Cox

Evolution, Adaptation and Spread of Influenza Viruses: What Have We Learned?: 

Evolution, Adaptation and Spread of Influenza Viruses: What Have We Learned? Nancy J. Cox, Ph.D. Chief, Influenza Branch National Center for Infectious Diseases Centers for Disease Control and Prevention

Influenza Historical Background: 

Influenza Historical Background Epidemics of influenza have occurred in humans since ancient times – recorded by Hippocrates in 412 BC “Influenza” – term dates from 15th century Italy when epidemics were attributed to the influence of the stars First pandemic clearly described in 1580

Background, continued: 

Background, continued Influenza A virus isolated in ferrets in 1933 by Smith et al. Virus first grown in embryonated eggs in 1936 Antigenic differences detected between viruses in 1937 Influenza B virus isolated in 1940 by Francis and McGill (independently) Inactivated influenza vaccine found to be effective in 1944 (U.S. military)

Influenza Viruses: 

Influenza Viruses Enveloped RNA viruses, 80-100 nm in diameter 8 single-stranded negative sense gene segments encoding 10 proteins Aerosol and droplet transmission Incubation period 1-4 days Viral shedding Adults <5 days Young children up to 10-14 days Two virus types: A and B, based on NP and M1 Three A subtypes: H1N1, H1N2 & H3N2, based on spike-like surface glyproteins

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of Human Influenza A Viruses Periods of Observed Circulation/Detection 1918 1977 1999 H2 H1 H5 1968 1957 H1 H3 H9 1997

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Influenza

Antigenic Change: 

Antigenic Change Antigenic ‘drift” occurs in HA and NA Associated with seasonal epidemics Continual development of new strains secondary to genetic mutations A viruses >> B viruses Antigenic “shift” occurs in HA and NA Associated with pandemics Appearance of novel influenza A viruses bearing new HA or HA & NA

Infection and Disease: 

Infection and Disease Highest infection & disease rates in children Highest rates of serious complications in elderly and persons with certain underlying chronic conditions, e.g.: Cardiopulmonary disease Metabolic disease (e.g. diabetes) Immune deficiency

Influenza Viruses Infect Several Animal Species: 

Influenza Viruses Infect Several Animal Species All influenza A subtypes found in wild birds Fecal transmission among wild birds Usually no illness Other animal species Domestic poultry (chickens and quail) Humans, swine, horses, seals, whales Humans usually infected by human influenza viruses

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Human virus Reassortant virus Non-human virus Post-1997 View of Transmission of Avian Influenza A Viruses to Humans 15 HAs 9 NAs

Pandemics and Pandemic “Scares”of the 20th Century: 

Pandemics and Pandemic “Scares”of the 20th Century 1918-19 “Spanish flu” H1N1 1957 “Asian flu” H2N2 1968 “Hong Kong flu” H3N2 1976 “swine flu” episode H1N1 1977 “Russian flu” H1N1 1997 “Bird flu” in HK H5N1 1999 “Bird flu” in HK H9N2

1918-19 Pandemic: 

1918-19 Pandemic Scattered outbreaks in U.S. in spring Relatively low activity in summer Peak of first wave in October Peak of second wave in mid-winter U.S. ~550,000 deaths nearly 1/2 in persons 20-40 years old Worldwide ~ 40 million deaths

1957-58 and 1968-69 Pandemics: 

1957-58 and 1968-69 Pandemics 1957 Emergence of influenza A (H2N2) virus 66,000 U.S. deaths 36% of deaths (24,000) in persons <65 years 1968 Emergence of influenza A (H3N2) virus 28,100 U.S. deaths 41% of deaths (11,600) in persons <65 years

The Next Pandemic : 

The Next Pandemic Another pandemic is expected. According to our CDC model, the projected health impact in the U.S. is: 89,000 – 207,000 …....deaths 314,000 – 734,000 ..…hospitalizations 18 million – 42 million…clinic visits

Grand Challenge of Modeling: Spread of an Influenza Pandemic: 

Grand Challenge of Modeling: Spread of an Influenza Pandemic Susceptibility of the population Person-to-person spread Mixing of global populations Traffic patterns, especially global travel Would we have time to produce vaccine and to ramp up production of antiviral drugs?

Average Annual Impact of Influenza on U.S.: 

Average Annual Impact of Influenza on U.S. >20,000 deaths >110,000 hospitalizations Currently >90% deaths in persons >65 years Higher numbers of deaths and hospitalizations in A(H3N2) years

P&I Mortality for 122 U.S. Cities& Associated Influenza Viruses: 

P&I Mortality for 122 U.S. Cities & Associated Influenza Viruses 91-92 92-93 93-94 94-95 95-96 96-97 97-98 98-99 99-00 00-01 01-02 A(H3N2) A(H1N1) B

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0 0.2 0.4 0.6 0.8 1 R e l a t i v e I n f l u e n z a A c t i v i t y JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC

WHO Recommendations for Vaccine Strains: 

WHO Recommendations for Vaccine Strains Formal recommendations for strains to be included in the currently licensed inactivated influenza vaccine are made each year during February for the following influenza season in the Northern Hemisphere and in September (since 1998) for the following season in the Southern Hemisphere.

Considerations for Recommendations: 

Considerations for Recommendations Are there new antigenic variants (HI and gene sequencing data)? Are new variants spreading and causing disease? Are current vaccines able to induce antibodies to the new variants? Are any new variants useful for vaccine production?

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Why Sequence HA Genes? Sequence analysis provides information on the molecular basis for antigenic drift. Sequence analysis provides precise data on evolutionary relationships between HA genes of Influenza viruses. Sequence data provide adjunct to HI data for vaccine strain selection. Rates of change at nucleotide/amino acid levels Types of amino acid changes Positions of amino acid changes Predictability in patterns of change Molecular correlates with epidemiology

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What Viruses are Sequenced? Variant viruses (HI test) Typical viruses (HI test) Geographic distribution (emphasis on viruses from Asia) Temporal distribution S. Hemisphere N. Hemisphere

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Panama/2007/99 Moscow/10/99 Sydney/5/97 Johannesburg/33/94 Beijing/32/92 Beijing/353/89 Sichuan/11/87 Mississippi/1/85 Philippines/2/82 Bangkok/1/79 Nanchang/933/95 H3 HA

Conclusions fromPrediction Attempts: 

Conclusions from Prediction Attempts We identified 18 codons in HA1 that are positively selected; sites A, B and RBP Strains with more mutations in these codons = progenitors of successful new lineages – retrospective testing Alternate hypotheses less successful in predicting Monitoring change in + selected codons may predict course of evolution and be useful for vaccine strain selection

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Antigenic Drift in H3N2 Viruses Demonstrated in an HI Assay A/Johannesburg/33/94 A/Nanchang/933/95 Viruses Joh/33/94 Nan/933/95 Post-infection ferret antisera raised against 640 40 A/Sydney/5/97 <10 Syd/5/97 <10 20 640 40 1280 40

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Signature Amino Acid Changes Observed Between the HAs of A/Johannesburg/33/94 and A/Nanchang/933/95-like Viruses Joh/33/94 Nan/933/95 G135T, N145K, N262S

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135 135 135 145 145 145 262 A B C D E Model of the Influenza A H3 HA Structure Top view Side view

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Approach Generate transfectant viruses with the Joh/HA containing single mutations alone G135T N145K N262S and combinations of double mutations G135T/ N145K G135T/ N262S N145K/ N262S Determine antigenicity of each mutant virus using post-infection ferret sera in an HI assay

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vRNA expressing plasmids PA PB1 PB2 NP M NS NP PB2 PB1 PA PR8 protein expression plasmids Plasmid-based Reverse Genetics PR8 wt or mutant Joh/94 Joh/94 HA 293T cell

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Summary of Reverse Genetics Experiments The presence of each mutation alone was insufficient to recreate the antigenic difference observed between Johannesburg/33/94 and Nanchang/933/95-like viruses. But, the combination of mutations G135T and N145K in site A of the Joh 94 HA were sufficient to re-create this example of antigenic drift in the H3 HA. Reverse genetics techniques allowed us to identify contribution of specific aa residues to antigenic drift in the H3 HA. Look at other examples and at contributions of 18 codons.

The Flu Brew: Is it Right?: 

The Flu Brew: Is it Right?

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Organization of WHO's Influenza Program National Influenza Centers (Approx. 110 Labs in 80 Countries) World Health Organization (Geneva) Vaccine Producers International Collaborating Centers (Atlanta, London, Melbourne, Tokyo) Isolate influenza viruses Identify viruses and send to International Collaborating Center(s) Collect epi information Collect information for the Weekly Epidemiological Record and WWW for distribution Make annual vaccine recommendations Analyze influenza viruses Provide data for vaccine recommendations Distribute vaccine viruses

Current Status of WHO System: 

Current Status of WHO System 175,000 isolates/yr (600 to 1200 M cases) WHO CCs receive 6,500 samples/yr. WHO CCs and NICs sequence 1,000 samples/yr. >240 M doses of influenza vaccine produced worldwide

Enhancing Surveillance for Pandemic and Epidemic Influenza: 

Enhancing Surveillance for Pandemic and Epidemic Influenza Need higher throughput labs Need better data collection Lab + Epi linked New algorithms for identifying key viruses Increase data based size for better predictive methods

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Match Between Epidemic and Vaccine Strains of Influenza, USA, 1992-2001 ++++ identical or minimal difference +++ substantial cross-reaction Season Epidemic Strain(s) Vaccine Strain(s) Antigenic Match A/Shangdong/09/93 (H3N2) A/Shangdong/09/93 (H3N2) 1995/96 1996/97 1997/98 A/Texas/36/91 (H1N1) A/Texas/36/91 (H1N1) A/Johannesburg/33/94 (H3N2) A/Nanchang/933/95 (H3N2) A/Sydney/5/97 (H3N2) A/Nanchang/933/95 (H3N2) A/Nanchang/933/95 (H3N2) A/Johannesburg/33/94 (H3N2) ++ moderate cross-reaction + some cross-reaction 1998/99 A/Sydney/5/97 (H3N2) A/Sydney/5/97 (H3N2) 1999/00 A/Sydney/5/97 (H3N2) A/Sydney/5/97 (H3N2) 2000/01 A/New Caledonia/20/99 (H1N1) B/Sichuan/379/99 A/New Caledonia/20/99 (H1N1) B/Yamamashi/166/98 ++ 2001/02 A/Panama/20/07 (H3N2) A/Panama/2007/99 (H3N2)

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Factors Contributing to Improved Influenza Vaccine Match Better Worldwide Surveillance Europe, Latin America and Asia Improved Communications Telephone and Fax E-mail Technical Advances Molecular Analyses Increasing International Cooperation Exchange of Viruses and Serum Panels Standardization of Reagents and Vaccines

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Success of WHO's Influenza Surveillance Network Action-orientated network Reagents updated annually Annual recommendations Collaborative spirit of participants

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of Influenza B Virus Periods of Observed Circulation in Humans 2001 1940 B B/Victoria/2/87-like 1987 B/Yamagata/16/88-like

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Genetic Reassortment in Influenza Viruses

Hemagglutination Inhibition Reactions of Influenza B Viruses of the Yamagata and Victoria Lineages: 

Hemagglutination Inhibition Reactions of Influenza B Viruses of the Yamagata and Victoria Lineages

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Lee/40 Shandong/7/97 Hong Kong/330/2001 Panama/45/90 Yamanashi/166/98 Sichuan/379/99 Yamagata/16/88 Singapore/222/79 USSR/100/83 Ann Arbor/1/86 Victoria/2/87 Harbin/7/94 Type B HA

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India/7526/2001 Maryland/1/2002 Oman/16296/2001 New York/1/2002 * Taiwan/1484/2001 Guangzhou/7/1997 Osaka/547/1997 Beijing/243/1997 Shandong/7/1997 Nanchang/1/2000 Sichuan/34/2001 Bangkok/34/1999 Bangkok/54/1999 Canada/464/2001 CNIC/27/2001 Hong Kong/335/2001 Yunnan/123/2001 Hong Kong/330/2001 Hawaii/38/2001 Hawaii/10/2001 (egg-grown) Chongqing/3/2000 Hawaii/35/2001 Hawaii/10/2001 (tissue culture) Hawaii/26/2001 Hong Kong/329/2001 Hawaii/9/2001 Hong Kong/112/2001 Hawaii/37/2001 Hawaii/36/2001 Hong Kong/123/2001 Philippines/93079/2001 Hong Kong/336/2001 Hong Kong/6/2001 Hong Kong/22/2001 Taiwan/217/1997 Guangdong/5/1994 Victoria/2/1987 Brazil/952/2001 Alaska/16/2000 Texas/1/2000 Sichuan/379/1999 Yamanashi/166/1998 Beijing/184/1993 Harbin/7/1994 Hong Kong/557/2000 Wuhan/356/2000 Sichuan/317/2001 Argentina/69/2001 Wuhan/2/2001 Yamagata/16/1988 Hong Kong/70/1996 HK330 Clade Oman16296 Clade H 116 R I 121 N D 164 E I 121 T

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Multiple Genotypes of Influenza B Viruses Identified in 2001/2002 Yamagata/16/88 Victoria/2/87

Hemagglutination Inhibition Antibody Responses to the B Component of the 2001-2002 Influenza VaccinesADULT POPULATION: 

Hemagglutination Inhibition Antibody Responses to the B Component of the 2001-2002 Influenza Vaccines ADULT POPULATION

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Canada Switzerland Israel Oman India Thailand Hong Kong China Japan Philippines Hawaii Geographic Distribution of B/Victoria/02/87 Lineage Viruses April 2001 to Present USA Italy Netherlands Australia

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Conclusions Influenza B/Victoria/2/87 lineage viruses have reemerged in North America and Europe after a 10 year absence. Reassortment has occurred between 2 lineages of B viruses. Genetic reassortants bearing a B/Shandong/7/94-like HA and B/Sichuan/379/99-like NA were identified – vaccine choices. A B/Victoria lineage virus has been recommended for vaccines. Children under 10 expected to be most susceptible.

Questions About B Viruses: 

Questions About B Viruses Why did B/Vic lineage viruses survive only in China after B/Yamagata lineage viruses spread in the late 1980s? Why are B/Vic lineage viruses spreading to North America,Europe and Australia now after a 10 year absence? Will both lineages of influenza B viruses continue to circulate?

Other Key Questions: 

Other Key Questions Can we explain the seasonality of influenza? Why do so many epidemic variants appear first in China? Is there real spread or only apparent spread of epidemic variants? Is it possible to predict the next antigenic variant?

Acknowledgements: 

Acknowledgements WHO National Influenza Centers WHO Collaborating Centers in London, Melbourne and Tokyo Robin Bush and Walter Fitch Members of the Influenza Branch (A.Klimov, H. Hall, C. Smith, K. Subbarao, S. Tong, M. Shaw and X. Xu)