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Chair of Microbiology, Virology, and Immunology
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
Differences between orthomyxoviruses and
paramyxoviruses
Feature Orthomyxoviruses Paramyxoviruses
Viruses and diseases Influenza A,B,C Mumps, measles, respiratory syncytial, parainfluenza
Genome Single-stranded RNA in 8 pieces, MW 2-4x106 Single-stranded RNA in single piece, MW 5-8x106
Inner ribonucleo-protein helix 9-nm diameter 18-nm diameter
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Influenza

• La malatia per linfluenza della stella(the
  disease caused by the influence of the stars)
• In French grippe, from French verb agripper
  (clinging)

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INFLUENZA

• severe respiratory disease
• 20-50 million respiratory illnesses each year in
  the U.S.
• 30 million visits to physicians, 200,000
  hospitalizations
• 20,000 deaths
• new influenza virus strains associated with
  severe pandemics and high mortality.

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ORTHOMYXOVIRUSES (INFLUENZA VIRUSES)

• Classification
• Type A viruses cause the most cases of influenza
  in humans and undergo mutations more frequently
  than the other type viruses
• Type B viruses are endemic in USA and associated
  with local epidemics
• Type C viruses rarely cause disease

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Orthomyxoviruses. Nomenclature
Human influenza virus Influenza
A/Bangkok/1/79(H3N2) Influenza A/Singapore/1/57(H2
N2) Influenza B/Ann Arbor/1/86
Influenza type
Hemagglutinin subtype
Year of isolation
A/Sydney/5/97 (H3N2)
Geographic source
Isolate number
Neuraminidase subtype
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Influenza virus A
Orthomyxoviruses medium-sized, enveloped, (-)
sense that vary in shape from spherical to
helical. Their genome is segmented into eight
pieces
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Influenza Virus Structure

• Influenza viral genome
• (-) ssRNA
• 8 segments (pieces)
• One gene per segment
• nucleoprotein
• matrix proteins
• NS (nonstructural proteins, that are not
  incorporated into viral particles) gene encodes
  two different non-structural proteins
• subunits of RNA polymerase
• spikes (about 500)
• Flu viruses are named by the type of surface
  proteins
• Hemagglutinin - trimer (HA)
• Helps virus enter cell
• Type A infects humans, birds and pigs
• Type A has 20 different sub types
• Neuraminidase - tetramer (NA)
• Helps virus exit cell
• 9 subtypes

• Flu Viruses Currently infecting...
• Humans H1N1, H1N2, and H3N2
• Avian Flu Virus H5N1

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ORTHOMYXOVIRUSES
HA - hemagglutinin
NA - neuraminidase
helical nucleocapsid (RNA plus NP protein)
lipid bilayer membrane
polymerase complex
M1 protein
type A, B, C NP, M1 protein sub-types HA or
NA protein
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Haemagglutinin (HA) Encoded by RNA segment 4 Can agglutinate red blood cells - hence the nomenclature Cleavage by host-cell protease is required (resulting in HA1 and HA2) for infection to occur Hemagglutinin glycoprotein is the viral attachment protein and fusion protein, and it elicits neutralizing, protective antibody responses Neuraminadase (NA) Encoded by RNA segment 6 Removes neuraminic (sialic) acid from cell and permits dissemination of viruses Important in releasing mature virus from cells Stimulates production of protective antibodies
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Subtype Hemagglutinin (N) Hemagglutinin (N) Hemagglutinin (N) Hemagglutinin (N) Neuraminidase (H) Neuraminidase (H) Neuraminidase (H) Neuraminidase (H)
Subtype Human Swine Horse Bird Human Swine Horse Bird
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5
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Influenzavirus B

• Virions enveloped
• About 500 spikes
• Nucleocapsid enclosed within lipoprotein membrane
• Virions contain 8 segments of linear
  negative-sense single stranded RNA
• Total genome length is 13588 nt
• The largest segment 2341 nt

• Infect much man and birds.
• Cause human disease but generally not as severe
  as A types.
• Believed to be epidemiologically important -
  reassortment with type A leads to epidemics.

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Influenzavirus C

• Virions enveloped
• Many spikes
• Nucleocapsid enclosed within lipoprotein membrane
• Virions contain 7 segments of linear
  negative-sense single stranded RNA
• Total genome length is 12900 nt
• Glycoprotein
• -hemagglutinin esterase fusion (HEF)
• esterase -gt receptor destroying enzyme

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Antigen
Influenza viruses are divided into 3 groups
determined by the ribonucleoprotein (RNP) antigen
and M antigen

• Soluble antigens include ribonucleoprotein and M
  protein which are much stable in antigenicity.
• Surface antigens include HA and NA which are
  much variable in antigenicity.

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Features of viral genera
TYPE A yes yes yes shift,
drift yes sensitive sensitive 2
severity of illness animal reservoir human
pandemics human epidemics antigenic
changes segmented genome amantadine,
rimantidine zanamivir surface glycoproteins
TYPE B no no yes drift yes no
effect sensitive 2
TYPE C no no no (sporadic) drift yes no
effect (1)
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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.
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Influenza virus reproduction
Insert figure 25.1 Influenza cycle
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Single-cell reproductive cycle

• 1. Attachment to the epithalial cells of the host
  through hemagglutinin.
• 2. Endocytosis
• 3. Uncoating - gt This exposes the contents of the
  virus to the cytosol.
• 4.The RNA enter the nucleus of the cell where
  fresh copies are made.
• 5. These copies return to the cytosol where some
  serve as mRNA molecules to be translated into the
  proteins of fresh virus particles.
• 6. Progeny virions are formed and released by
  budding from the plasma membrane of the cell
  (aided by the neuraminidase) thus spreading the
  infection to new cells.

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Need to make mRNA
MINUS (NEGATIVE) SENSE RNA GENOMES
proteins
(ve) sense mRNA
(-ve) sense genomic RNA
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Need to make mRNA
MINUS (NEGATIVE) SENSE RNA GENOMES RNA
polymerase must be packaged in virion.
proteins
(ve) sense mRNA
If used, RNA modifying enzymes are packaged in
virion.
(-ve) sense genomic RNA
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Influenza Type A Viruses antigenic Shift
1889-1977
Year Subtype Common Name Type of variation
1889 H2N2
1900 H3N8
1918-1957 H1N1 Spanish flu antigenic drift
1957 H2N2 Asian flu antigenic shift
1957-1968 H2N2 Asian flu antigenic drift
1968 H3N2 Hong Kong flu antigenic shift
1968-1990 H3N2 Hong Kong flu antigenic drift
1977-1989 H1N1 Russian flu reappearance of viruses from 1918,1950 antigenic drift
Ukraine to-day ? Brisben-1(H1N1), Brisben-2(H1N2), Brisben- 1007" (?3N2), B Florida
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1918 Influenza epidemic
gt 20 million died of the flu during WW I A new
influenza vaccine must be developed yearly
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Mechanisms of Influenza Virus Antigenic Shift
15 HAs 9 NAs
Non-human virus
Human virus
Reassortant virus
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Antigenic changes of Influenza A

• Viruses can undergo frequent changes due to
  recombination, reassortment, insertions and point
  mutations
• Antigenic drift
• Antigenic shift occurs every 8-10 yrs
• Minor antigenic changes favor persistence of the
  viruses in the population and allow recombination
  that can eventually lead to severe epidemics
  and/or pandemics

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ANTIGENIC DRIFT

• Gradual accumulation of mutations that allow the
  hemagglutinin to escape neutralizing antibodies
• Epidemic strains thought to have changes in
  three or more antigenic sites

• GRADUAL ANTIGENIC CHANGE WITHOUT A CHANGE IN
  SUBTYPE

H3N2 1968 HONG KONG
H3N2 1975 VICTORIA
H3N2 1993 BEJING
H3N2 2004 FUJIAN
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Antigenic drift

• Antigenic differences can result from changes in
  one amino acid
• Can involve any antigenic protein
• Can occurs every year

• RNA replication is error prone
• New HA types are created frequently
• Requires new vaccine every season

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Antigenic shift

• Occurs every 8-10 yrs
• Major antigenic change of either H or N antigens
  or both H and N
• Occurs by gene reassortment after simultaneous
  infection of a cell with two different viruses
• Three different H proteins and 2 major N proteins
  have evolved

H1N1 Spanish flu H2N2 Avian flu H2N2 Asian flu




NA NA



HA
HA
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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?
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

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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

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Why do we not have influenza B pandemics?

• so far no shifts have been recorded
• no animal reservoir known

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Epidemiology

• Source of infection patients and carriers.
• AEROSOL
• 100,000 TO 1,000,000 VIRIONS PER DROPLET
• Common large droplets (sneezing, coughing,
  contact with saliva)
• Probably common contact
• Direct
• Fomite
• Rare airborne over long distance
• 18-72 HR INCUBATION

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SYMPTOMS

• FEVER
• HEADACHE
• MYALGIA
• COUGH
• RHINITIS
• OCULAR SYMPTOMS
• CHILLS and/or SWEATS

Infection may be very mild, even asymptomatic,
moderate or very severe
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Clinical Responses

• Acute Symptoms last one week
• Abrupt onset of fever, myalgia, headache and
  non-productive cough
• Fatigue and weakness can last 2-3 weeks.
• Infected individual predisposed to bacterial
  infections Staphylococcus, Streptococcus,
  Hempohilus
• Other complications - Reyes Syndrome
• Immunity dependent upon localized anti-viral
  secretory IgA ( strain specific)
• Develop long lasting circulating anti-viral IgG

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NORMAL TRACHEAL MUCOSA
3 DAYS POST-INFECTION
7 DAYS POST-INFECTION
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Immunity to influenza

• Antibody to HA - gtprotective
• Antibody to NA - gt decrease severeity
• Serum antibody - gt years
• Secretory antibody - gt months

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Laboratory Diagnosis

• VIROLOGICAL
• Respiratory secretions (direct aspirate , gargle
  , nasal
• washings)
• Virus isolation and growth in embryonated eggs
• Cell culture in primary monkey kidney or
  madindarby canine kidney cells
• Hemagglutination (inhibition)
• Hemadsorption (inhibition)
• IFA/ ELISA
• Direct immunofluorescence

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Laboratory Diagnosis

• Serodiagnosis
• Four-fold or greater increase in hemagglutination
  inhibition antibody titers between acute and
  convalescent specimens
• Hemagglutination inhibition
• Hemadsorption inhibition
• ELISA
• Complement fixation test
• NT

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Masks and Hand Washing
Prophylaxis

• To be Continued

• Hand washing
• Generally perceived to be useful
• No studies specifically performed for influenza
• Easy to recommend
• Masks
• Effectiveness not shown for influenza
• However, could reduce transmission associated
  with large droplets

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Types of Vaccine

• Killed Whole Virusinactivated virus vaccine
  grown in embryonated eggs 70-90 effective in
  healthy persons lt65 years of age, 30-70 in
  persons 65 years
• Live VirusAttenuated strains were widely used in
  Russia but not elsewhere.
• Virus SubunitHA extracted from recombinant virus
  forms the basis of today's vaccines.
• SyntheticMuch research is being done to try and
  find a neutralising epitope that is more stable,
  and can therefore be used for a universal
  vaccine.

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Trivalent Influenza virus vaccines

• 1999-2000
• A/Sydney/05/97 (H3N2)
• A/Beijing/262/95 (H1N1)
• B/Yamanashi/166/98

• 2000-2001
• A/Moscow/10/99(H3N2)-like
• A/New Caledonia/20/99 (H1N1)-like
• B/Beijing/184/93-like

• To day
• A/Brisben/59/2007 (H1N1)
• A/Brisben/10/2007 (H3N2)
• B/Florida/4/2006

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Prevention and Treatment
70-90 effective in preventing illness

• RIMANTADINE (blocks the M2 ion channel) (M2)
• type A only, needs to be given early
• AMANTADINE (blocks the M2 ion channel) (M2)
• type A only, needs to be given early
• ZANAMIVIR (neuraminidase inhibitors) (NA)
• types A and B, needs to be given early
• OSELTAMIVIR (neuraminidase inhibitors) (NA)
• types A and B, needs to be given early

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Antivirals Adamantanes and Neuraminidase
Inhibitors
NA inhibitors
HA blockers
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Avian Influenza Poultry Outbreaks, Asia, 2003-06

• Hong Kong 1997 18 cases of influenza in humans
  caused by a highly pathogenic avian influenza
  virus (H5N1) 30 fatality rate
• 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
• H9N2 subtype also detected among infected poultry
• It has infected humans in 10 countries.

In the future reassortment between H9N2 or H5N1
avian viruses and H1N1 or H3N2 human viruses???
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Avian Flu

• Avian influenza, or bird flu, is a contagious
  disease of animals caused by viruses that
  normally infect only birds and, less commonly,
  pigs. Avian influenza viruses are highly
  species-specific, but have, on rare occasions,
  crossed the species barrier to infect humans.
• Pandemic viruses appear as the result of
  antigenic shift, which causes new combinations of
  proteins on the surface of the virus. If the new
  virus spreads easily from person to person a
  pandemic can result.

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Emergence of New Influenza Subtypes
H5N1 Antigenic shift due to genome reassortment
within intermediate hosts drives flu epidemics
and pandemics
Solid lines transmission demonstrated Dotted
lines transmission postulated but not
demonstrated.

1. Nonpathogenic H5 influenza virus Wild fowl ?
   domestic ducks and geese, ? domestic chickens.
2. H5 virus became highly pathogenic in chickens ?
   domestic ducks and geese.
3. Highly Pathogenic H5 virus reassorted its genome
   with those of other influenza viruses in aquatic
   birds, ? spread to poultry farms, humans, and
   occasionally to pigs.

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Avian influenza

• Wild birds are the reservoir.
• Circulation of low pathogenic avian flu in
  domestic poultry leads to mutations to highly
  pathogenic forms over time.
• Co-infection with swine or humans infected with
  human influenza can result in genetic
  reassortment and highly pathogenic strains.

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Avian influenza
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Why do new strains of influenza and bird flu
arise in Asia?
In 2003, an outbreak of chicken flu
necessitated killing tens of millions of birds
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H5N1 in wild birds, poultry humans 5/19/06

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• Family Paramyxoviridae
• Subfamily Paramyxovirinae
• Genera
• Morbillivirus measles virus,
• Respirovirus (earlier Paramyxovirus)
  parainfluenza virus serotypes 1 and 3
• Rubulavirus - parainfluenza virus serotypes 2,
  4?, 4b, mumps virus
• Henivirus Australian Hendra-virus (diseases of
  human and horses), Malasian Nipah-virus (diseases
  of human and swine)
• Subfamily Pneumovirinae
• Genera
• Pneumovirus RS-virus
• Metapneumovirus human metapmeumovirus
  (diseases in children)

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PARAMYXOVIRUS FAMILYproperties of attachment
protein
GENUS GLYCOPROTEINS TYPICAL MEMBERS
Paramyxovirus genus HN, F HPIV1, HPIV3
Rubulavirus Genus HN, F HPIV2, HPIV4 mumps virus
Morbillivirus genus H, F measles virus
Pneumovirus genus G, F respiratory syncytial virus
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Virion

• Large virion consists of a negative RNA genome in
  a helical nucleocapsid surrounded by an enevlope
  containing a viral attachment protein
• HN of paramyxovirus and mumps virus has
  hemagglutinin and neuraminidase.
• H of measles virus has hemagglutinin activity
• G of RSV lacks these activities

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PARAMYXOVIRUSES
pleomorphic
HN/H/G glycoprotein SPIKES
F glycoprotein SPIKES
helical nucleocapsid (RNA minus NP protein)
lipid bilayer membrane
polymerase complex
M protein
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Insert figure 25.5 Effects of paramyxoviruses
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MUMPS (Epidemic Parotitis) Mumps is an acute
contagious disease characterized by a
nonsuppurative enlargement of one or both of the
parotid glands, although other organs may also be
involved. Properties of the Virus Mumps virus is
a typical paramyxovirus. It has typical
hemagglutination, neuraminidase, and hemolysin
activities. Hemagglutination can be inhibited by
specific antisera to mumps virus, and this
inhibition can be used to measure antibody
responses. Similarly, the nucleocapsid of the
virus particle forms the major component of the
"S" (soluble) complement-fixing antigen.
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Mumps virus

• Droplets spread the infection via saliva and
  secretions from the respiratory tract.
• Incubation period of 2-3 weeks
• Malaise and fever is followed within a day by
  painful enlargement of one or both of the parotid
  (salivary) glands
• A possible complication in males after puberty is
  orchitis - painful swelling of one or both
  testicles.
• Inflammation of the ovary and pancreas can also
  occur.
• Disease is usually self-limiting within a few
  days
• Aseptic meningitis (usually resolving without
  problems) or postexposure encephalitis (can prove
  fatal) are the most serious complications
  associated with mumps.

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Mumps pathogenesis
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Mumps virus CPE
Epidemic parotitis
Orchitis
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Prevention and treatment

• Treatment none (passive immunization has been
  used).
• Prevention one invariant serotype therefore
  vaccines are viable - both formalin-inactivated
  and live attenuated exist, the latter now being
  widely used- see MMR.

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PARAINFLUENZA VIRUS INFECTIONS The parainfluenza
viruses are paramyxoviruses with morphologic and
biologic properties typical of the genus. They
grow welt in primary monkey or human epithelial
cell culture but poorly or not at all in the
embryonated egg. They produce a minimal
cytopathic effect in cell culture but are
recognized by the hemadsorption method.
Laboratory diagnosis may be made by the HI, CF,
and Nt tests.
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Important Characteristics

• Typing Four types (1-4) distinguished
  antigenically, by cytopathic effect, and
  pathogenically
• Haemagglutinin and fusion F protein is found in
  the envelope

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Pathogenesis and Immunity

• Cause acute respiratory infections of man ranging
  from relatively mild influenza-like illness to
  bronchitis, croup (narrowing of airways which can
  result in respiratory distress) and pneumonia
  common infection of children.
• Transmitted by aerosols.

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Lab Diagnosis

• Nasopharynx specimen is culture in a surrogate
  cell line in AGMK. Infected cell are detected by
  hemeadsorption or DFA
• DFA also can be done rapidly to identify the
  agent in direct specimen
• Serotypes 1-3 are comfirmed by hemeagglutination
  inhibition using standardized antisera

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• MEASLES (Rubeola)
• Measles is an acute, highly infectious disease
  characterized by a maculopapular rash, fever, and
  respiratory symptoms.
• Properties of the Virus. Measles virus is a
  typical paramyxovirus, related to canine
  distemper and bovine rinderpest. All 3 lack
  neuraminidase activity. Measles agglutinates
  monkey erythrocytes at 37 C but does not elute,
  and it interacts with a distinct cell receptor.
  Measles virus also causes hemolysis, and this
  activity can be separated from that of the
  hemagglutinin.
• In culture, produces characteristic intranuclear
  inclusion bodies and syncytial giant cells.

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Pathogenesis and Immunity

• Childhood infection almost universal, protection
  resulting from this is probably lifelong. Both
  man and wild monkeys are commonly infected
• Transmission and initial stages of disease
  similar to mumps, but this virus can also infect
  via the eye and multiply in the conjunctivae.
  Viraemia following primary local multiplication
  results in widespread distribution to many
  organs.
• After a 10-12 day incubation period
• Disease
• Fever,
• Respiratory tract syndrom (dry cough, rhinorrhea,
  sore throat)
• conjunctivitis (virus may be excreted during this
  phase!), followed a few days later by the
  characteristic red,
• maculopapular rash,
• Koplik's spots
• Towards the end of the disease, there is
  extensive, generalized virus infection in
  lymphoid tissues and skin

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Measles

• most serious complication is subacute sclerosing
  panencephalitis (SSPE), a progressive
  neurological degeneration of the cerebral cortex,
  white matter and brain stem
• 1 case in a million infections
• involves a defective virus spreading through the
  brain by cell fusion and destroys cells
• mental disorders
• leads to coma and death in months or years
• attenuated viral vaccine MMR

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Subacute sclerosing panencephalitis
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Prevention

• Both live and killed vaccines exist. Vaccination
  with the live attenuated vaccine has been
  practised since the 1960's with a dramatic
  decline in the incidence of the disease .
• Trivalent live attenuated vaccine (MMR) usually
  given - all of these viruses best avoided during
  pregnancy!

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The particle is slightly smaller (80-120 nm) than
other paramyxoviruses, and the nucleocapsid
measures 11-15 nm. Although RS is one of the most
labile of viruses, it can be stabilized by molar
MgSCgt4 (like measles and other paramyxoviruses).
RS virus does not hemagglutinate. A soluble
complement-fixing antigen can be separated from
the virus particle. This labile paramyxovirus
produces a characteristic syncytial effect, the
fusion of cells in human cell culture. It is the
single most serious cause of bronchiolitis and
pneumonitis in infants.
RESPIRATORY SYNCYTIAL (RS) VIRUS
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Important Characteristics

• RSV is highly infectious, transmission by
  respiratory secretions.
• Primary multiplication occurs in epithelial cells
  of URT producing a mild illness. In 50 children
  less than 8 months old, virus subsequently
  spreads into the L.R.T. causing bronchitis,
  pneumonia and croup.
• Has been suggested as a possible factor in cot
  death and asthma.

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RSV- syncytium formation

Immunofluoresent stain
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• RSV is a viral disease.
• Respiratory Syncytial Virus (RSV)is a very
  serious virus often found in children and infants
  under the age of three.
• Adults are at very low risk of catching RSV.
• RSV Bronchiolitis - clinical features

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How is this disease transmitted?

• This disease is transmitted by
• coughing
• sneezing
• sharing wash cloths towels and other things with
  someone with RSV.
• Most people with RSV get it in fall and winter.

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Risk Group
Risk Group

• Babies and elders are most at risk of catching
  RSV.
• Although adults do catch it, it appears to them
  as being a common cold
• This disease is extremely serious when it comes
  to children and infants under the age of 3 and
  elders.
• This disease can result in death.

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• Symptoms for this disease are
• sneezing
• runny nose
• sore throat
• low fever
• common cold symptoms just more severe.

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Treatments for Disease

• The main thing a doctor will tell you if you ask
  about treatments for RSV he will most likely say
  to let it run its course.Things you can do for
  the person that has RSV are
• comfort that person.
• Things you can do if you have it are
• Drink plenty of fluids.
• Get lots of rest
• Antiviral Agents
• Ribavirin (Virazole), a synthetic guanosine
  analogue, given as an aerosol
• Supportive
• Fluids, oxygen, respiratory support,
  bronchodilators.

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Disease Prevention
Disease Prevention

• Wash hands before you eat and after you use the
  bathroom.
• Dont share towels or wash cloths with others.
• Eat healthy.
• Keep clean.
• Stay clear of people who are ill
• Disinfection of surfaces
• Gloves, masks, goggles, gowns
• Isolation, and cohort nursing
• Immunization
• Active Immunization
• Formalin inactivated vaccine resulted in
  enhanced disease
• Subunit vaccines being studied
• Passive immunization (immunoprophylaxis)
• Pooled hyperimmune globulin (RespiGam)
• Monoclonal antibody to F protein- Palivizumab
  (Synagis)