1. Influenza:

2. ORTHOMYXOVIRIDAE Nomenclature and Classification
1. Influenza family which is subdivided into 3 genera: Influenza A, Influenza B, and Influenza C, based on antigenic differences in the nucleoprotein (NP) and matrix (M) protein.
2. Influenza viruses are further characterized within type by antigenic differences associated with the H and N glycoproteins; there are at least 14 subtypes of H and 9 subtypes of N proteins in influenza A virus.
3. All subtypes have been described in birds and some of them have been found in mammals.
4. To facilitate epidemiological studies, individual viruses are coded in the following manner:

3. Nomenclature A/equine/Prague/1/56(H7N7) A/fowl/Hong Kong/1/98(H5N1)
A/equine/Saskatoon/1/90(H3N8)
Serotype of
HA and N
group
year
species
Isolate number
location
A/equine/Prague/1/56(H7N7)
A/fowl/Hong Kong/1/98(H5N1)
A/swine/Lincoln/1/86(H1N1)

4. Virus characteristics
1. Medium sized ( nm diameter), enveloped, spherical to slightly pleomorphic in shape.
2. Genome consists of 8 segments of ss RNA which code for 10 proteins (5 structural, 3 associated with polymerase and 2 non-structural); genetic reassortment can occur
3. The envelope contains 2 glycoproteins: H (hemagglutinin), and N (neuraminidase)
4\ genetic reassortment occur frequently
5. Can infect humans, birds, swine, equine, seals, mink and whales

5. Influenza virions nucleocapsid envelope (RNA fragments
wrapped in protein)
envelope
haemagglutinin and
neuraminidase “spikes”
In envelope
100 nm

7. Hemagglutinin and Neuraminidase
sialic acid
on receptor HA N
active site
receptor
binding
site
variable
loops
variable
loops

8. Influenza infection: humans Influenzavirus A horses pigs birds
marine mammals
Influenzavirus B
humans
Influenzavirus C
swine
humans

9. Distribution of HA serotypes in nature
Birds
Horses
Pigs
Humans
HA1
yes
yes
yes
HA2
yes
yes
HA3
yes
yes
yes
yes
HA4
yes
HA5
yes
yes
HA6
yes
HA7
yes
yes
HA8-14
yes

10. Distribution of N serotypes in nature
Birds
Horses
Pigs N1
yes
yes
yes N2
yes
yes
yes N3
yes N4
yes N5
yes N6
yes N7
yes
yes N8
yes
yes N9
yes

11. Evolution and Spread of flu viruses
H1N1
H3N2
pigs
poultry
H1N1
H2N2
H3N2
(H5N1, H9N2)
humans
aquatic birds
fecal/oral
H3N8
horses
All HA and N
serotypes
H7N7
respiratory

12. Factors that sustain epizootics/epidemics
Antigenic drift
Reassortment and antigenic shift
Short term immunity
Cross species transfer

13. ORTHOMYXOVIRIDAE Antigenic Variation
1. Periodic epidemics of influenza Type A are due to antigenic shifts in the virus which are believed to occur through genetic re-assortment between human and animal (or mammalian and avian) viruses. For example, the amino acid sequence of H3 protein of human isolates is more similar to the amino acid sequence of any Hx of mammalian or avian isolates than to the amino acid sequence of H2 proteins of human isolates.
Prior to 1957 H1N1
1957 (Asian flu) H2N2
1968 (Hong Kong) H3N2
1977 H1N1
2. Between epidemics, the influenza virus undergoes minor changes in the HA and N proteins These differences are due to multiple point mutations. These minor changes are referred to as antigenic drifts.

14. Reassortment

15. Pathogenesis of Influenza Viruses - Respiratory Form
Day 1
1. Infection initiated by aerosol route; however, birds can also be infected by fecal/oral route b.entrapment of virions in mucus and their removal by the mucociliary transport system.
2. Non-specific neutralization of virus by receptors mimicking glycoprotein present in the mucus.
3. Interferon production.
Day 1 - 3
1. Infection of individual epithelial cells of trachea
2. Spread to contiguous cells resulting in loss of ciliary activity
3. Destruction of goblet cells and mucus glands further compromises the mucociliary transport system.
4. Destruction of cells initiates a local inflammatory response which results in increased amount of exudates and transudates.
5. The surface of the trachea becomes increasingly anaerobic which provides optimum conditions for bacterial attachment and colonization.

16. Pathogenesis of Influenza Viruses - Respiratory Form
Day 5-9
1. Infection of the lung resulting in destruction of type I and II pneumocytes
2. Increasing accumulation of exudates and transudates, loss of surfactant produced by type II pneumocytes, blockage of airways and local hypoxia.
3. Concomitant reduction of macrophage and PMN activity. Secondary bacterial infections become established resulting in bronchial pneumonia.
4. IgA and IgG begin to appear in the upper and lower respiratory tracts, respectively.
5. Secretory IgA appears 8 dpi, reaching a peak in 11 days but declines rapidly.
6. Serum antibodies, detectable by HI and VN tests appear 3 to 7 days after infection, peaking in 14 days; may persist up to 18 months.
7. This is followed by recovery or by systemic spread of the virus
8. Antibody response in young animals is slower and less pronounced

17. Pathogenesis of Influenza Viruses - Respiratory Form
1. The best model to illustrate why some influenza infections become systemic and others do not is illustrated by the avian influenza virus. Essentially virulence is determined by the ease by which the HA protein can be cleaved.
2. The pathogenesis of avian influenza is quite different from that of mammals. The virus replicates in the intestinal tract as well as the respiratory tract and is readily isolated from the cloaca.
3. Virulent strains (H5 and H7) of AI cause viremia and generalized infection which is often complicated by secondary bacterial or viral infections.
4. Antibody assays in birds are complicated because the adult birds may have experienced infections with many different subtypes.
5. Antibody titers are often low, especially in ducks.

18. Epidemiology
1. In general, orthomyxoviruses are not stable in the environment; sensitive to heat (56C, 30 min), acid pH, and lipid solvents. Require close contact for transmission.
2. Exceptions are avian influenza viruses. Avian influenza is unique in that it can retain its infectivity for several weeks outside of its host.
3. Can spread by the aerosol route
4. Epidemiology of AI is poorly understood because of the role of wild birds, the great variety of different strains and the variable effects in different host species.
5. Wild ducks and geese are refractory to disease but wild ducks probably represent the most important reservoir for AI viruses. Virus is shed in secretions of respiratory tract and in feces. Survives for long time in feces.

19. Replication of Influenza Virus
(1) Adsorption: the virus interacts with sialic acid-containing cell receptors via its hemagglutinin and enters via endocytosis. (2) Fusion and uncoating: the hemagglutinin undergoes a conformational change mediated by the acid environment of the endosome, which leads to the fusion of viral and cellular membranes. The ribonucleoprotein complexes are then transported into the nucleus. (3) Transcription and replication: the viral RNA is transcribed and replicated in the nucleus by the viral RNA polymerase (Two different species of RNA are synthesized from the viral RNA template: (a) full-length, positive-sense replicative intermediate RNAs, which are used by the polymerase to produce virion RNA and (b) mRNAs.

20. Influenza virus replication
HA cleaved by
proteases
HA binds to
receptor
virus
buds
virus in
phagolysosome
lowered
pH, HA
fuses
membranes
RNA
released
N releases
virus

21. Cleavage of HA Clara (mucus), extracellular, serum, bacterial
binds
receptor
binds
receptor
penetrates
cell
Clara (mucus),
extracellular,
serum,
bacterial
proteases
HA1
HA0
HA2

22. HA cleavage and virulence
low
virulence
low
cleavability
May ‘94 ->
June ‘94
P Q - - R E T R
respiratory
infection
high
virulence
Dec ‘94 ->
Jan ‘95
P Q R K R K T R
high
cleavability
systemic
infecton

23. Secondary effects of HA
Turns on genes for
TNFa
IL-1
IL-2
IL-6
high fever
cell damage
cachexia
shock
oxygen
free
radicals
activates
NFKB HA
anti
oxidants
bacterial
products

24. Influenza species specificity
Receptor binding target cell
Avian influenza a2,3, sialic acid and gal intestinal epithelial
Human influenza a2,6 sialic acid-gal tracheal epithelial
Pigs a2,3 and tracheal epithelial
a2,6 sialic acid-gal
Pigs are the mixing vessel for Influenza

25. The big pandemic of 1918

26. Major influenza subtypes that have circulated in humans and swine since the 1918 “Spanish Flu” pandemic.
Richard Webby and Robert Webster, “Influenza in Humans: Impact, Evolution and Surveillance.” 2001)

27. Relative frequency of influenza subtypes isolated from humans in recent years.*
Richard Webby and Robert Webster, “Influenza in Humans: Impact, Evolution and Surveillance.” , 2001)
* The data represent worldwide frequencies that were obtained from: The WHO influenza surveillance network homepage.

28. Schematic representation of the genetic reassortment events that lead to the development of the 1957 and 1968 pandemic strains of human influenza A viruses.
1918
H1N1
1957
H2N2
1968
H3N2
H2 HA,
N2 NA,
PB1
H3 HA,
Christopher Olsen, “The emergence of novel swine influenza viruses in North America,” Elsevier Science B.V., Article in Press, 2002.

29. ORTHOMYXOVIRIDAE Human Influenza Viruses
1. Humans are susceptible to Types A, B, and C.
2. Type A causes the classical influenza with which we are familiar.
3. Type B causes a mild to severe influenza. Reye's syndrome which is characterized by rapidly progressive non-inflammatory encephalopathy and fatty infiltration of the liver leading to its dysfunction, has been associated with type B influenza virus (also with other viral-induced respiratory and enteric infections).
4. Type C influenza virus is associated with mild upper respiratory disease.

30. Clinical Signs and Symptoms of Human Influenza
(1) Influenza viruses are spread from person-to-person primarily through the coughing and sneezing of infected persons.
(2) The incubation period for influenza is 1--4 days, with an average of 2 days.
(3) Persons can be infectious starting the day before symptoms begin through approximately 5 days after illness onset; children can be infectious for a longer period.
(4) Uncomplicated influenza illness is characterized by the abrupt onset of constitutional and respiratory signs and symptoms (e.g., fever, myalgia, headache, severe malaise, nonproductive cough, sore throat, and rhinitis). Reported sensitivity and specificity of clinical definitions for influenza-like illness that include fever and cough have ranged from 63% to 78% and 55% to 71%, respectively, compared with viral culture . Sensitivity and predictive value of clinical definitions can vary, depending on the degree of co-circulation of other respiratory pathogens and the level of influenza activity (28).
(5) Influenza illness typically resolves after several days for most persons, although cough and malaise can persist for >2 weeks.
(6) In some persons, influenza can exacerbate underlying medical conditions (e.g., pulmonary or cardiac disease), lead to secondary bacterial pneumonia or primary influenza viral pneumonia, or occur as part of a co-infection with other viral or bacterial pathogens .
(7) Influenza infection has also been associated with encephalopathy, transverse myelitis, Reye syndrome, myositis, myocarditis, and pericarditis .

31. Equine influenza A1 H7N7 rare pockets in central Europe??
A2 H3N8 annual epizootics
World wide except - Australia, New Zealand, Iceland
Highly contagious, rapid spread

32. ORTHOMYXOVIRIDAE Equine Influenza
1. Mild to acute upper respiratory disease. 2 subtypes: A/equine/1 and A/equine/2
2. Antigenic drift has been detected periodically:
3. Virus strain of avian origin was suspected in the outbreak in China.
4. Clinically, the disease is similar to that caused by equine herpesvirus type 4 (EHV-4) and equine rhinoviruses (Picornaviridae).
5. Interstitial myocarditis has been reported in horses suffering from acute influenza

33. Pathogenesis inhalation (infected animal or fomites) replication in
epithelial cells
upper RT

34. Clinical signs-EIV Sudden onset Fever (39-42), biphasic
Loss of appetite
Muscle soreness
Dry cough
Nasal discharge (serous ->mucopurulent)
Clinical signs are similar to other respiratory diseases such as equine rhinopneumonities and viral arteritis
Need lab testing

35. Exercise or Rest Gross et al. 1998. Equine Vet. Jn. 30:489
Exercised group
More severe disease
More weight loss
No difference in recovery time
Long term effects??

36. Diagnosis Clinical signs Virus isolation Directagen Flu-A
Serological tests
HAI
Single radial haemolysis

37. EIV Conventional vaccines
Inactivated, H7N7 and H3N8 isolates
Adjuvant
Most -> short lived protection
Revaccinate at 6 week intervals

38. EIV Intranasal, attenuated vaccine
Heska Co.

39. Avian Influenza (AI) 1. Clinical signs/host:
a. Inapparent to acute systemic disease in chickens
b. Clinically affected birds may show respiratory, CNS and enteric signs of the disease
c. The more severe form of the disease is sometimes referred to as "fowl plague."
d. Various species of wild birds, mainly waterfowl, constitute an important reservoir.
e. Among domestic birds, chickens and turkeys are most likely to develop disease
f. Pheasants, quail, guinea fowl and partridges are also susceptible

40. Avian Influenza (AI) 2. Virus types: 3. Economic losses:
a. All virulent strains that cause disease in chickens are of the H5 and H7 subtypes.
b. However, not all subtypes of H5 or H7 cause the same severity of disease in chickens
c. The amino acid sequence in the hinge region of the HA molecule determines virulence. A change in 1 to 4 amino acids in this region results in increased virulence.
3. Economic losses:
a. Since 1980, infection of turkeys has become an economically important disease in many parts of USA.
b. Losses arise from condemnation at processing plant due to air saculitis caused by secondary bacterial infections (E. coli) and losses in egg production.
c. An outbreak in Pennsylvania and Virginia in , caused by an H5N2 virus, resulted in the slaughter of > 17 million birds, with compensation and other costs in excess of $60 million.
d. Again, all 14 known H subtypes and 9 N subtypes in all possible combinations have been isolated from poultry and wild birds.

41. Avian influenza Pennsylvania - 1983 - $61,000,000
Mexico $$?
Asymptomatic to fatal (sudden death)
Kristi Askin, Tina Tuason, Elisabeth Ludlage

42. The emergence of H5N1 influenza in Hong Kong.
Robert G. Webster, “Influenza: An Emerging Disease,” Emerging Infectious Diseases,” Vol. 4 No. 3, July-Sept., 1998
(URL:

43. Swine Influenza
1. One principal subtype (H1N1) but 2 variants within this subtype in United States up until 1998; one is common in Europe and the other in the USA.
2. Swine were infected with H3N2 strains – 1998, reassorted virus from humans and birds
3. Clinically, upper respiratory disease which generally runs its course within a week.
4. Avoiding stress during infection usually results in lower mortality rate (< 1%).
5. Recovered animals either lose weight or their weight gains are reduced, attributing to economic loss to producers.
6. Outbreaks in swine occur in late fall and early winter.
7. Swine influenza virus (H1N1) can infect turkeys and humans. In turkeys, it causes drop in egg production and increased number of abnormal eggs.

44. SIV in North America
Influenza recognized clinically in pigs (Koen al 1918)
1930 First swine influenza isolated. classical H1N1(Shope 1931)
’s Classical H1N1 in North America (Hinshaw , )
1976 Swine influenza vaccine in humans
Appearance of H3N2 (Zhou et al 1999)
seropositive to H3 (Olsen 2000)
2000 H1N2 reassortmant of H1N1 and H3N2 (Karasin 2000)
2000 H4N6 avian isolated from pigs (Karasin 2000)

45. Swine influenza H1N1 - two variants H3N2 Ontario (1989-92) 53% H1N1
17% H3N2 (similar to human virus)
4% H1N2 (similar to human virus)

46. Genotypes of H3N2 influenza A viruses isolated from pigs in North America since 1997.
Christopher Olsen, “The emergence of novel swine influenza viruses in North America,” Elsevier Science B.V., Article in Press, 2002.

47. Genotype of the H1N2 influenza A viruses isolated from pigs in the United States since 1999.
Christopher Olsen, “The emergence of novel swine influenza viruses in North America,” Elsevier Science B.V., Article in Press, 2002.

48. Swine Influenza:Zoonosis
20 million deaths , due to swine influenza
Continued reports of humans with swine influenza death by H1N1 strain of swine influenza
(Kimura et al 1998 Mayo Clin Proc 73:243)
All 10 islet recipients had antibodies to swine influenza (Butler Nature 391: )

49. Swine Influenza-zoonosis
Classical swine influenza viruses can also be directly transmitted to humans as zoonotic infections, sometimes with fatal consequences. Human infections with swine influenza viruses have been documented in the U.S. at least 10 times since 1974, including fatal infections, as well as in Europe and in New Zealand. In addition, data suggest that zoonotic swine influenza virus infections may actually occur more routinely among people in regular contact with pigs than the relatively small number of documented cases would suggest.

50. Swine Influenza Disease
Respiratory Disease in epizootic
Fever, lethargy, coughing, nasal/ocular discharge, off feed
PRDC porcine respiratory disease complex
PRRSV, Mycoplasma

51. Swine Influenza-disease
8. The pathogenesis of swine and equine influenza virus infections resembles that in man.
Influenza Virus-Induced Pneumonia in a Pig

52. Body temperature course after intratracheal inoculation withH1N1, H3N2 or H1N2 subtypes of SIV
Kristen Van Reeth, “A New Look at Swine Influenza in Europe.” 2001)

53. Relationship between H1 antibody titer and protection against lung lesions.*
Terri Wasmoen, “Immune Response to Swine Influenza Vaccination.” 2001)
* No data is available for H3N2 at 10 and 20 or H1N1 at 320 and 640 antibody titers (at publication.)

54. Diagnosis of SIV Virus detection Virus isolation egg inoculation (EI)
Virus isolation-cell culture
Membrane enzyme immunoassay-hu flu (EIA)
Microwell enzyme immunoassay-hu flu
IFA
Immunohistochemistry(IHC)
PCR
Antibodies
Hemagglutination Inhibition
ELISA to H1 or H3

55. MaxiVac®-FLU Pharmaceutical Name
Swine Influenza Vaccine, Killed VirusProduct
Features and Benefits
Inactivated virus vaccine provides strong, durable immunity against swine flu.
Special oil-in-water adjuvant system helps stimulate strong immune response.
Protection against clinical disease shown in vaccinates challenged with highly virulent SIV.
Significantly reduces circulating virus in lungs after severe challenge which helps prevent costly setbacks due to lung tissue damage, consolidation, and opportunistic bacterial infections.
Reduced viral shedding helps prevent virulent virus spread.
Convenient vaccination schedule fits easily into any herd  health program.

56. MMWR Prevention and Control of Influenza
Recommendations and Reports April 20, 2001 / 50(RR04);1-46
Prevention and Control of Influenza
Recommendations of the Advisory Committee on Immunization Practices (ACIP)
Advisory Committee on Immunization Practices Membership List, February 2001
CHAIRMAN
John F. Modlin, M.D. Professor of Pediatrics and Medicine Dartmouth Medical School Lebanon, New Hampshire
EXECUTIVE SECRETARY
Dixie E. Snider, Jr., M.D., M.P.H. Associate Director for Science Centers for Disease Control and Prevention Atlanta, Georgia

57. Process for human influenza vaccines
surveillance
February meeting
Commonwealth Serum Labs (Australia)
CDC (USA)
Natl. Inst. For Medical Research (UK)
European Inst. For Biological Standardization (EU)
Food and Drug Admin. (USA)

58. Process for human influenza vaccines
March-April
Genetic and antigenic characterization of approved strains
Distribution by WHO to manufacturers
Production of seed stock
Tests for contaminants (bacteria, mycoplasma, viruses)

59. Process for human influenza vaccines
April-August
Vaccine production
License application made
Clinical trials (to be submitted before vaccination season)

60. Process for human influenza vaccines
August-September
Distribution begins

61. Human Influenza Vaccine Composition (2001-2002)
Influenza vaccine contains three strains (i.e., two type A and one type B.
The vaccine is made from highly purified, egg-grown viruses that have been made noninfectious (i.e., inactivated). Subvirion and purified surface-antigen preparations are available.
The trivalent influenza vaccine prepared for the season will include A/Moscow/10/99 (H3N2)-like, A/New Caledonia/20/99 (H1N1)-like, and B/Sichuan/379/99-like antigens.
To be administered IM

62. Flu vaccine recommendations
1. Persons at Increased Risk for Complications
Vaccination is recommended for the following groups of persons who are at increased risk for complications from influenza:
persons aged >65 years;
residents of nursing homes and other chronic-care facilities that house persons of any age who have chronic medical conditions;
adults and children who have chronic disorders of the pulmonary or cardiovascular systems, including asthma;
adults and children who have required regular medical follow-up or hospitalization during the preceding year because of chronic metabolic diseases (including diabetes mellitus), renal dysfunction, hemoglobinopathies, or immunosuppression (including immunosuppression caused by medications or by human immunodeficiency [HIV] virus);
children and teenagers (aged 6 months--18 years) who are receiving long-term aspirin therapy and, therefore, might be at risk for developing Reye syndrome after influenza infection; and
women who will be in the second or third trimester of pregnancy during the influenza season.
2. Persons Aged Years
Vaccination is recommended for persons aged years because this group has an increased prevalence of persons with high-risk conditions.
3. Persons Who Can Transmit Influenza to Those at High Risk
Persons who are clinically or subclinically infected can transmit influenza virus to persons at high risk for complications from influenza.. The following groups should be vaccinated:
physicians, nurses, and other personnel in both hospital and outpatient-care settings, including emergency response workers;
employees of nursing homes and chronic-care facilities who have contact with patients or residents;
employees of assisted living and other residences for persons in groups at high risk;
persons who provide home care to persons in groups at high risk; and
household members (including children) of persons in groups at high risk.

63. Chemotherapy-humans Prevent membrane fusion Neuraminidase inhibitors
Amantidine (Symmetrel)
Remantidine (Flumadine)
Neuraminidase inhibitors
Zanamivir (Relenza)
Oseltamivir (Tamiflu)

65. Human flu vaccine used
Pasteur Merieux Connaught