1. (-) strand segmented RNA viruses : Orthomyxoviruses (flu)
Orthomyxoviruses: (-) strand segmented RNA viruses
Influenza A, B, C
- Flu, risk of secondary pneumonia
- Genetic drift/shift
- Vaccines
- H5N1 (bird flu)
- Novel H1N1 (swine flu)
- H7N9 infections in China

2. Negative strand viruses: general definition
1. Genomic RNA cannot function as mRNA
2. Genomic RNA serves as a template for two kinds of RNA:
mRNA and complementary, genomic RNA (cRNA)
3. Since cells do not have RNA dependent RNA polymerases,
negative strand viruses package the enzyme in the virion
All negative strand viruses infecting humans:
 Are enveloped
 Enter cells by fusion of envelope with cellular membranes
 Egress the infected cell by budding
 Become infectious on egress
 Are sensitive to lipid solvents, drying, etc.

3. Differentiation of Influenza A, B, and C.
Genetically related: all orthomyxoviruses
Influenza A: infect humans, poultry, pigs, etc.,
wild aquatic birds are usually the natural host
re-assort
most virulent
associated with pandemics
Influenza B: predominantly infects humans (& seals)
less virulent & common than A
Influenza C: humans, dogs & pigs
least common
Vaccine: A – H1N1
A – H3N2 B

4. Differentiation of Influenza A, B, and C
Genetically related: all orthomyxoviruses
Influenza A: infect humans, poultry, pigs, etc.,
wild aquatic birds are usually the natural host
re-assort
most virulent
associated with pandemics
Influenza B: predominantly infects humans (and seals)
less virulent and less common than A
No reassortment so far
Seasonal epidemics
Co-circulates with A virus
Influenza C: humans, dogs and pigs
least common
Seasonal Tetravalent Vaccine:
A – H1N1
A – H3N2
B – Yamagata-lineage
B – Victoria-lineage

5. Orthomyxoviruses Virion structure: Segmented (-) strand RNA genome
Enveloped with two protein spikes on its surface:
Hemagglutinin (H) fuses envelope to cell membranes
Neuraminidase (N) cleaves terminal sialic acid

6. Influenza virus life cycle
Fusion and
uncoating
Packaging
Low pH
Nuclear replication (unusual for RNA viruses) - cap stealing, mRNA splicing

7. The Influenza Hemagglutinin
Binds cellular receptor, sialic acid.
After acidification in endosomes, has a conformational change promoting fusion of viral and cellular membranes.

8. The Influenza Neuraminidase
-cleaves N-acetyl neuraminic acid/ NANA
Two functions:
1. Removing NANA from mucoproteins (mucin) enhances viral evasion of antiviral mucosa
2. Removing NANA from viral and cellular glycoproteins facilitiates virion release

9. Entry of Influenza virus into cells
1. Attachment to sialic acid
Low pH results in a conformational change and exposure of fusogenic peptide
pH 7.4
2. Uptake into endosomes
3. Fusion with lysosomes pH
4. Release of RNP
Cytoplasm
Nucleus

10. 10 Proteins Orthomyxoviruses: flow of events during replication mRNAs
Virion-carried
polymerase
Virion negative
strand RNA
mRNAs
10 Proteins
Complementary
Genomic RNA
Progeny negative
strand RNA
Virus

11. The replicative cycle of influenza viruses
pH 5-6
endosome
coated vesicle
coated pit M1
mRNA
Np, Ns, Pol
Synthesis and
glycosylation of
H, N
Viral RNA
synthesis
Golgi
RNP
binding
Budding
Insertion of
membrane
proteins
Entry
Replication
Nuclear replication (unusual for RNA viruses) - cap stealing

12. Assembly of Influenza virions
Packaging of distinct RNPs at the membrane can result in reassortment when two types of virus are present

13. Influenza Pathogenesis
S. aureus
pneumococcus
Group A streptococci
H. influenzae

15. pandemic influenza

16. Some definitions:
Endemic: infection/disease maintained in a population without external inputs
- infections rates can be high, but are relatively constant
Epidemic: new cases of infection within a population
- can be localized or global
Pandemic: world-wide epidemic

17. Why do new strains of influenza appear every year?
Three reasons:
Humans are infected by avian or swine influenza
Genetic reassortants arise
High mutation rate

18. Influenza A distribution

19. Chronology of Influenza Viruses in Humans
pH1N1
H3N2 (Hong Kong flu)
H2N2 (Asian flu)
H1N1 (Spanish flu)
H1N1 (Russian flu)
H2N?
1890
1918
1940
1957
1968
1977
2009
Isolation of flu virus
1976 Swine H1N1 outbreak at Fort Dix-New Jersey
- Vaccination of 40 million people in the U.S

20. Antigenic shift and pandemics
1. Genetic Reassortment: Antigenic shift
Acquisition of a novel HA segment by reassortment between human and animal viruses (Segmented genome)
No immunity in humans against new HA
Pandemics are caused by antigenic shift
Genetic Reassortment
Parent
Viruses
Antigenic shift and pandemics
Reassortants

21. 2. Minor antigenic drift Prototype Duration Major Derivative Genotype
Epidemics Strains
A/SW/ A/Weiss H0N1
A/PR8/
1946
A/Cam/ A/Scan/ H1N1
A/FM1/ A/Liverpool/51
1955
A/Sing/ A/Eng/ H2N2
A/Alch/ A/Eng/64
A/Eng/68
(1890)
A/Hong Kong/ current A/Hong Kong H3N2
(1900) A/England
A/Port Chalmers
A/Victoria
A/Bangkok
Swine Flu (1976) Hsw/Nsw
A/USSR/ current H1N1
A/Hong Kong (1998) H5N1

22. The significance of minor antigenic drift.
Viruses are sequential isolations of A/Hong Kong-68.
Table shows serum neutralization titers obtained in ferrets.
Virus Hong Eng./ Port Victoria
Kong Chalmers
Hong Kong , ,
Eng ,
Port Chalmers-C
Victoria < < ,280

23. Treatment Neuraminidase activity is essential for virion release
Oseltamivir (Tamiflu) and Zanamivir inhibits NA activity
Effective only if taken within 3 days of infection

24. Vaccines are quadrivalent:
Prevention
Yolk sac
Allantoic
fluid
Majority of the vaccines are made
in embryonated eggs
Vaccines are quadrivalent:
Influenza A H1N1
Influenza A H3N2
Influenza B Yamagata
Influenza B Victoria
Strains for making vaccines are selected in the Spring by WHO Committee; vaccines are made during Summer for administration in the Fall
Beware of egg allergies

25. Vaccines Currently licensed Effectiveness Cost*
Killed vaccine Poor $10
Live vaccine High $45
attenuated
(Flumist)
- Recommended for high risk individuals, adults >65, healthcare workers, etc.
- FDA approved for uses in all age groups
- Poorly immunogenic;
requires a good
adjuvant
- FDA approved for use in
Healthy 2 to 49 years old
- Does not require
an adjuvant
*2003 prices, currently higher

26. Selection of annual vaccine by genetic reassortment
Attenuated
parent
Virulent new
virus X 
6:2 Selection
Progeny

27. Revisiting the 1918 Spanish Flu
million dead in
Unusual: lethal in young adults

28. Resurrecting the 1918 virus
Jurassic park???
Virus did not reassort in pigs - it jumped directly from birds

29. National Preparedness
$7.1 billion flu pandemic plan
$251 million to help foreign partners train medical personnel, develop monitoring capabilities to detect outbreaks and draw up preparedness plans.
$1.2 billion to purchase enough vaccine developed by the National Institutes of Health against current bird flu strain to vaccinate 20 million people.
$1 billion to stockpile additional antiviral medications to provide enough to treat first responders and other key personnel.
$2.8 billion for a "crash program" to accelerate cell culture technology, which Bush said would produce enough vaccine for all Americans within six months.
Relief from the "burden of litigation" for vaccine manufacturers.
$583 million for pandemic preparedness, including $100 million to help states complete and exercise their pandemic plans.

30. 2009 H1N1 (Swine Flu) Why called swine flu?
Quandruple reassortment: genes from influenza viruses infecting Asian/European pigs, avian, and humans further reassorted in pig
Now thought to have emerged in Asia and traveled to N. America in infected human

31. 2009 H1N1 timeline
Mid March- 60% of La Gloria, Veracruz Mexico get respiratory illness
Mar. 17- confirmed novel H1N1
Mar. 28- New H1N1 in U.S.
April 1- H1N1 isolated from boy is later fully sequenced
April 22, U.S. declares public health emergency
June 11, Level 6 pandemic (reflects virus spread, not disease severity), >70 countries affected
Aug Pandemic declared over: ~15% of population infected,
~ 0.02% mortality
Replaced the older H1N1 virus
Now the 2009 virus circulates as a seasonal virus

33. Transmission

34. Age groups infected
Elderly have pre-existing immunity?

35. Old H1N1 viruses protect against 2009 H1N1 virus
1940
1977
1918
1957
1968
2009
H1N1 (Spanish flu)
H1N1 (Russian flu)
Protection
No Protection
Complete Protection
1977 – 2007 (Partial Protection)
Manicassamy et al, PLoS Pathogens, 2010

36. Treatment & Prevention
rest, supportive care
call doctor if trouble breathing, chest pain, dizziness, persistent nausea, dizziness
Medication: oseltamivir (most flu now resistant), zanamivir
Vaccine
Hygiene: wash hands, cover nose & mouth when sneeze, don’t touch mouth, eyes, or nose
stay home until 24 hours after fever subsides.

37. Entry of new viruses into human population

38. Definition of Pandemic
Interepidemic period
Current
Pandemic alert period
Pandemic

39. The Onion on Bird Flu Preparations
Tamiflu: stockpiled by countries & individuals for fear of pandemic flu
- questionable efficacy and limited production.
October 26, November 1, 2005 | Issue 41•43

40. Avian Influenza viruses
H5N1:
Detected in humans during a domestic poultry outbreak (HK 1997)
Human cases from direct contact; >350 deaths (mortality rate >60%)
Transmission studies: 4-5 mutations sufficient for mammals
HPAIV:
Highly pathogenic avian influenza viruses (H5N1 and H7N7)
Multibasic cleavage site: cleaved by ubiquitous furin-like proteases
Systemic dissemination of HPAIV due to ubiquitous cleavage of HA
Seasonal viruses (H1N1, H3N2) restricted to upper respiratory tract - Trypsin dependent cleavage of HA
LPAIV:
H9N2: few human cases
Trypsin-like cleavage site: proteases restricted to the respiratory tract

41. Will H5N1 become pandemic?
Seroprevalence suggests bird-human H5N1transmission for decades without significant human-to-human spread
Human-to-human transmission is low
H5N1 only infects cells of lower respiratory tract in humans due to receptor specificity; transmission much better from upper respiratory tract
Can acquire mammalian adaptive mutations or reassort with circulating human strain; recent studies indicate that 4-5 mutations are sufficient for transmission in ferrets

42. Human infection with Influenza A (H7N9) virus in China
Reassortant of three avian
viruses
Reservoir not known
Virus has been isolated from
poultry and pigeons
Limited human-to-human
transmission
Acquiring mammalian
adaptive mutations
Sensitive to Tamiflu