonia, myocarditis, pericarditis, and central
nervous system complications occur rarely. The latter include
encephalomyelitis, polyneuritis, Guillain-Barre syndrome, and
Reye's syndrome (see below).
When influenza appears in epidemic form, the clinical findings
are consistent enough so that the disease can be diagnosed in
most cases. Sporadic cases cannot be diagnosed on clinical
grounds. Mild as well as asymptomatic infections occur. The
severity of the pandemic of 1918-1919 has been attributed to the
fact that bacterial pneumonia often developed.
The lethal impact of an influenza epidemic is reflected in the
excess deaths due to pneumonia and cardiovascular and renal
diseases. Pregnant women and elderly persons with chronic
illnesses have a higher risk of complications and death.
Reye's syndrome occurs mainly in children. It is characterized by
encephalopathy and fatty degeneration of the liver, and the
mortality rate is high. In 1979-1980, more than 400 cases were
reported in the USA, with a mortality rate near 30%. Reye's
syndrome is associated with influenza B, rarely with influenza A,
and sometimes with other viral diseases such as chickenpox and
zoster.
Virus-neutralizing antibody occurs earlier in nasal
secretions and rises to high liters sooner among those
already possessing high concentrations of IgA in their
nasal washings prior to the infection. Even though
infected with influenza virus, such individuals remain well.
In contrast, those with low nasal wash IgA levels prior to
infection are highly susceptible not only to infection but
also to clinical illness.
Immunity
Three immunologically unrelated types of influenza virus are
known and are referred to as influenza A, B, and C. In
addition, the swine, equine, and avian influenza viruses are
antigenically related to the human influenza A virus. Influenza
C virus exists as a single and stable antigenic type.
At least 18 different antigenic components have been
determined in type A strains of influenza virus by quantitative
adsorption methods. More undoubtedly exist. Strains share
their antigenic components, but in varying proportions. A strain
generally shares its antigens with strains prevalent within a
few years of its isolation.
Influenza Vaccines




Whole virus vaccine: inactivated virus vaccine grown
in embryonated eggs; 70-90% effective in healthy
persons <65 years of age, 30-70% in persons ≥65 years
Split virus vaccine: previously associated with fewer
systemic reactions among the elderly and children <12
years
Subunit vaccine: composed of HA and NA
Live, attenutated influenza virus vaccines under
development
Prevention and Treatment by Drugs
Amantadine hydrochloride and its analog rimantadine are
antiviral drugs for systemic use in the prevention of
influenza A. The drugs block penetration of or uncoat
influenza A virus in the host cell and prevent virus
replication. The established effect is prophylaxis, and
amantadine (200 mg/d) must be given to high-risk persons
during epidemics of influenza A if protection is to result.
Amantadine is relatively nontoxic but may produce central
nervous system stimulation with dizziness and insomnia,
particularly in the elderly. It should be considered for
persons with chronic obstructive respiratory disease, cardiac
insufficiency, or renal disease, particularly if they have not
been vaccinated yearly or if a new influenza A strain is
epidemic. Amantadine may also modify the severity of
influenza A if started within 24-48 hours after onset of
illness.
Influenza: Chemoprophylaxis
 Amantadine
and rimantadine: effective
against type A, but not type B, influenza
viruses; block the M2 ion channel
 70-90% effective in preventing illness
 Administered to individuals at high risk of
complications who are vaccinated after
outbreak of infection, persons with
immune defficiency
Influenza: Chemotherapy
 Amantadine
(adults and children ≥ 1 year)
and rimantadine (adults)
 Zanamivir and oseltamivir: neuraminidase
inhibitors active against both type A and B
influenza viruses
 Reduce duration of illness by ~1 day when
administered within 2 days of the onset of
illness (uncomplicated influenza)
Control Through Immunization
In the 1940s, it appeared that killed influenza vaccine given by
subcutaneous injection might pro vide protection against
epidemic influenza. This hope was dashed when the H1N1
strain appeared in 1947, making existing vaccines useless
because of major antigenic changes. Each subsequent major
antigenic shift that appeared every 10-15 years made existing
vaccines useless.
It is possible that the number of antigens of influenza viruses
might be finite but might vary in proportion from one strain to the
next. If several strains of broad antigenic composition were
combined, such a vaccine might yield an antigenic mass capable
of protecting against present and future epidemics. On the other
hand, the number of antigenic shifts and the possibility of
antigenic drift among influenza viruses might be infinite,
rendering the outlook for future control dubious.
A. Who should be vaccinated? At present, it is
recommended in the USA that vaccination be limited to
those at high risk — the elderly and persons with chronic
bronchopulmonary or cardiac disease, or metabolic and
renal disorders. They should be vaccinated every year,
according to dosage directions provided by the
manufacturer. However, if a major antigenic shift
becomes apparent, the entire population might be
considered for vaccination — as was the case in the
1976 "swine flu" epidemic.
B. When should vaccination be done? At present,
yearly vaccinations should be given before the influenza
season begins, ie, in early fall.
C. How are antigens for killed influenza vaccines
selected and prepared?
1. If only minor antigenic drift is expected for the next
influenza season, the most recent strains of A and B
viruses representative of the main antigens are included.
They are grown in embryonated eggs, harvested,
purified, inactivated, concentrated to a standard
hemagglutinin content, and stored for administration in
the tall.
2. if a strain representing a major antigenic shift has been
isolated (usually in Southeast Asia, where influenza
occurs 6 months before it becomes epidemic in Europe or
the USA), then ways must be found to grow the important
new antigen in bulk. This is accomplished by the
recombination method. Stable hybrids can be made of
the low-yield new antigen strain and a high-yield eggadapted influenza virus. By co-cultivation of the new,
different isolate with an established high-egg-yield virus,
recombinant progeny are obtained. These hybrids are
selected out, grown in bulk, and incorporated into the
"vaccine for the next season." Whenever such
recombinant vaccines have been tested, their potency
was equal to that of wild-strain vaccines.
3. From either of these 2 methods, subviral antigens
can be prepared, Such "split viruses" result from
chemical treatment of virion suspensions, with subsequent purification and concentration, and they contain
the most important antigenic proteins. The split-virus
vaccines produce fewer side-effects than whole-virus
vaccines. They are preferred for that reason but may
require several injections instead of a single one,
because of lower antigenicity. They are recommended
for children.
D. What are the major risks of and untoward reactions
associated with influenza vaccines?
1. All killed vaccines can produce fever, local inflammation at the
site of subcutaneous injection, and systemic toxicity with poorly
defined nonspecific symptoms of illness for 1-2 days.
2. Since the vaccine strains are grown in eggs, some egg protein
antigens are present in the vaccine. Persons allergic to eggs may
develop symptoms and signs of hypersensitivity.
3. Whatever immunity results from an inactivated vaccine appears
to be of short duration — probably 1-3 years against the
homologous virus.
4. Guillain-Barre syndrome, an ascending paralysis, has been
statistically associated with mass vaccination programs, eg, the
"swine flu" vaccination of 1976. It occurred 5-7 times more
frequently in vaccinated than in matched, unvaccinated persons.
While most persons affected by this syndrome recover completely,
5-10% have residual muscle weakness and 3-5% a fatal outcome.
However, no such increased risk of contracting Guillain-Barre
syndrome has been associated with subsequent standard influenza
vaccines.
E. Current research approaches to better influenza
vaccines.
1. A neuraminidase-specific vaccine, which induces
antibodies only to the neuraminidase antigen of the
prevailing influenza virus. Antibody to neuraminidase
reduces the amount of virus replicating in the respiratory
tract and the ability to transmit virus to contacts. It reduces
clinical symptoms in the infected person but permits
subclinical infection that may give rise to more lasting
immunity.
E. Current research approaches to better influenza
vaccines.
2. A live vaccine using temperature-sensitive (ts) mutants.
Such ts- mutants grow well at the cooler (33 °C) temperature
of the upper respiratory tract but fait to grow at the higher
(37 °C) temperature of the lung. Mutants selected for this ts
property appear to be attenuated or avirulent. Thus, they
might be given as a live vaccine into the respiratory tract,
stimulating local as well as systemic immunity. By
recombination of the ts gene with the gene for the current
major antigen, potent live vaccines could theoretically be
produced and rapidly administered to cope with an influenza
epidemic.
Attenuated live influenza virus vaccine has been used in many
coutries with reported success. The attenuated virus was
selected by serial transfer through embryonated eggs rather
than by genetic manipulation.
E. Current research approaches to better influenza
vaccines
3. Combined yearly vaccination of persons at high risk,
using the best mix of important antigens, and
administration of amantadine or other anti-influenza drugs
at times of particular stress, eg, surgery, hospitalization.
Laboratory Diagnosis
Influenza is readily diagnosed by laboratory procedures.
For antibody determinations, the first serum should be
taken less than 5 days after onset and the second 10-14
days later.
For rapid detection of influenza virus in clinical
specimens, positive smears from nasal swabs may be
demonstrated by specific staining with fluoresceinlabeled antibody.
A. Recovery of Virus: Throat washings or garglings are
obtained within 3 days after onset and should be tested at once
or stored frozen. Penicillin and streptomycin are added to limit
bacterial contamination, and embryonated eggs are inoculated
by the amniotic route. Amniotic and allantoic fluids are
harvested 2-4 days later and tested for hemagglutinins. If
results are negative, passage is made to fresh embryos. If
hemagglutinins are not detected after 2 such passages, the
result is negative.
If a strain of virus is isolated – as demonstrated by the presence
of hemagglutinins — it is titrated in the presence of type-specific
influenza sera to determine its type. The new virus belongs to
the same type as the serum that inhibits its hemagglutinating
power.
Primate cell cultures (human or monkey) are susceptible to
certain human strains of influenza virus. Rapid diagnosis can be
made by growing the virus from the clinical specimen in cell
culture and then staining the cultured cells with fluorescent
influenza antibody 24 hours later, when infected cells are rich in
antigen even though they may appear normal.
The phenomenon of hemadsorption is utilized for the early
detection of virus growth in cell cultures. Guinea pig red cells or
human 0 cells are added to the cultures 24-48 hours after the
clinical specimens have been inoculated and are viewed under
the low power lens. Positive hemadsorption shows red blood
cells firmly attached to the cell culture sheets as rosettes or
chains. The cytopathogenic effects of the influenza viruses are
often negligible. Hemadsorption provides a more sensitive
testing procedure.
B. Typing of New Isolates: A double immunodiffusion (DID) test
is used for typing influenza virus isolates. The allantoic fluid
content of a single infected embryonated egg may be used for
the DID test. Reference antisera are placed in the outer wells,
and the virus harvest, after disruption by detergent, is added to
the center well. The plates are incubated overnight in a moist
atmosphere, and precipitin lines are read the following morning.
Membrane iminunofluorescence has also been recommended
as a simple, rapid, and accurate method for typing current
influenza A isolates. Surface antigens of infected, unfixed
monkey kidney cells are stained in suspension by the indirect
immunofluorescence method using anti-H3N2 and anti-H1N1
antisera.
C. Serology: Paired sera are used to detect rises in HI, CF, or Nt
antibodies. The HI antibody is used most often. Normal sera
often contain nonspecific mucoprotein inhibitors that must first be
destroyed by treatment with RDE (receptor-destroying enzyme of
Vibrio cholerae cultures), trypsin, or periodate. Because normal
persons usually have influenza antibodies, a 4-fold or greater
increase in titer is necessary to indicate influenza infection. Peak
levels of antibodies are present 2—4 weeks after onset, persist
for about 4 weeks, and then gradually fall during the course of a
year to preinfection levels.
Within one type of influenza virus, strains may differ markedly in
antigenicity. It is best to use recently isolated strains.
Complement-fixing antigens are of 2 types. One is soluble (S
antigen) and type-specific but not strain-specific. The other is part
of the virus particle (V antigen) and is highly strain-specific. It is
useful for demonstrating antibody rise when the first serum
specimen was not taken early in the disease, because the peak
CF titer occurs in the fourth week.
Emerging Influenza Viruses
Hong Kong 1997: 18 cases of influenza in
humans caused by a highly pathogenic avian
influenza virus (H5N1); 30% fatality rate
 H9N2 subtype also detected among infected
poultry
 1999: H9N2 influenza viruses isolated from
two patients in Hong Kong; reassortant
between quail H9N2 virus and H5N1
chicken/duck virus
 In the future: reassortment between H9N2 or
H5N1 avian viruses and H1N1 or H3N2
human viruses???

Emerging Influenza Viruses

Avian influenza outbreak in the Netherlands in
early 2003
-
-
Epidemic of avian influenza virus in poultry
farms in the Netherlands
>80 poultry workers developed conjunctivitis
from this virus (H7N7)
One death from primary influenzal pneumonia
Person-to-person spread documented in three
cases
Antibodies to the avian virus found in pigs in the
Netherlands
Avian Influenza Poultry Outbreaks, Asia, 2003-06
• 2 yrs ago first case of HPAI in poultry H5N1 was
reported from Korea (12/03)
Has spread from E Asia, SE Asia and Pacific to
Eurasia, Near East, Europe and to Africa
H5N1 has been identified in migratory water birds
and /or poultry in 55 countries as of 5/29/06
It has infected humans in 10 countries.
Avian Influenza Poultry
Outbreaks, Asia, 2003-06
• No sustained person-to-person transmission
identified
Current signs are disturbing
• The number of human and animal infections
continue to increase
• Small clusters of human cases have been
documented, suggesting the virus is close to
sustained human to human transmission
• H5N1 continues to evolve in the “reassortment”
laboratory provided by the unprecedented number
of people, pigs & poultry in Asia- the population
explosion provides an enormous mixing vessel for
the virus
Cumulative # of confirmed
human cases of Avian flu (H5N1)
reported to WHO (5/29/06)
Onset
Date
Azerb
Cambod
Egypt
China
Indones
Iraq
Thailnd
Turkey
V.Nam
Djibouti
Total
2003
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
3/3
0/0
3/3
2004
0/0
0/0
0/0
0/0
0/0
0/0
12/17
0/0
20/29
0/0
32/46
2005
0/0
4/4
0/0
5/8
11/17
0/0
2/5
0/0
19/61
0/0
41/95
2006
5/8
2/2
6/14
7/10
25/31
2/2
0/0
4/12
0/0
0/0
32/47
Tota
l
5/8
6/6
6/14
12/
18
36/48
2/2
14/22
4/12
42/93
0/1
127/
224
Blue = Deaths White = Cases
Local Practices
Avian Influenza – H5N1
H5N1 in wild birds, poultry &
humans – 5/19/06
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