1. Chair of Microbiology, Virology, and Immunology
Paramyxoviruses: Mumps virus, Measles virus.

2. 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

3. Influenza
“La malatia per l’influenza della stella” (the disease caused by the influence of the stars)
In French: grippe, from French verb “agripper” (clinging)

4. 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.
A severe respiratory disease of humans, other mammals, and birds. Influenza remains an important public health problem, responsible for million respiratory illnesses each year in the U.S. involving about 30 million visits to physicians, more than 200,000 hospitalizations, and 20,000 deaths. Emergence of new influenza virus strains that have undergone major genetic changes are associated with severe pandemics, which occur every years, and are associated with high mortality.

5. 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

6. Orthomyxoviruses. Nomenclature
Human influenza virus
Influenza A/Bangkok/1/79(H3N2)
Influenza A/Singapore/1/57(H2N2)
Influenza B/Ann Arbor/1/86
Influenza type
Year of isolation
Hemagglutinin subtype
A/Sydney/5/97 (H3N2)
Geographic source
Isolate number
Neuraminidase subtype

7. Influenza virus A
Orthomyxoviruses: medium-sized, enveloped, (-) sense that vary in shape from spherical to helical. Their genome is segmented into eight pieces

8. 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
Influenza Virus Structure
Flu Viruses Currently infecting...
Humans: H1N1, H1N2, and H3N2
Avian Flu Virus: H5N1

9. ORTHOMYXOVIRUSES type A, B, C : NP, M1 protein
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

10. Haemagglutinin (HA) Neuraminadase (NA) 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

11. Hemagglutinin (N) Neuraminidase (H)
Subtype
Hemagglutinin (N)
Neuraminidase (H)
Human
Swine
Horse
Bird 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

12. 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 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.

13. 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 nt
Glycoprotein
-hemagglutinin esterase fusion (HEF)
esterase -> receptor destroying enzyme

14. 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.

15. Features of viral genera
severity of illness
animal reservoir
human pandemics
human epidemics
antigenic changes
segmented genome
amantadine, rimantidine
zanamivir
surface glycoproteins
TYPE A
++++
yes
shift, drift
sensitive 2
TYPE B ++ no
yes
drift
no effect
sensitive 2
TYPE C + no
no (sporadic)
drift
yes
no effect
(1)

16. 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.

17. Influenza virus reproduction
Insert figure 25.1
Influenza cycle

18. Single-cell reproductive cycle
1. Attachment to the epithalial cells of the host through hemagglutinin.
2. Endocytosis
3. Uncoating - > 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.

19. Need to make mRNA MINUS (NEGATIVE) SENSE RNA GENOMES proteins
(+ve) sense mRNA
AAA
(-ve) sense genomic RNA

20. Need to make mRNA MINUS (NEGATIVE) SENSE RNA GENOMES
RNA polymerase must be
packaged in virion.
proteins
(+ve) sense mRNA
AAA
If used, RNA modifying enzymes are
packaged in virion.
(-ve) sense genomic RNA

22. Influenza Type A Viruses: antigenic Shift 1889-1977
Year
Subtype
Common Name
Type of variation
1889
H2N2
1900
H3N8
H1N1
Spanish flu
antigenic drift
1957
Asian flu
antigenic shift
1968
H3N2
Hong Kong flu
Russian flu reappearance of viruses from 1918,1950
Ukraine to-day
А “Brisben-1“(H1N1), “Brisben-2“(H1N2), “Brisben- 1007" (Н3N2), B “Florida”

23. 1918 Influenza epidemic > 20 million died of the flu during WW I
A new influenza vaccine must be developed yearly

24. Mechanisms of Influenza Virus Antigenic “Shift”
DIRECT
15 HAs
9 NAs
Non-human
virus
Human
virus
Reassortant
virus

25. 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

26. GRADUAL ANTIGENIC CHANGE WITHOUT A CHANGE IN SUBTYPE
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

27. 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”

28. Antigenic shift NA HA HA 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 HA HA

29. What is an Epidemic? What is a Pandemic? Epidemic Pandemic
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

30. 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
We don’t know for sure where influenza comes from, but we do know influenza causes significant illness not only in humans but is a wide variety of other animals.
In ducks and waterfowl, the influenza virus targets the gastro-intestinal tracks, but the infection remains sub- clinical.

31. Why do we not have influenza B pandemics?
so far no shifts have been recorded
no animal reservoir known

32. 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

33. SYMPTOMS FEVER HEADACHE MYALGIA COUGH RHINITIS OCULAR SYMPTOMS
CHILLS and/or SWEATS
Infection may be very mild, even asymptomatic, moderate or very severe

34. 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

35. NORMAL TRACHEAL MUCOSA
3 DAYS POST-INFECTION
7 DAYS POST-INFECTION

36. Immunity to influenza Antibody to HA - >protective
Antibody to NA - > decrease severeity
Serum antibody - > years
Secretory antibody - > months

37. 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

38. 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

39. Prophylaxis Masks and Hand Washing
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

40. Types of Vaccine
Killed Whole Virus inactivated virus vaccine grown in embryonated eggs; 70-90% effective in healthy persons <65 years of age, 30-70% in persons ≥65 years
Live Virus Attenuated strains were widely used in Russia but not elsewhere.
Virus Subunit HA extracted from recombinant virus forms the basis of today's vaccines.
Synthetic Much research is being done to try and find a neutralising epitope that is more stable, and can therefore be used for a universal vaccine.

41. Trivalent Influenza virus vaccines
A/Sydney/05/97 (H3N2)
A/Beijing/262/95 (H1N1)
B/Yamanashi/166/98
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

42. 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)
ZANAMIVIR (neuraminidase inhibitors) (NA)
types A and B, needs to be given early
OSELTAMIVIR (neuraminidase inhibitors) (NA)

43. Adamantanes and Neuraminidase Inhibitors
Antivirals:
Adamantanes and Neuraminidase Inhibitors
NA inhibitors
HA blockers

44. 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???

45. 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.

46. 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.
Nonpathogenic H5 influenza virus: Wild fowl  domestic ducks and geese,  domestic chickens.
H5 virus became highly pathogenic in chickens  domestic ducks and geese.
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.

47. 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.

48. Avian influenza

49. 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

50. H5N1 in wild birds, poultry & humans – 5/19/06

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

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

53. 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

54. PARAMYXOVIRUSES pleomorphic HN/H/G glycoprotein SPIKES F glycoprotein
helical nucleocapsid (RNA minus
NP protein)
lipid bilayer membrane
polymerase complex
M protein

55. Effects of paramyxoviruses
Insert figure 25.5
Effects of paramyxoviruses

56. 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.

57. 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.

58. Mumps pathogenesis

59. Mumps virus CPE
Epidemic parotitis
Orchitis

60. 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.

61. 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.

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

63. 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.

64. 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

65. 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.

66. 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 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

68. 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

69. Subacute sclerosing panencephalitis

70. 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!

71. RESPIRATORY SYNCYTIAL (RS) VIRUS
The particle is slightly smaller ( nm) than other paramyxoviruses, and the nucleocapsid measures nm. Although RS is one of the most labile of viruses, it can be stabilized by molar MgSC>4 (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

72. 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.

73. RSV- syncytium formation
Immunofluoresent stain

74. 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

75. 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.

76. 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.

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

78. 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 it’s 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.

79. Disease Prevention Disease Prevention
Wash hands before you eat and after you use the bathroom.
Don’t 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)
Disease Prevention