Avian Influenza (H5N1) & Pandemic Risk

Dr C M ChuMD, MSc (Lond), FRCP (Lond, Edin, Glasg), PDipID

Senior Medical OfficerDepartment of Medicine & GeriatricsUnited Christian Hospital

Influenza virus

Photo: courtesy of Dr W Lim, PHLC.Influenza virus type A, B, C (nucleoprotein)

Lipid envelope

RNA/nucleoprotein

H & N

H and N subtypes of influenza A isolated in animals and human

Host of origin

Humans Pigs Horses Birds*

Haemagglutinin H1/1934/PR H1 H3 H1subtypes H2/1957/Sing H3 H7(H1 - H16) H3/1968/HK

H5/1997/HK H16H7/2003/NLH9/1999/HK

Neuraminidase N1/1934/PR N1 N7 N1subtypes N2/1957/Sing N2 N8(N1 - N9) N3/2004/Can

N7/2003/NL N9

* Highly pathogenic H5 and H7 - outbreaks in poultry.

Pandemic potential

1889 1918 1957 1968 1977 1997 20031999 2005

0.5M 0.07M 0.03M 6/18 0/2 H7N3 0/2

H7N7 1/89

H5N1 60/117H1N1 H2N2 H3N2H1N1 H5N1

1900

H9N2?

H3N8

?H2N?H3N?

Excessdeath (US)

Historical timeline of influenza pandemic and recent avian influenza activity in human

Seroepidemiology ???aviangenesequence

H2, N2,PB1genes fromavian virus

H3, PB1genes fromavian virus

H1N1 ?LeakagefromRussianlab

Poultry to human transmission

Pandemic influenza: overall estimated attack rate 10-20% and mortality rate 1.3%.

Human to human transmission

Chen H, et al. PNAS 2006

H NPGD

Vietnam

Hunan

Yunan

Indonesia

Chen H, et al. PNAS 2006

H5N1 surveillance in birds of southern China (2002–2005)

H5N1 non-H5N1

(H3, 6, 9, 11)

Migratory birds

n=13115 6 (0.34%) 38 (2%)

Market poultry

n=51121 512 (1%) 3051 (6%)

HA sublineages

1. Guangdong, Hong Kong (GD)

2. Vietnam, Thailand, Malaysia (VTM)

3. Hunan, Yunnan, Indonesia (HN, YN, IDN)

To Europe

? migratory birds

? poultry trafficking

H5N1 in poultry and wild birds

?

Migratory birds

Local wild birds

Backyard farms or farms with poor biosecurity and erratic vaccination

Environment: farms, homes, markets, transport, contaminated by virus

Proposed model for control of bird flu

?

90% of H5N1 isolates are from ducks and geese

0

5

10

15

20

25

30

35

40

45

H5N

1 is

olat

es (%

)

Goose Duck chicken Minorpoultry

Yen et al., J Infect Dis 2005

a Virus titres in eggs (log10EID50/mL), MDCK cells (log10PFU/mL), and mice (log10mouse 50% LD/mL).b Number of infectious units (EID50 and PFU) in one MLD50.

Higher viral load Higher infectivity

H5N1 infection

Clinical feature

In 20032 isolated human cases of H5N1 infections (imported)1 person died subsequently

Farm chicken outbreak(Mar/Apr)

Farm / market chicken outbreak(Oct/Nov)

Culling (1.5 M)

Table 1. Impact of avian influenza on human and birds in the HKSAR.

033.835 millions720Total

0112 (imported)2003

0.919 million2002

1.372 million2001

021999

1.544 millions6181997

Number ofmortality

Number of documented human cases

Number of Poultry affected or stamped out

Number ofmortality

Number of documentedhuman cases

Year

H9N2H5N1

H5N1 pneumonia = 13 Death = 7 (53.8%)

Clinical features of human infection by avian influenza A virus(Up to 20/10/2005)

Conj, ILICAP, ILI, diarrh, enceph

Conj, ILI, CAP

CAPILICAP, ILIConjConjInitial synd

0 (0%)67 (51.5%)1 (%)1 (50%)0 (0%)6 (33.3%)0 (0%)0 (0%)No.of cases (fatal)

2130892 (imp)21813No. of cases

H7N3H5N1H7N7H5N1H9N2H5N1H7N7H7N7Agent

AvianAvianAvianAvianAvianAvianAvianSealAnimal source

Canada2004

SE Asia, 2003to now

NL2003

HK2003

HK1999

HK1997

UK1995

USA1980

ILI: acute onset of fever, cough ± sorethroat ± myalgia

CAP: fever + new pulmonary infiltrate + respiratory symptoms (SOB, cough) withno recent hospitalization

Analysis of Clinical Features (1)ILI(7) CAP (11) p value

1. Mean age ± SD (yrs) 2.86 ± 1.35 26.3 ± 18.5 0.002(range) (1 - 5) (3 - 60)

2. M : F ratio 5 : 2 3 : 8 NS

3. Underlying illness 2 4 NS

4. Mean duration of symptoms 3.3 ± 3.3 5.0 ± 3.3 NSto admission ± SD (days)

5. Persistent fever before 3 10 < 0.05admission

6. Mean admission 38.9 ± 0.68 39.2 ± 0.81 NStemperature ± SD (°C)

7. URTI symptoms 5 10 NS8. LRTI signs 0 11 < 0.0019. GI symptoms 2 8 NS

Analysis of Clinical Features (2)

* Virological diagnosis: 16: culture +; 2: 4x rise anti-H5 nt Ab

ILI (7) CAP(11) p value10. Mean initial peripheral 11.2 ± 5.0 3.9 ± 1.9 < 0.015

WBC count ± SD (x 109/L)11. Lymphopenia 0 11 < 0.00112. ALC 2.57±1.71 0.48±0.26 < 0.0513. Raised ALT 1 10 < 0.00414. Impaired renal function 0 4 NS15. Hypoxaemia 0 10 < 0.00116. CXR changes 0 11 < 0.00117. Intensive care 0 10 < 0.00118. Ventilatory support 0 8 < 0.00419 Death 0 6 < 0.05

F/24 (D2 after onset of symptoms) D5 ICU admission

Photos courtesy of Dr Edward Ho

D11 D13

Vietnam (Feb 2004)

The WHO consultancy group

Prof K Y Yuen, HKUDr David S C Hui, CUHKDr Y K Ng, QMHDr C M Chu, UCH

Typical case: M 31 yrs, consumed dead chicken 5 days before illness (onset 3/1/04). Fever 40C, malaise, dry cough, SOB, headache for 2 days. His 2 sisters died of confirmed H5N1 2 weeks later.

5/1/04 6/1/04

6/1/04 Died 9/1/04

WBC 2.5, L=0.6

Plt 57, ALT 109, AST 322

Fortum, Amikacin

Shock

ARDS

Rapid progression to death: M/52 yrs old, poultry farm worker hx of contact with dead chicken. Fever 5 days/ dry cough, runny nose & SOB for 2 days. CPK 15820. Rx: Fortum & Amikacin.

9/2/04 Died 10/2/04

Atypical presentations of AI

Diarrhoea ( EID July, 2004)39-year-old woman presented with fever for 1 week, diarrhea, nausea, and vomiting, with no early respiratory symptoms, she later developed rapidly progressive pneumonia.

Encephalitis ( NEJM Feb, 2005)A four-year-old boy presented with severe diarrhea, followed by seizures, coma, and death. Viruses were isolated from cerebrospinal fluid, fecal, throat, and serum specimens. The patient's nine-year-old sister had died from a similar syndrome two weeks earlier. In both siblings, the clinical diagnosis was acute encephalitis. Neither patient had respiratory symptoms at presentation.

Influenza virus H5N1 nucleoprotein antigen expression in tissues of infected cats 7 days after inoculation

Rimmelzwaan et al., Am J Pathol 2006

Cats fed on virus-infected chicks, ganglioneuritis in submucosal & myentericplexi of small intestine: direct infection from lumen.

All cats excreted virus at respiratory & digestive tract.

H5N1: cytokine dys-regulation

Cheung CY et al. Lancet 2002.

PNAS 2004

Journal of Medical Virology 2001

PathogenesisMulti-organ involvementMarked up-regulation of pro-inflammatory cytokinesReactive haemophagocytosis demonstrated in some cases

Laboratory diagnosisClinical features and radiological features are non-specificEpidemiological link important

Contact with sick/dead birds/poultryInefficient human-to-human transmission

Rapid diagnosis of H5N1 (2)IF IF RT-PCR

Specimens flu A NP H5*

Culture – 0 2 ??? 0†n=145

Culture + H3N2 74 0 0n=81

Culture + H5N1 7 5 (+2) 10n=11* Mab against H5 (R Webster, St. Jude’s Children’s Hospital)† 2 specimens had nonspecific band, negative on hybridization

What specimens and tests should be analyzed?

VIRAL DETECTION on AB DETECTION on acute andTS, NPA, NPS, ETA, convalescent sera (TAT)BAL ± CSF, serum, stool(TAT)

GOLD STANDARD: viral culture 4x increase in microneutralizingon MDCK cells or chick embryo Ab titre (P3) using outbreak(>3 d) strain (3 d)

RAPID RT-PCR for H5/N1 or EIA/WB by baculovirus-expressedM gene of all subtypes (1 d) recombinant H5

Antigen detection for NP by ICmembrane EIA /immunofluorescence ofall subtypes (0.5–4 h)

Potential treatment options

PB2 - cap binding, endonuclease, 759 aa

PB1 - RNA polymerase, 757 aa

(ribavirin, ?viramidine)

PB1-F2 - mitochondrial toxin, 87 aa

(cellular apoptosis)

PA - RNA polymerase subunit / proteolysis, 716 aa

H (HA) - attachment to sialic acid receptor and membrane fusion, 560 aa

(neutralizing antibody, ? Convalescent plasma)

NP - complex with RNA genome, 498 aa

N (NA) - sialidase for viral release, 450 aa

(oseltamivir, zanamivir, ?peramivir)

M1 - structural / nuclear export of RNA, 252 aa

M2 - ion channel, 96 aa

(Amantadine, rimantadine)

NS1 - interferon antagonist, 230 aa

(???Interferon alpha-2b, beta)

NS2 (NEP) - nuclear export factor, 121 aa

Options of antivirals

?Efficay of antivirals in H5N1:No RCT data for H5N1 Rx & Px

Available Human H5N1 Animal data Cell-basedoptions data assay dataOseltamivir Case reports H5N1, mice +(75 mg po bd)Zanamivir – H5N1, mice +(oro-inh 10 mg bd)Ribavirin – B, mice +(0.5 g iv q8h)Adamantanes Case reports A, B; mice +/–(100 mg bd)Interferon alpha – – + (IC50 100X of

CoV & rhinovirus) Convalescent Ab (?) – MnAb +

Are these antivirals active in vitro?Is clinically useful [serum] achievable?

Antiviral IC50/EC50 (μg/L) Serum level afternormal doses (μg/L)

Oseltamivir 3 (S)1 200–40028 (R)1

339 (H274Y clone)1

31.24 (S)2

Zanamivir 0.27 (S)3 39–54 (17–142)299.07 (S)2

Ribavirin 2300–4300 (EC50) 17000 [500 mg iv]

Correlation between clinical efficacy and serum [drug] to IC50 /EC50 unknown.1 IC50 as determined by NA activity assays (Hanoi 2005).2 EC50 as determined by MDCK-SIAT1 and HAI assays (VN1203/2004).3 IC50 as determined by NA activity assays (VN1203/2004).

Amantadine

Anti-influenza treatment,1997 HKSAR experience

N = 18No antiviral in 8Antiviral given in10

Amantadine alone D1 (1)Amantadine alone D3 (1)Amantadine alone D4 (1)Amantadine alone D5 (1)Amantadine alone D6 (2)* died D13Amantadine alone D7 (2)** died D8 and D28Amantadine + iv ribavirin D11(1)* died D29Amantadine alone D12 (1)

OutcomeNo antiviral: 2 died (SLE [died D17] and Reye’s [died D12])Antiviral given: 4 died*

0

20

40

60

80

100

2003 2004 20052001 -2002

1997 -2001

2002 2003 2004 -2005

2003 2004 2005

30/37

54/54

68/82

0/2 0/61

27/1216/45

0/4

4/9

4/355/76

Freq

uenc

y (%

)

Vietnam Hong Kong SAR China

Ser31Asn only

Ser31Asn + Leu26Ile

0/3

1997-2002

Prevalence of amantadine-resistant mutants among H5N1 viruses isolated in Vietnam, Hong Kong, and China.

Cheung et al. J Infect Dis 2006 In press

Oseltamivir (Tamiflu®)

Location Year % Rx Mortality Commentof treated

Vietnam 2004 5/10 4/5 Significant delay of Rx

Thailand 2005 7/12 5/7 Both survivors completed 5 dof Rx. Median day of start =4.5 vs 9.5 (survived vs dead)

Vietnam 2005 10/10 8/10 No details

17/22

? Delay of antivirals in patients with ILI / CAP with Hx of contact with sick / dead birds

Treatment outcome of H5N1 infection by oseltamivir

? Under-dosing oseltamivir for the very young, diarrhoea, or shock

Kiso et al., Lancet 2005 (weight-based dosing)Children dosed 2 mg/kg bd in Japan18% H3N2 developed resistance

Other countries (unit-based dosing)≤15 kg = 30 mg bd>15-23 kg = 45 mg bd>23-40 kg = 60 mg bd>40 kg = 75 mg bd

Ribavirin in influenza B (mice)

Oseltamivir (5 mg/kg/day) and ribavirin (40 mg/kg/day) were used alone and in combination.

Ribavirin alone effective (90–100% survival of mice) when Rx started as late as 3 days after infection.

40% survival even when Rx started 4 days post-infection.

Oseltamivir only effective if started within 24 h.

Ribavirin + oseltamivir no better than ribavirin alone.

Smee DF, et al. Antivir Chem Chemother 2004;15:261-8.

Zanamivir

Can we just dissolve zanamivir in saline, esp for patients on intubation or CPAP/BIPAP?

Zanamivir 16 mg qid (as 16 mg/mL in normal saline) administered by disposable nebulizer at an airflow of 6–7 L/min for 10 min (Ison et al., AntivirTher 2003)

No data on serum/sputum drug concentrations in pneumonic patients or by nebulization???

Zanamivir (mcg/L) IC50 = 0.27 (S)EC50 = 299.07 (S)[serum] = 39–54 (17–142)[sputum] = 47–1336

Possible options and RCTs

Z + R?Z?RR

A + R?A?RS

High dose O

WHOSR

WHO + AWHOSS

StudyControlOseltamivir (O)Amantidine (A)

Copyright J Clin Pharmacol 2000

The dosage of oseltamivir used for Rx ? Neurotoxicity?

Copyright Drug Metab Disp 2002

Clinical pharmacokinetics of oseltamivir with probenicid

Can we enhance [serum] of oseltamivir in patients with no problems of the heart, kidney, fluid overload, and polypharmacyproblems?

The use of Neuraminidase inhibitor

? Pre-exposure Px with std dose oseltamivir (HCW)? Post-exposure Px of household contacts with std dose oseltamivir for incubation period? For Rx, durations = 14 dBut oseltamivir resistance

Le et al, Nature, 2005: 1 survivedde Jong et al, NEJM, 2005: 2 diedChen et al (unpublished): quasi-species and spontaneous oseltamivir-resistant mutant not uncommon

? Zanamivir Px: all HCW caring those on oseltamvirRx (?transmission of oseltamivir resistance)

D 9ETAD 8ETAUCH*F/60 (thymoma)

D 9LungD 8BALQEH*M/54 (old MI)

D 16ETAQEH*F/34 (SLE)

D 13ETAD 8 (D 9)NPATMH*F/25

D 5 (D 8)ETAYCHF/24

D 5ETAD 3NPAYCHF/19

D 6NPAD 3 (D 4)NPAPMHF/14

D 7ETAPWH*F/13

D 9NPAD 5 (D 7)NPAQMHF/5

D 6NPAYCHM/4

D 8NPAD 4ETATMHF/3

D 11NPAD 5TSPMHM/3

D 11ETAQEH*M/3 (index)

D 7NPAD 1 (D 4)ETAQMHM/2

D 2NPAQMHM/2

D 1NPATMHM/1

First -ve cultureSampleFirst (last) +ve cultureSampleHospitalGender/ Age

? Duration of antiviral Rx: viral culture positivity /conversion in clinical specimens

* Death

Serum samples from 16 H5N1 case patients were tested in a microneutralization assay by use of the A/Hong Kong/156/97 virus. Values represent the log2 mean titre of duplicate assays.

J Infect Dis 1999

? Duration of antiviral Rx: onset of Neutralizing antibody

Immunomodulators?

?Immunomodulators(IVIG, anti-TNF, steroid) for severe H5N1 disease:Experience from 1997 HKSAR (6/18).

IndicationsNonresponsive ARDS in 5. Sudden desaturation in 1*.

IVIG 1 dose, 9 g, D10 Died D12.

MP (500 mg x 1 D8) + HC (100 mg q8h D12-15) Died D17.

MP 1 g D11-12 Died D13.

MP 500 mg D22, tailing 53 d. Survived.CT: bilateral ground glass. L consolidation.

HC 200 mg q6h D20, tailing 8 d.* Died D29.

MP 500 mg x 1 D22, tailing 40 d SurvivedCT: diffuse fibrosis, cystic changes, ground glass appearance.

HLH

Rx of EBV-related HLH improves survivalVP-16 induces apoptosis & ⇓ cytokinesDexamethasone pro-apoptotic

N-acetylcysteineAnti-oxidantReduce influenza symptomsReduce influenza mortality in miceAnti-inflammatoryUsed with Zinc, vitamin E, selenium

Prevention

?

Migratory birds

Local wild birds

Environment: farms, homes, markets, transport, contaminated by virus

Proposed model for control of bird flu

?

Improvement of the biosecurity of industrial farms

Moratorium at hottest month for 2 weeks

Segregation &immunization

of ducks & geeseat industrial farms

Human-bird segregation: central slaughtering, personal hygiene

H5N2vaccine

Containing an emerging pandemic?? [Ferguson N. Nature 2005]

Expected pattern of spread of an uncontrolled

epidemicRo = 1.5

Successful containment of emergent pandemic influenza

R0=1.8 + social+5km prophylaxis + 5km quarantine

Containment failure due to single-country policy implementation

Strategies to contain an emerging pandemic [Ferguson N. Nature 2005]

Rapid identification of original case clusterRapid, sensitive case detection and delivery of treatmentEffective delivery of treatment to targeted populationSufficient stockpiles of drugPopulation cooperation with containing strategy (social distancing)International cooperation in policy development, epidemic surveillance and control strategy implementation

Vaccine development

Luke et al., Emerg Infect Dis 2006

Pandemic flu vaccine for all?

Global population: 6481 million

Manufacturing capacity: 300 million doses (trivalent)

900 million doses (monovalent)

2-dose regimen

1/5 dose for intradermal

Available doses for the world = 900 x 5/2 = 2250 million doses of H5 (~1/3 world population)

Adjuvants should improve immunogenicity

Altered HA cleavage site sequence with ↓ basic amino acids to ↓ virulence & ↑viral titre in chick embryo

Subvirion H5N1 vaccine (Haemagglutinin antigen)

90 µg IMI x 2 doses28 days apartHaemagglutination-inhibition titre > 1:40 in 58%Neutralisation antibody titre > 1:40 in 54%Adjuvant (Aluminium)?Intra-dermal injection?

Hae

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Mic

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Anything that can go wrong will go wrong

Murphy’s 3rd law

If there is a possibility of several things going wrong, the one that will cause the most damage will be the one to go wrong.

Murphy’s 4th law

AcknowledgementProf K Y Yuen & Department of Microbiology, HKUDr K S Chan, HOHHProf David Hui, CUHKDr S T Lai, PMHDr W C Yu, PMHDr C W Leung, PMHDr K Y Lai, QEHDr Vivian Wong, HAHO

HA?