Download HAI, NtT, CFT.

RESPIRATORY VIRUSES
Viruses that initiate infection via respiratory tract
Site of infection
Family
Viruses
Local respiratory Orthomyxoviridae Influenza А and В viruses
infection
Paramyxoviridae Parainfluenza viruses (4 types),
respiratory-syncytial virus RSV (3 types)
Picornaviridae
Rinoviruses (113 types)
Reoviridae
Reoviruses (3 types)
Coronaviridae
Types 1-4
Adenoviridae
Types 1-7, 14, 21
Generalized
Herpesviridae
diseases, usually
with initial
Paramyxoviridae
respiratory
Togaviridae
symptoms
Picornaviridae
Varicella virus, Epstain-Barr virus (EBV),
cytomegalovirus
Mumps and measles viruses
Rubella virus
Some enteroviruses
Bunyaviridae
Hantaviruses
Arenaviridae
Lassa fever virus
Family Orthomyxoviridae.
Influenza virus
• (-) RNA enveloped virus with haemagglutinin
and neuraminidase spikes
• Pleomorphic
• Helical symmetry
• Diameter 80-120 nm
• 3 types: A, B and C
Structure of influenza virus
• The internal antigens (matrix and RNP) are the typespecific proteins and are used to determine whether a
particular virus is A, B, or C.
• The external antigens (hemagglutinin and
neuraminidase) are
subtype and strainspecific antigens of
influenza A virus:
H1N1, H2N2, H3N2
Comparison of influenza A, B and C viruses
Severity of illness
Animal reservoir
Human
pandemics
Human
epidemics
Surface antigens
Antigenic
changes
Amantadine,
rimantadine
Zanamivir
A
B
C
++++
++
+
yes
yes
no
no
no
no
yes
yes
no
2
shift, drift
2
drift
1
drift
sensitive
no effect
no effect
sensitive
sensitive
Influenza virus replication
• Hemagglutinin (HA) protein is involved in
attachment and membrane fusion in the
endosome of the infected cell.
The neuraminidase protein
• digests sialic acid (neuraminic acid) on
the surface of cells, when the virus
binds to the cell, and it is internalized
(endocytosed).
• Promotes the release of virions from
infected cells.
Types of
hemagglutinin and neuraminidase
Antigenic variations
• Antigenic drift is due to mutation. Both HA and NA after a few
years may accumulate changes in genome 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 is due to reassortment. It is the process by
which at least two different strains of a virus combine to form a
new subtype having a mixture of the surface antigens of the two
original strains. As a result, the population has no immunity
against the new strain.
Antigenic shift results in
an epidemic or pandemic.
Antigenic drift creates influenza
viruses with slightly modified
antigens, while antigenic shift
generates viruses with entirely novel
antigens.
Past antigenic shift
1918 H1N1 “Spanish Influenza”
20-40 million deaths
1957 H2N2 “Asian Flu”
1-2 million deaths
1968 H3N2 “Hong Kong Flu”
700,000 deaths
1977 H1N1 Re-emergence
No pandemic
2009 H1N1 “Swine Flu
Mild Pandemic
At least 15 HA subtypes and 9 NA subtypes occur in nature.
Up until 1997, only viruses of H1, H2, and H3 are known to infect
and cause disease in humans.
Theories behind antigenic shift
• 1. Reassortment of the H and N genes between
human and avian influenza viruses trough a third
host.
Coinfection with human
and animal strains of virus
can generate very different
virus strains by genetic
reassortment
• 2. Recycling of pre-existing strains (this probably
occurred in 1977 when H1N1 re-surfaced).
• 3. Gradual adaptation of avian influenza viruses to
human transmission.
Reassortment of
H and N genes
between human
and avian
influenza viruses
Sometimes pigs can be
infected with more than
one virus type at a time,
which can allow the
genes from these viruses
to mix. This can result in
an influenza virus
containing genes from a
number of sources,
called a “reassortant”
virus.
Bird or avian flu (2006)
• The outbreaks affecting some Asian countries
was caused by influenza A/H5N1 virus.
• It caused severe infection in humans.
• While avian influenza spreads rapidly among
birds, it does not infect humans easily, and
there is no confirmed evidence of human-tohuman transmission.
2009 Influenza A (H1N1) Virus
The 2009 Swine flu
outbreak in humans was
due to a new strain of
influenza A virus subtype
H1N1 that derived in part
from human influenza,
avian influenza, and two
separate strains of swine
influenza (American swine
and Eurasian swine viruses).
Swine influenza viruses
are most commonly of
the H1N1 subtype, but
other subtypes are also
circulating in pigs (e.g.,
H1N2, H3N1, H3N2).
Epidemiology and pathogenesis
Spread: via small particle aerosols.
• The incubation period is about 18 to 96
hours.
• Site of infection: the epithelial cells of
the respiratory tract:
trachea and bronchi
Pathogenesis
• Infection of mucosal cells
results in cellular
destruction and
desquamation of the
superficial mucosa.
• The resulting edema and
mononuclear cell
infiltration are
accompanied by local
symptoms: nonproductive
cough, sore throat, and
nasal discharge.
• Systemic symptoms:
fever, muscle aches,
headache, and general
prostration.
Symptoms
Pathogenesis and immunity
Immunity to an influenza
virus is type-specific and lasts
for many years.
Recurrent cases of influenza
are caused primarily by
antigenically different strains.
LABORATORY DIAGNOSIS OF INFLUENZA
Specimen: the nose and the throat swab.
1. Detection of antigen by
immunofluorescence (IF) and ELISA.
2. The virus isolation in cell culture or
chicken eggs.
Identification of influenza strain and type:
hemagglutination inhibition (HAI) and
hemadsorption inhibition (HadsI).
3. Serology: HAI, neutralization test (NtT),
ELISA, CFT in pared sera.
Immunofluorescence (IF) to detect virus into host
cells
direct IF
indirect IF
Hemadsorption (Hads)
• Virus growth in cell cultures is detected by testing for
hemadsorption: red cells are added to the culture
and adhere to virus budding from infected cells.
• If the culture tests positive, hemadsorption inhibition
test with specific antisera is used to identify the virus.
cell culture
positive Hads
Chicken embryo structure
Chick embryo culture method
Inoculation of chick embryo
Removing allantoic fluid
• Fluid from the amniotic or allantoic cavity of chick
embryos is tested for the presence of newly formed
viruses by haemagglutination test;
• the virus in positive fluids is then identified by
haemagglutination inhibition test with specific
antisera.
Haemagglutination inhibition test (HAI)
Influenza viruses bind to red blood cells using the
haemagglutinin causing the formation of a lattice.
HA: two-fold serial dilutions of a virus are prepared, mixed
with red blood cells, and added to the wells of a plastic plate.
The red blood cells that are attached to virus particles form a
lattice that coats the well. The red blood cells that are not
bound by virus sink to the bottom of a well and form a button.
negative HA positive HA
The basis of the HAI assay is that antibodies to influenza
virus will prevent attachment of the virus to red blood cells.
By adding specific antibodies to the virus it is possible to block
this interaction and detect the virus. If antibodies to the virus
are specific, hemagglutination will not be observed.
negative HAI positive HAI
TREATMENT
Class
M2 inhibitors
(adamantane
derivatives)
Effective
against
Influenza A
Drug name
Amantadine,
rimantadine
Neuraminidase Influenza A & B Zanamivir,
inhibitors
oseltamivir
Mechanism of Action of Neuraminidase Inhibitors
Interferon
• The innate immune system forms the first line of
defence against viruses - Interferons (IFNs),
glycoproteins known as cytokines.
• Interferons are produced by:
– the cells of the immune system (leukocytes, fibroblasts, or
lymphocytes) in response to challenges by foreign agents
such as viruses, parasites and tumor cells;
– cells infected with a virus.
• Interferons assist the immune response by:
– inhibiting viral replication within host cells,
– activating natural killer cells and macrophages,
– inducing the resistance of host cells to viral
infection.
VACCINES
• The vaccine is multivalent and the current
one is to two strains of influenza A and one
of influenza B.
• Inactivated preparation of egg-grown virus.
• Live, attenuated vaccine.
• Split-vaccine consists of all viral antigenes.
• Sub-united vaccine consists of
hemagglutinines and neuraminidases
(H1N1, H3N2 and B).
Unique features of Adenoviruses
• Double-stranded linear(+)DNA.
• Naked icosahedral capsid has
fibers (viral attachment
proteins) and vertices.
• 47 human serotypes
• Viruses cause:
– lytic,
– persistent,
– latent infections in humans,
– some strain can immortalize
certain animal cells.
Life cycle of adenoviruses
Pathogenesis of adenoviruses infections
• Virus is spread:
– by aerosol,
– direct contact,
– fecal-oral.
• Virus infects: epithelial cells in respiratory
and gastrointestinal tract, conjunctiva and
cornea.
• Virus persists in lymphoid tissue (tonsils,
adenoids, Peyer’s patches).
Mechanism of adenovirus spread within the body
Time course of adenovirus respiratory infection
ILLNESS ASSOCIATED WITH ADENOVIRUSES
• Incubation period is 2-14 days.
Clinical syndromes:
• Eye
Epidemic keratoconjunctivitis, acute follicular
conjunctivitis, pharyngoconjunctival fever.
• Respiratory system
Common cold (rhinitis), pharyngitis, tonsillitis, bronchitis,
pneumonia.
• Genitourinary
Acute hemorrhagic cystitis, orchitis, nephritis.
• Gastrointestinal
Gastroenteritis, mesenteric adenitis, appendicitis.
• Rare results of adenovirus infections:
Meningitis, encephalitis, arthritis, myocarditis, hepatitis.
Fatal disease may occur in immunocompromised patients, as a result
of a new infection or reactivation of latent virus
Respiratory consequences of adenovirus infections
IMMUNITY
Strong, type-specific
PREVENTION
A vaccine is available against Adult
Respiratory Distress Syndrome.
Oral, live-attenuated vaccines against
serotypes 4 and 7 of adenovirus is
administered in tablet form.
It is given to new recruits into various
arm forces around the world.
LABORATORY DIAGNOSIS OF ADENOVIRUSES
INFECTION
1. Detection of antigen from pharyngeal and eye
secretions and feces by:
IF, ELISA and polymerase chain reaction.
2. The virus isolation in cell culture.
Cytopathic effects (CPE) include
swelling and rounding of cells.
Identification:
HAI, CFT; type virus – by NtT.
3. Serology (rise in antibody titer): HAI, NtT, CFT.
Family Paramyxoviridae
• Genus Respirovirus (parainfluenza viruses types1 and 3)
• Genus Rubulavirus (parainfluenza viruses types 2 and 4,
mumps virus)
• Genus Morbillivirus (measles virus)
• Genus Pneumovirus (respiratory syncytial virus)
Parainfluenza viruses
• ss (-) RNA viruses
• Enveloped viruses with hemagglutinin and
neuraminidase spikes and fusion (F) protein
• Helical symmetry
• 4 types: 1, 2, 3, 4a, 4b
Clinical features of parainfluenza (PIV)
• Incubation period is 2 to 6 days.
• Clinical symptoms:
• Rhinitis, pharyngitis, cough, fever, croup
(laryngotracheobronchitis), bronchiolitis, and pneumonia.
• Croup - the subglottic region becomes narrower and
results in difficulty with breathing, a seal bark-like cough
and hoarseness.
• There is clinical variation between the different PIV
types.
• PIV-1 and 2: croup in children ages 2-6 years in
autumn/early winter.
• PIV-3: bronchiolitis and pneumonia, and croup
sporadically, without a particular seasonal occurrence.
• PIV-4: mild upper respiratory infections.
LABORATORY DIAGNOSIS OF PARAINFLUENZA
1. Detection of antigen from nasopharyngeal
aspirates and throat swab by IF and PCR.
2. The virus isolation in cell culture.
Indication:
Haemadsorption of erythrocytes
on the surface of cells infected with virus.
Identification: HadsI, HAI, NtT, CFT.
3. Serology – detection of rise in titer
of IgG in pared sera:
NtT, ELISA, CFT, HAI.
Respiratory Syncytial Virus (RSV)
• ss RNA enveloped virus
• Considerable strain variation exists,
may be classified into subgroup A and B.
•
CPE - the formation of multi-nucleate syncytium
Clinical features:
MUMPS
• Mumps is a viral infection that primarily affects the salivary
glands causing them to become inflamed, resulting in the
characteristic “chipmuck cheeks”
• Transmission by:
– salivary gland secretions, mainly just before and shortly
after clinical onset
– direct and close person-to-person contact and
– airborne route.
The virus enters the body
through the pharynx or
the conjunctiva.
Clinical symptoms of mumps
• The incubation period usually is 18 to 21 days, but
may extend from 12 to 35 days.
• Prodromal phase: fever, anorexia, malaise, myalgia.
The symptoms:
Fever.
Parotitis. Pain from parotitis
swelling persists for 7 - 10 days.
It may be unilateral or bilateral.
Up to 20% infections result in no
symptoms.
Complications of mumps
• Orchitis
20-50 %
• Meningitis and
meningoencephalitis 15 %
• Ovaritis
5%
• Pancreatitis
2-5 %
• Rare complications: polyarthritis,
diabetes, nephritis, thyroiditis,
deafness, myocarditis.
LABORATORY DIAGNOSIS OF MUMPS
1. The virus isolation from the saliva, liquor
or urine in cell culture (or chicken eggs).
CPE: giant multinucleated
cells formation.
Identification:
HAI, NtT, IF, CFT.
2. Serology: HAI, NtT, ELISA, CFT
(demonstrating IgM in the first serum and
detecting IgG rise in paired sera).
Measles virus
• Family Paramyxoviridae
• Genus Morbillivirus
• enveloped RNA virus which is composed of a helical nucleocapsid, and
matrix protein. The envelope surface is covered with hemagglutinin and
fusion proteins which are embedded in a lipid bilayer.
Electron Micrograph
Age distribution of measles cases
• Measles is one of the most contagious of all human viruses,
with about 40 million infections world wide each year, and
one to two million deaths.
Measles
• a systemic infection, disseminated by viremia, with acute
disease manifestations involving the lymphatic and
respiratory systems, the skin, and sometimes the brain.
• Measles virus enters through the oropharynx and the
conjunctiva.
• Incubation period is 10 days until the onset of fever and
14 days to rash.
• The clinical symptoms:
– Fever, cough, laryngitis, coryza, conjunctivitis, Koplick’s spots,
and maculopapular rash.
• Complications:
– Pneumonia, otitis media, optic neuritis
– Encephalitis, subacute sclerosing panencephalitis (SSPE)
– Hemorrhagic measles
Measles pathogenesis
Koplick’s spots
Conjunctivitis.
Maculopapular
rash
Subacute Sclerosing Panencephalitis
LABORATORY DIAGNOSIS OF MEASLES
Clinically Koplick’s spots are pathognomonic.
1.Detection of antigen from nasopharyngeal
aspirates and throat swab by IF.
2. The virus isolation in cell culture.
CPE: giant multinucleated
cells formation.
Identification: HAI, IF, NtT.
3. Serology: HAI, NtT, CFT.
Family Togaviridae. Rubella virus
• It is an enveloped (toga=cloak),
RNA virus with icosahedral symmetry.
• There is only one major antigenic type.
Pathogenesis and clinical features of rubella
• Spread via an aerosol route through infected
droplets.
• Replication: in the epithelium and lymph nodes of
the upper respiratory tract leading to viremia
and spread to other tissues.
• Incubation period is 12 to 23 days (the average 18
days).
• Enlarged tender lymph nodes (post-auricular, postcervical, and suboccipital) usually develop 4-6 days
before rash.
• Maculopapular rash begins on the face and lasts
from 12 hours to 5 days.
Rubella rash
Complications of rubella
•
•
•
•
Encephalitis
Arthritis
Thrombocytopenic purpura
Congenital syndromes:
•
•
•
•
Laboratory diagnosis of rubella
Serology:
Rising Ig G antibody titres - 4-fold or greater:
HAI, CFT, NtT, IF.
Presence of rubella-specific IgM: ELISA.
Isolation the virus in cell cultures from
respiratory tract secretions and, in infants with
congenital infection, from urine, cerebrospinal
fluid, and blood. Identification:
– viral interference,
– HAI,
– IF,
– ELISA.
Vaccination
• Combined measles-mumps-rubella live-virus
vaccine (MMR) is recommended for all healthy
12 to 18-month-old children.
• The second dose of:
– rubella vaccine is given either at school entry or at
entry to high school.
– mumps vaccine is recommended either at 6 or 12
to 13 years of age.
– Live measles vaccine should be given to anyone
who does not have a history of measles or has not
received vaccine after the age of 15 months.