Virus threats worldwide Albert Osterhaus - 1996-2003, WHO outbreak reports - Bornem 24-03-05
Emerging viruses
“in a changing world”
West Nile Yellow fever Dengue Avian influenza Lassa Meningitis Japanese encephalitis Ebola/Marburg Rift valley Polio Nipah SARS Venez. Eq. encephalitisCrimean-Congo HF VHFMonkeypox Hanta Enterovirus
Emerging virus infections in the last decennia Emerging virus infections in the last decennia - Part 1 - - Part 2 -
Facilitated by changes in: Facilitated by changes in:
Social environment Ecology: - Behaviour (taboos, mores, i.v. drug abuse) - Animal contacts (wildlife, zoo, pets…) - Mobility (air travel, wars …) - Agriculture (deforestation…) - Demography (population density, urbanisation…) - Fisheries (predator migration…) - Socio-ecomic status (poverty, wealth) - Environmental pollution (immune suppression…) - Public health measures (breakdown…) - Global warming (flora and fauna…)
Technology Virus: - Medical (transfusion, transplantation, vaccination) - Mutation (host range, virulence…) - Food production (recycling…) - Recombination/reassortment
Bioterrorism?
Emerging virus infections in the last decennia Lentiviruses - Part 2 - - Host range -
Facilitated by changes in:
Ecology: - Animal contacts (wildlife, zoo, pets…) - Agriculture (deforestation…) - Fisheries (predator migration…) - Environmental pollution (immune suppression…) - Global warming (flora and fauna…)
Virus: - Mutation (host range, virulence…) - Recombination/reassortment
Bioterrorism? Nature is the main bioterrorist!!!
1 Vectors for transmission of virus infections
Aedes spec. Sandfly spec. Ixodus spec.
- Yellow fever virus - Phlebovirus - Tick-borne - Dengue virus encephalitis - Omsk hemorrhagic fever virus
Arbo-virus infections DHF in the Americas - Vector distribution - - 1970-1999 -
60
Distribution of Aedes aegypti Dengue hemorrhagic fever 50 1970 1997 <‘81 ‘81-’97 Aedes Aegypti 40
30
20 (Thousands)
Reported Cases 10
0 1970s 1980s 1990s*
West Nile Virus West Nile Virus - Hosts - - USA 1999-2002 -
2 West Nile Virus West Nile Virus - Symptoms - - Cases -
Fatal: <0.1% of infections 1 CNS disease <1% CNS Year # Cases # States # Counties Onset Date Range = disease ~150 infections 1999 62 1 6 2 AUG – 24 SEP ~20% “West Nile Fever” 2000 21 3 10 20 JUL – 27 SEP
2001 66 10 39 13 JUL – 7 DEC
~80% 2002 4,156* 39 708 19 MAY – 14 DEC Asymptomatic
* 284 deaths
Virus infections Animals and animal products - Host range differences -
? blood ? Storage ? MBM Incinerate Food ? Burial Human (other species) Human
Fat Cow Destructor Cosmetics Food ? Drugs Farm Gelatin ? Calf
Milk Sewage Water
MEAT Abattoir Meatproducts SRM
Influenza pandemics of the 20th century Influenza A viruses
Subtype divisions of influenza A viruses Virus Subtype Name Est. # deaths made on basis of HA (1-16) and NA (1-9) antigens 1918 H1N1 “Spanish Flu” > 40 million Genomic nucleic acid consists of 8 RNA segments, allowing gene reassortment during mixed infection 1957 H2N2 “Asian Flu” > 1 million Virulence of influenza virus depends on presence of presence of series of basic 1968 H3N2 “Hong Kong Flu” > 1 million amino acids adjacent to cleavage site of HA polypeptide
3 Influenza A virus Bird markets - Antigenic drift and antigenic shift - -Asia -
Antigenic drift
Antigenic shift
Influenza A virus Influenza A virus - Man as a “mixing vessel” - - Surveillance in wild birds -
Reassortant virus Human virus
Avian virus Avian virus
De Jong et al., Nature 1997 Claas & Osterhaus, Nature Medicine 1998
Influenza A virus surveillance Influenza A virus surveillance - Results 1999 & 2000 - - HA and NA subtypes -
Order Family # Species # Samples HA Species NA Species Procellariiformes Procellariidae Fulmar 1 101 Anseriformes Anatidae Goose 6 1387 1 Mallard, Teal 1 Mallard, Teal Duck 12 2232 2 Mallard, White-fronted Goose 2 Mallard, Wigeon, Greylag Goose, Swan 2 73 3 Mallard, Teal White-fronted Goose, Guillemot Falconiformes Accipitridae Hawk, Eagle, Buzzard 3 26 4 Mallard 3 Black-headed Gull Gruiformes Rallidae Rail, Coot 4 281 Charadriiformes Charadriidae Plover, Lapwing 5 166 5 Mallard 4 Mallard, Wigeon Scolopacidae Sandpiper, Dunlin, Snipe 23 1977 6 Mallard, Wigeon, Greylag Goose, 5 Mallard Laridae Gull 5 886 White-fronted Goose, Guillemot 6 Mallard, Black-headed Gull Alcidae Auk 2 28 7 Mallard 7 Shoveler Burhinidae Oystercatcher 1 31 8 --- 8 Mallard, Teal, White-fronted Goose Passeriformes Alaudidae Lark 1 61 Prunellidae Accentor 2 67 9 --- 9 --- Muscicapidae Thrush, Warbler, etc. 26 740 10 Mallard Paridae Tit 4 99 11 Mallard, Teal, Shoveler Sturnidae Starling 1 52 12 --- Fringillidae Finch 5 99 13 Black-headed Gull Motacillidae Wagtail, Pipit 3 40 Others Others Others 17 35 14 --- Poultry 406 15 --- 123 ++8787 XX Black-headed Gull
4 Highly pathogenic avian influenza (HPAI) seriously suspected clinically: February 28th 2003
H7N7 in The Netherlands H7N7 in The Netherlands - Phylogeny of the HA gene - - Phylogeny of the NA gene -
NL 2003 NL 2003
Chick/Germany/49 Mallard/NL/2/00 Mallard/NL/12/00
FPV/Weybridge
duck/Manitoba/53
Teal/TW/98 Italy ‘99-’00 Swine/England/97 Equine/Praque/1/56 0.01 0.1 Equine ‘74
H7N7 in The Netherlands H7N7 in The Netherlands - Human cases - - Fatal case chest X-ray upon admission -
10
# cases 78 conjunctivitis 5 conjunctivitis + ILI 2 ILI (1 fatal) 5 4 no symptoms
86 primary cases 3 secondary cases
0 Mar 15 Mar 22 Mar 29 Mar 5 Apr 12 Apr Mar 1 Mar 8 Mar 19 Apr
Date of onset of disease
5 H7N7 in The Netherlands Influenza A virus - Sequence analysis of full-length genomes - - Recent zoonoses -
Virus # substitutions in Gene A/Netherlands/33/03 A/Netherlands/219/03 Subtype Country Year # Cases # Dead Segment Nucleotide Amino acid Nucleotide Amino acid 1 (PB2) 2 0 8 5 (S79I,V297I,R355K,Q563R,E627K) H7N7 UK 1996 1 0 2 (PB1) 0 0 2 0 H5N1 Hong Kong 1997 18 6 3 (PA) 0 0 5 1(F666L) H9N2 SE Asia 1999 >2 0 4 (HA) 0 0 4 3(I13S,A143T,K416R) H5N1 Hong Kong 2003 2? 1? 5 (NP) 0 0 1 0 H7N7 Netherlands 2003 89 1 6 (NA) 0 0 5 4(N308S, A346V, T442A, P458S) H7N2 USA 2003 1 0 7 (MA) 0 0 0 0 H7N3 Canada 2004 2 0 8 (NS) 1 1 (K126R)2 1 1(V137I)2 H5N1 SE Asia 2004 34 23 1. Sequences were compared to those of A/Chicken/Netherlands/1/03 2. Both mutations in NS1 Fouchier et al., PNAS 2004
Influenza A virus (H5N1) pathogenic for cats (Kuiken et al. 2004, Science)
Avian influenza A (H5N1) virus fatal for tigers and leopards: J. Keawcharoen et al,submitted, 2004
Morbilliviruses Morbilliviruses - Seals - - Seals - Another phocid distemper outbreak in Europe
1988: Outbreak of seal disease in Northern Europe Jensen et al., Osterhaus, ADME, Nature 334: 301-302 (1988) Science 297:209, 2002 Osterhaus, ADME, Vedder, EJ, Nature 335: 20 (1988) Osterhaus, ADME et al, Nature 335: 403 (1988) Osterhaus, ADME et al, Nature 337: 21 (1989)
6 Order Mononegavirales, family Paramyxoviridae Newly discovered paramyxovirus - Electron microscopy -
DNA Maximum likelihood, Polymerase ORF Morbillivirus PDVRPV TuV CDV Henipahvirus MV hPIV3 HeV bPIV3 Respirovirus - Pleiomorph Paramyxovirinae NiV SeV hPIV1 - Average size: 100-600 nm Pneumovirinae - Envelope projections: 13-17 nm hRSV NDV Avulavirus Pneumovirus - Paramyxovirus bRSV LPMV MuV Rubulavirus Metapneumovirus APV hPIV2 SV5 SV41 v.d. Hoogen et al., Nature Med. 2001 0.1 hMPV
Human metapneumovirus Severe acute respiratory syndrome (SARS) - Risk groups - - A world-wide threat -
- (Young) children ~ 10 % of children with RTI
- Immunocompromised individuals (fatal cases!)
-Elderly
- Normal individuals > 2-3 % of RTI in community surveillance studies
Osterhaus and Fouchier, The Lancet 2003 v.d. Hoogen et al., JID 2003
Novel coronavirus hMPV in SARS patients identified in SARS patients
Hong Kong researchers announce fingings of Paramyxoviruses in SARS patients. Similar findings communicated from Canada through SARS etiology network
7 SARS coronavirus phylogeny HCoV-NL: a fourth coronavirus of humans (Rota et al., Science 2003) - phylogeny - Fouchier et al., PNAS 2004 Spike Full genome Replicase 1ab Matrix Nucelocapsid TM glycoprotein E TM glycoprotein
SARS and the Koch’s postulates
Cynomolgus monkeys (Macaca fascicularis) Two viruses have been isolated from SARS patients: SCV and hMPV Simultaneous SCV-hMPV Koch’s postulates modified for viruses by Rivers (1937): infection experiments: 1. Isolation from diseased hosts 2. Cultivation in host cells •SCV alone 3. Proof of filterability 4. Production of comparable disease in host or related species •hMPV alone 5. Reisolation of the virus •SCV followed by hMPV 6. Detection of specific immune response
Fouchier et al.,Nature 2003
SARS coronavirus (SCV) in macaques Detection SCV in pulmonary lesions by IHC and TEM (Kuiken et al. 2003, Lancet)
Histological lesions in lungs from cynomolgus macaques infected with SCV
Kuiken et al. The Lancet 2003
8 SARS coronavirus in macaques Exclusion of hMPV as the primary cause of (Fouchier et al.,Nature 2003) SARS by macaque infection experiment
Time after Macaque # 3 Macaque # 4 inoculation Koch’s postulates modified for viruses by Rivers: (days) Throat Nose Faeces IF Throat Nose Faeces IF 1.Isolation from diseased hosts 0 - - N.T. - - - N.T. - 2.Cultivation in host cells 2 + + N.T. N.T. + + N.T. N.T. 3.Proof of filterability 4 + + - N.T. + + + N.T. 4.No production of comparable disease in related species 6 - - - N.T. + + - N.T. 8 - - - N.T. + + - N.T. 5.Reisolation of the virus 10 - - - + - + - + 6.Detection of specific immune response 12 - - - N.T. - - - N.T. 14 - - - N.T. - - - N.T. SCV infected macaques subsequently inoculated with hMPV did not show 16 - - - + - - - + disease exacerbation + : IFA and VN titer >20
April 16, 2003 WHO Geneva Search for natural reservoir of SARS-CoV (Guan Yi et al. 2003 Science) Conference of SARS etiology network
Official declaration of Species N PCR/ Antibody SARS-CoV as etiologic isolation agent of SARS Himalayan palm 6 6 3 civet Chinese ferret 2 0 1 badger Short- and mid-term objectives: Raccoon dog 1 1 1 Clarification of transmission routes and natural history Establishment and evaluation of diagnostic tools Hog badgers (3), beavers (3), domestic cats (3), Chinese hares (4), and Chinese muntjacs (2) all tested negative.
Daily excretion of SARS-CoV (Martina et al. 2003 Nature) Conclusion SARS animal infection experiments
Inoculated cats Inoculated ferrets
SCV and not hMPV fulfills all the Koch’s postulates for the cause of SARS.
SCV is therefore the aetiological agent for the recently identified disease
Non-inoculated cats Non-inoculated ferrets entity SARS.
The SCV-macaque, ferret, cat and rodent models may be used for
pathogenesis studies, as well as testing of vaccination strategies,
and therapeutic interventions.
9 Treatment of SARS-CoV infected macaques with pegylated interferon-α (Haagmans et al. 2004, Nature Medicine) SARS-CoV excretion from pharynx
350
3 groups of cynomolgus macaques 300 Control (n = 4) Prophylactic (n = 4) 250 Post-exposure (n = 4) 200 Treated with 3 mg/kg IFN, at -3, -1, +1, +3 dpi (prophylactic) 150 +1, +3 (post-exposure) SCV eq/ml 100 Same infection protocol 50 Euthanasia at 4 dpi 0 * * * Examination by: Day: 0 2 4 0 2 4 0 2 4 Pathology (gross, histology) Control Prophylactic Post exposure Immunohistochemistry group g ro u p g ro u p RT-PCR Virus isolation Haagmans et al., Nature Med. 2004
SARS-CoV replication, Ag-expression and histopathological lesions in lungs Antiviral effect of purified human SARS immunoglobulins in a Macaques: protection and no indication for AME 10 8
10 7 Rotterdam studies /ml 50 10 6 * * 10 5 SCV TCID 10 4 * * 1400
10 3 1200 Human anti-SARS antibody ELISA titers in b macaques 0 and 1 day after i.p. injection of different 1000 100 amounts of human SARS antiserum. 800 80
60 titer Ab 600
microscopic fields) 40 400 + SCV infected cells infected SCV 20 200
(% SCV (% * 0 0 c 40 1e+7
30 SARS CoV titres in the lungs of 1e+6 macaques treated 5 days earlier with 20 different amounts of human SARS antiserum 1e+5 10 and infected 4 days earlier with SARS CoV Histopathology score * 1e+4 0 SCV TCID50/ml C ontrol Prophylactic Post exposure group g ro u p g ro u p 1e+3
1e+2 Haagmans et al., Nature Med. 2004 control 0.5 ml 5 ml 50 ml
A B ter Meulen et al, Lancet 2004 Prophylactic treatment of ferrets with huMab C3104: Binding of CR 3014 to SARS - CoV SARS-CoV replication, shedding and pathology
Lung AB 10e7 10e6 Pharynx
10e6 10e5
10e5 10e4 /mL 50 10e4 10e3 TCID 10e3 SARS-CoV eq / mL eq SARS-CoV 10e2 10e2
10 p< 0.001 <10
day 4 0 2 4 0 2 4 days . C D 100 C D 240 Lung Pathology CR3014 10 HEK/293 T cells 8 75 * 6
percentage 4 50 2 *
counts 0 p=0.013 25 CONTROL day 4 percentage neutralization percentage
0 ter Meulen et al, Lancet 2004 0 0 1 2 3 4 10 10 10 10 10 .03 .05 .1 .2 .4 .8 1.6 3.2 6.3 12.5 25 50 relative fluorescence intensity μ g/mL
10 Vaccination of macaques with inactivated SARS CoV: Vaccination of macaques with inactivated SARS CoV: Virus neutralizing antibody titers after 2 vaccinations SARS CoV PCR titers in the lungs 5 days after challenge Rotterdam studies Rotterdam studies
256 10 6
128 10 5
4 64 10 10 3 32 10 2 * SCV TCID50 /g TCID50 SCV * D.L. 10 1 D.L. SARS CoV VN titer VN CoV SARS 16 10 0 8 Vaccine - + ++ - + ++ Vaccine - + ++ - + ++ Adjuvant - - - + + + Adjuvant - - - + + + Dose and adjuvant dependent protection! Dose and adjuvant dependent VN antibody induction
Emerging virus infections Emerging virus infections - Conclusions 1 - - Conclusions 2 - Expect the unexpected! Expect the unexpected!
A complex mix of social, technological, Extensive diagnostic and surveillance networks ecological and viral changes (also for wild animals)
As well as novel vaccine-and antiviral development Predisposes for the emergence of virus vaccine antiviral strategies infections Should provide early warnings and safeguards to Originating from animal reservoirs limit their impact
Acknowledgements Department of Virology
Dept. Virology, WHO & Members of the WHO Erasmus MC SARS aetiology team Theo Bestebroer Centers for Disease Control & Prevention, Atlanta, USA Bé Niemeyer Central Public Health Laboratory, London, UK Georgina Aron Public Health Laboratory Centre, Hongkong, SAR China Robert Dias-d’Ullois Emerging Prince of Wales Hospital, SAR China Geert van Amerongen Queen Mary Hospital, SAR China Gerard van Doornum Singapore General Hospital, Singapore Martin Schutten Federal Laboratories for Health Canada, Winnipeg, Canada Bert Niesters Health Canada, Ottawa, Canada virus infections Bart Haagmans Bernhard-Nocht Institute, Hamburg, Germany Byron Martina Institut Pasteur, Paris, France Thijs Kuiken National Institute of Infectious Disease, Tokyo, Japan Ron Fouchier Ab Osterhaus
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