Orthomyxoviridae

Influenza viruses
Orthomyxoviridae

 There are influenza viruses that cause

worlwide epidemics of acute respiratory
disease.
 They are subclassified as immunologic types
A, B and C on the basis of antigenic
properties of their major nucleocapsid protein
(NP) and viral envelorpe matrix protein (M
protein).
Orthomyxoviridae

 The antigens of each type are unique and do

not cross-react with those of the other types.
 Influenza A cause far more serious illness
than the other types;
 influenza C has been associated only with
mild, nonepidemic illness.
Structure
Structure

 The orthomyxoviruses are enveloped, single-

stranded, segmented RNA viruses with
negative polarity genomes.
 The virion usually are 80-120 nm in diameter.
12.04
Structure
Structure

 The genome of influenza A and B viruses

consists of eight individual segments of RNA.
 Each segment encodes a different viral
protein.
 One segment is a precursor of two
nonstructural proteins (NS1 and NS2), which
exist only in infected cells.
 The other segments encode virions proteins.
Structure

(a). The M protein, which is located on the inner

surface of the viral membrane, is the most abundant
protein in the viral particle.
(b). The NP is a single phosphoprotein species
associated with the RNA.
(c). Hemagglutinin and neuraminidase are surface
spike glycoproteins.
(d). Three large internal peptides (P1, P2 and P3) are
associated with virus transcription and replication
and exist in very small numbers in the virion.
Structure
EM
Replication:

 Occurs primarily in the cytoplasm of infected

cells.
 The nucleus is obligatorily involved, however,
and appears to be the site of viral RNA
synthesis.
 The host apparently must constantly supply
fresh RNA transcripts.
Antigenic variation:

 In addition to the NP and M protein variations that

produce influenza A, B, and C, the influenza viruses
are characterized by a high frequency of
immunologic variations within the subtypes.
 These variations are caused by the surface spike
glycoproteins, hemagglutinin and neuraminidase,
and they lead to the appearance of new serologic
types that are subject to epidemic spread.
 Two types of antigenic variation have been
demonstrated.
Antigenic variation:

 Antigenic drift
 Antigenic shift
Antigenic drift

 Antigenic drift is due to mutation.

 Antibodies to the HA protein are the most important
in protection, although those to NA also play a role.
 Both proteins undergo antigenic drift (i.e. accumulate
mutations) and accumulate changes such that an
individual immune to the original strain is not immune
to the drifted one.
 Antigenic drift results in sporadic outbreaks and
limited epidemics.
Antigenic shift

 Antigenic shift is due to reassortment.

 In the case of influenza A,  antigenic shift periodically
occurs.
 Apparently "new" HA and/or NA are found in the
circulating viral strains.
 There is little immunity (particularly if both proteins
change, or if new HA is present) and an
epidemic/pandemic is seen.
  
Antigenic shift

 Antigenic shift probably results from genetic

recombination between a human and an animal
strain of influenza.
 Influenza A viruses have undergone four major
antigenic shifts since 1933.
 The first stain isolated from a human (in 1933) has
been designated H0N1.
 In 1947, a new hemagglutinin type, H1N1 emerged.
 Variants designated H1N1 (1977) and H3N2 (1968)
have emerged in the past 30 years.
H5N1
Classification of influenza strains:

 Type A, B or C/place isolated/number of isolate/year

isolated
 In the case of influenza A, also: HA subtype (H) and
NA subtype (N)
 For example, the three strains for the 2007/2008
vaccine are:
 A/Solomon Islands/3/2006 (H1N1)
 A/Wisconsin/67/2005 (H3N2)
 B/Malaysia/2506/2004
PATHOGENESIS AND DISEASE

 Spread
 The virus is spread person to person via small particle aerosols
(less than 10μm diameter) that can get into respiratory tract.
 It can also survive for a short time on surfaces and can be
spread by this route if the virus is introduced into the nasal
mucosa before it loses infectivity.
 The incubation period is short, about 18 to 72 hours.

Virus concentration in nasal and tracheal secretions remains
high for 24 to 48 hours after symptoms start and may last
longer in children.
 Titers are usually high and so there are enough infectious
virions in a small droplet to start a new infection.
Site of infection

 Influenza virus infects the epithelial cells of

the respiratory tract.
 The cells die, in part due to the direct effects
of the virus on the cell, and also possibly due
to the effects of interferon.
Site of infection

 Cell death at later times may also result from the

actions of cytotoxic T-cells.
 As a result, the efficiency of ciliary clearance is
reduced, leading to impaired function of the mucus
elevator; thus there is reduced clearance of
infectious agents from the respiratory tract.
 Gaps in the protective epithelium provide other
pathogens with access to other cells; however,
viremia is very rare.
Symptoms and complications

1. Uncomplicated influenza
 Fever (38 - 40 degrees C)
 Myalgias, headache
 Ocular symptoms - photophobia, tears, ache
 Dry cough, nasal discharge
2. Pulmonary complications, sequelae:

 Croup (acute laryngotracheobronchitis) in

young children - symptoms include cough
(like a barking seal), difficulty breathing,
stridor (crowing sound during inspiration)
 Primary influenza virus pneumonia
2. Pulmonary complications, sequelae

 Secondary bacterial infection: This often involves

Streptococcus pneumoniae, Staphylococcus aureus,
Hemophilus influenzae
 The build up of fluids and lack of mucociliary
clearance in the respiratory tract provide a good
environment for bacterial growth.
 Complications often occur in patients with underlying
chronic obstructive pulmonary or heart disease.
 The underlying problems may not have been
recognized prior to the influenza infection.
3. Non-pulmonary complications of
influenza:

 Myositis: this is rare and more likely to be

seen in children after influenza type B
infection
 Cardiac complications
 Encephalopathy: even when not fatal,
encephalopathy can have serious sequelae
and this emphasizes the importance of
vaccination.
3. Non-pulmonary complications of
influenza:

 Reye’s syndrome: The effects of influenza

virus infection on the liver and brain are
particularly serious. In the liver fatty deposits
are seen while in the brain edema occurs.
 Reye's syndrome includes vomiting, lethargy
and may result in coma. It is rare, but
approximately 40% of cases are fatal.
3. Non-pulmonary complications of
influenza:

 The origin of Reye's syndrome is unclear but seems

to follow certain viral infections such as influenza
(eg. B type) or chicken pox (varicella zoster/herpes
zoster), especially if they are in the young and
especially if they have been treated with aspirin.
 Aspirin is contraindicated for childhood or adolescent
fevers because it is a risk factor in the development
Reye's syndrome.
 Acetaminophen and Ibuprofen are apparently not
associated with Reye's syndrome.
3. Non-pulmonary complications of
influenza:

 Guillan-Barré syndrome(acute idiopathic

polyneuritis):
 The cause of this syndrome in the central
nervous system is mysterious.
 It is an autoimmune disease that can follow a
viral or bacterial infection.
 Recent attenuated anti-influenza vaccines do
not seem to increase the risk of developing
Guillain-Barre Syndrome.
Recovery

 Interferon may play a role by decreasing virus production.

 Many of the symptoms of uncomplicated influenza (muscle
aches, fatigue, fever) are associated with the efficient induction
of interferon.
 The cell-mediated immune response is important in viral
clearance.
 The antibody response is usually not significant until after virus
has been cleared.
 Repair of the respiratory epithelium begins rapidly, but may
take some time to complete.
  Protection

 A humoral antibody response is the main

source of protection.
 IgG and IgA are important in protection
against reinfection.
 Antibody to the HA protein is most important
since this can neutralize the virus and
prevent the virus initiating the infection.
  Protection

 Neutralization frequently involves blocking of

the binding of the virus to host cells and may
work at other steps involved in the entry and
uncoating of the virus.
 Antibody to the NA protein has some
protective effect since it seems to slow the
spread of the virus.
 IgG persists longer than IgA and so plays a
more important role in long term immunity.
Epidemiology and immunity:

 Both influenza A and B may cause epidemic illness

when they are established in a nonimmune or only
partially immune population.
 Transmission occurs primarily by aerosolization.
 Illness spread rapidly in confined populations where
crowded conditions prevail.
 Immunity to a new influenza variant depends on
immunity to the previous variant circulating in the
population and on the relatedness of the two
variants.
Epidemiology and immunity:

 Most major epidemics and pandemics are

due to antigenic shifts that produce new
influenza subtypes antigenically and distantly
related to types prevailing in the past.
 Antigenic drifts may cause any degree of
illness from subclinical to severe.
 Individual resistance is governed by the
presence of IgA and serum IgG antibodies as
well as cellular immunity.
Laboratory diagnosis

 (see lab work also):

 Influenza is difficult to diagnose because it
produces viral respiratory syndromes similar
to those produced by many other viruses.
Laboratory diagnosis

 Samples: nasal secretions or epithelial cells

(throat swab).
 Direct diagnosis: RT-PCR, ELISA, IF
 isolation (culture cells or egss) +
identification)
 Recently, rapid tests that can be used in a
physician's office have been approved.
Indirect diagnosis (Ab)

 Serology: CFT, IHT (surveillance of

influenza)
Therapy and prevention:

 Immunization and chemical prevention are

available and partially successful.
 Treatment offers only relief of symptoms and
generally does not change the course of the
illness.
 Amantadine is useful both for relief of
symptoms and for prevention.
 It enhance the effectiveness of immunization.
Vaccine

 Immunization Practices and Recommendations for

the Prevention and Control of Influenza is published
in the Morbidity and Mortality Weekly Report
(MMWR)
 This updated annually and includes extensive advice
about who should or should not receive vaccine,
when it should be administered, and antiviral drugs
 
PREVENTION Vaccines

 A new vaccine is formulated annually with

the types and strains of influenza predicted
to be the major problems for that year
(predictions are based on worldwide
monitoring of influenza).
 The vaccine is multivalent and the current
one is to two strains of influenza A and one
of influenza B (trivalent).
Vaccine

 Immunization is about 80% effective.

 Difficulties stem from the mechanisms of vaccine
production and selection of the appropriate
immunizing strain.
(a). Inactivated vaccine produced from purified, egg-
grown virus is expensive to produce and causes
allergic reactions in people with hypersensitivity to
hens' eggs.
(b). Live virus vaccines
Live virus vaccine

 In 2003, a live, attenuated (much less pathogenic

than wild-type virus) influenza vaccine (LAIV,
marketed as FluMist) was approved for use in the
United States.
 It is only approved for healthy individuals (those not
at risk for complications from influenza infection)
from five to forty nine years of age.
 In 2007, it was also approved for healthy children
ages 24 - 59 months without a history of recurrent
wheezing.
Live virus vaccines

 It is given nasally and should provide

mucosal, humoral and cell-mediated
immunity.
 In this vaccine, the vaccine virus is a cold-
adapted strain which can grow in the upper
respiratory tract where it is cooler, but grows
poorly in the lower respiratory tract.
 It is attenuated due to multiple changes in
the various genome segments.
Live virus vaccines

 FluMist vaccine virus is grown on eggs and

so is contraindicated for people with an egg
allergy.
 Since this is a live viral vaccine, it is also
contraindicated for children and young
adolescents on any therapy containing
aspirin due to the potential risk of Reye's
syndrome.
Vaccination
Coronaviridae

 Coronaviruses, which are about 100nm in

diameter, are the largest positive strand RNA
viruses (indeed they have the largest
genomes of any RNA virus).
 They infect humans and animals in which
they cause respiratory and enteric disease in
a variety of animals.
 In humans, the major site of virus replication
is the epithelial cells of the respiratory tract
and about one-third of colds are caused by
coronaviruses.
 The symptoms are similar to those of
rhinovirus colds (runny nose, sore throat,
cough, headache, fever, chills etc.) with an
incubation time of about 3 days.
 Viral spread is limited by the immune
response of most patients but this immunity
is short-lived.
 Symptoms may last about a week with
considerable variation between patients.
 Often there are no apparent symptoms but
the patient still sheds infectious virus
Diagnosis

 Most coronavirus infections go undiagnosed

and the disease is self-limiting.
 Diagnosis can be carried out using immuno-
electron microscopy and serology.
 There are no anti-virals for routine
coronavirus infections but over-the-counter
remedies to alleviate symptoms are useful.
Severe acute respiratory syndrome
(SARS)

 In late 2002, a new syndrome was observed

in southern China (Guangdong Province).
 It was named severe acute respiratory
syndrome (SARS).
 This disease, which has now been reported
in Asia, North America, and Europe, is
characterized by a fever above 38 degrees
accompanied by headache, general malaise
and aches.
 SARSControl
The 2002/2003 SARS Outbreak
Severe acute respiratory syndrome
(SARS)

 In fact, respiratory symptoms are initially usually mild

but after a few days (or a week), the patient may
develop a dry non-productive cough and breathing
may become difficult (dyspnea).
 Respiratory distress leads to death in 3-30% of
cases.
 Laboratory tests show a reduction in lymphocyte
numbers and a rise in aminotransferase activity
which indicates damage to the liver. 
Rx
SARS - diagnosis

 The virus was grown on monkey Vero E6

cells in tissue culture
 EM
 RT-PCR
 Serology: ELISA, IF
ROTAVIRUSES AND OTHER VIRAL
AGENTS OF GASTROENTERITIS
Classification

 Family Reoviridae, which includes genus:

 Rotavirus,
 Reovirus
 Colorado Tick Fever virus.
Structure

 Rotaviruses are non-enveloped, icosahedral,

with double capsid.
 Their electron microscopic appearance
shows a 60-80nm wheel with radiating
spokes (Latin, rota = wheel).
 The rotavirus genome contains double
stranded (ds) RNA in 11 segments that can
be separated by polyacrylamide gel
electrophoresis (PAGE).
Pathogenesis

 Affected host cells are mature enterocytes

lining the middle and upper end of the
intestinal villi.
 In laboratory animals, hepatocytes are also
infected.
 The infectious particle is thought to be an
"intermediate sub-viral particle" (ISVP).
Pathogenesis

 Viral attachment protein is probably exposed after

protease digestion in the GI tract removes some or
all of the outer capsid (VP4).
 Virus replicates in the host cell cytoplasm.
 Virions enter the host cell by endocytosis and viral
mRNA is transcribed using the viral RNA polymerase
that is already present in the virion to form structural
protein units of the capsid.
 The mRNA segments are assembled into the
immature capsid and then replicated to form the
double stranded RNA genome.
 Large amounts of viral particles are shed in diarrheal
stools.
Pathogenesis

 Histopathology of infected intestines shows villous

atrophy and blunting, due to death of the mature
enterocytes and infiltration of lamina propria with
mononuclear cells.
 Subsequently there is repopulation of the villous tips
with immature secretory cells [crypt hyperplasia].
 Cell dysfunction and death results in a net secretion
of intestinal fluid, hence the watery diarrhea.
 Activation of the enteric nervous system may also
play a role.
Rotaviruses infection

 Seasonality
 Winter months (November through May).
 Incubation period - thought to be <4 days
 Contagious Period - Before onset of diarrhea to a
few days after end of diarrhea
 Age - Rotaviruses infect children at a young age.
 Older infants and young children (4 months - 2
years) tend to be more symptomatic with diarrhea.
 Young infants may be protected due to trans-
placental transfer of antibody.
Rotaviruses infection

 Asymptomatic infections are common,

especially in adults. Many cases and
outbreaks are nosocomial
 Group A infections are most common.
 Group B has been associated with outbreaks
in adults in China
 Group C is responsible for sporadic cases of
diarrhea in infants around the world.
Rotaviruses infection

 Spread is mainly person to person via fecal -

oral route and through fomites.
 Spread by food and water is also possible.
Spread via respiratory route is speculated.

 High numbers of viral particles are shed in

diarrheal stools (1010/gm).
 Infective dose is only 10-100 pfu.
Clinical Features

 Fever- can be high grade (>102° F in 30% of

patients)
 Vomiting, nausea precedes diarrhea.
 Diarrhea is usually watery (no blood or leukocytes),
lasting 3-9 days, but longer in malnourished and
immune deficient individuals.
 Necrotizing enterocolitis and hemorrhagic
gastroenteritis is seen in neonates
 Dehydration is the main contributor to mortality.
 Secondary malabsorption of lactose and fat, and
chronic diarrhea are possible
Diagnosis

 Direct: LA, ELISA (Ag identification) – group

A
 Group A rotaviruses can be cultured in
monkey kidney cells.
Treatment

 Supportive - rehydration (oral / intravenous)

 Antiviral agents not known to be effective
Prevention of spread

 Handwashing with good technique

 Disinfection of surfaces, toilets, toys
 Adequate chlorination of water
SMALL ROUND RNA VIRAL AGENTS
CAUSING GASTROENTERITIS

 This group of RNA viruses morphologically is

subdivided in to 2 sub-groups:
 Structured - Small round structured viruses
(SRSV), Calicivirus, Astrovirus
 Other small viruses that are relatively
structureless or featureless
 SRSV- Norwalk virus and Norwalk-like
agents

 Non-enveloped, single stranded RNA

viruses. 27-35 nm in size (figure 7 and 8)
 Contain a single capsid protein.
 Norwalk virus was first detected in stools of
patients with gastroenteritis in Norwalk, Ohio
in 1972.
 27 - 32nm in size with a ragged surface.
Norwalk virus
Norwalk-like viruses
 Norwalk-like viruses are similar in size and are
named after the place where they were first isolated
in relation to outbreaks of diarrhea.
 Different serotypes are:
- Hawaii
- Snow Mountain (CO)
- MC (MD)
- Taunton (England)
- Otofuke (Japan)
- Sapporo (Japan)
Clinical Features

 Adults and children are affected

 Relatively short incubation period: <24 hours
 Illness is short (<3 days)
 Nausea, vomiting, abdominal cramping and watery
diarrhea accompanied by headache, fever and
malaise 
 Outbreaks often occur in institutions, cruise ships,
etc. through contaminated food or water
 Feco-oral spread, perhaps also spread through
vomitus
 
Diagnosis

 Immune EM
 Serology for epidemiologic purposes
CALICIVIRUS (Caliciviridae)

 Described in 1976
 Calciviruses are non- enveloped single stranded RNA viruses, 30-45nm in size (figure 9). They appear round in shape with
icosahedral symmetry and contain a single capsid protein. The viral surface has 32 cup-shaped depressions (‘calici’= chalice or
calyx i.e. cup-like) described as the ‘Star of David’ appearance. Otherwise they are similar to Norwalk group of agents and
belong to family caliciviridae
 The 5 antigenic types in humans are UK 1 to 4, and Japan/Sapporo
 Epidemiology
 Worldwide distribution
 Outbreaks can involve infants and school-age children
 Viral excretion in stool can continue beyond the cessation of symptoms
 Transmission-fecal-oral, through contamination of food and person to person (even asymptomatic persons)
 Outbreaks related to consumption of contaminated oyster and shellfish have been described. It is thought that the seafood or
water or ice becomes contaminated with fecal material from sewage or food handlers.
 Most people have had infections by age 4 years (by seroprevalence studies).
 Clinical Manifestations
 Incubation period - 12hrs. to 4 days
 Most infections are asymptomatic
 Mild diarrhea, vomiting, anorexia and fever
 1/3 have respiratory (flu-like) symptoms (esp. UK3 and UK4strains)
 Diagnosis
 EM - not widely available, requires higher level of viral excretion for detection
 Immune EM, ELISA
CALICIVIRUS (Caliciviridae)

 Described in 1976
 Calciviruses are non- enveloped single stranded RNA viruses,
30-45nm in size.
 They appear round in shape with icosahedral symmetry and
contain a single capsid protein.
 The viral surface has 32 cup-shaped depressions (‘calici’=
chalice or calyx i.e. cup-like) described as the ‘Star of David’
appearance.
 Otherwise they are similar to Norwalk group of agents and
belong to family caliciviridae
 The 5 antigenic types in humans are UK 1 to 4, and
Japan/Sapporo
Epidemiology

 Worldwide distribution
 Outbreaks can involve infants and school-age children
 Viral excretion in stool can continue beyond the cessation of
symptoms
 Transmission-fecal-oral, through contamination of food and
person to person (even asymptomatic persons)
 Outbreaks related to consumption of contaminated oyster and
shellfish have been described.
 It is thought that the seafood or water or ice becomes
contaminated with fecal material from sewage or food handlers.
 Most people have had infections by age 4 years (by
seroprevalence studies).
Clinical Manifestations

 Incubation period - 12hrs. to 4 days

 Most infections are asymptomatic
 Mild diarrhea, vomiting, anorexia and fever
 1/3 have respiratory (flu-like) symptoms (esp.
UK3 and UK4strains)
Diagnosis

 EM - not widely available, requires higher

level of viral excretion for detection
 Immune EM, ELISA
ASTROVIRUS (Astroviridae)

 Described in relation to an outbreak of gastroenteritis

in 1975.
 Small single stranded RNA, non-enveloped virus,
about 27 - 32nm in size.
 They are round with an unbroken surface (unlike
indented surface of calicivirus)
 EM appearance of a 5 or 6 pointed star; contain 3
structural proteins.
 Astroviruses are immunologically distinct from
Norwalk and Caliciviruses - belong to family
Astroviridae
 7 human serotypes are known
EM
Astroviruses
 Clinical Features
 Diarrhea, headache, nausea, low-grade fever,
vomiting (Iess common)
 Epidemiology
 Worldwide, mainly in children <7 years of age.
 Transmission person-to-person via fecal-oral route
 Outbreaks due to fecal contamination of
sea-food/water
 Diagnosis
 EM and Immune EM are especially useful since the
virus is often shed in large amounts in stool.
 Immunfluoresence detects all serotypes.
Adenoviruses

 Adenovirus serotypes implicated in

gastroenteritis are 40, 41 (rare: 31, 3, 7).
 They cause diarrheal disease in infants and
children <4 years of age.
 The virus is spread by the feco-oral route
and is not shed in the nasopharynx
 Incubation period is 8-10 days
 Diarrhea lasts 5-12 days, prolonged diarrhea
often seen with type 40 infections
 Diagnosis - Latex agglutination, ELISA, EM
EM
 http://pathmicro.med.sc.edu/book/virol-
sta.htm