SARS: the Virus and the Status of Virological Diagnostics Olav Hungnes Dr.scient. Department of Airborne Infections, Division of Infectious Disease Control Norwegian Inst.of Public Health

The hunt for the SARS agent •Initial findings: common flu viruses in Chinese samples Nov-02; Chlamydia spp China Feb-03; flu B Hanoi March; paramyxoviridae Germany/Hong Kong in March; hMPV (a paramyxovirus) Canada March •then: a Coronavirus? –CoV-like particles seen i suspect SARS, (Sweden),CDC, HK Univ: electron microscopy; culture; DNA array, "consensus" CoV-PCR " 'You may seek it with thimbles--and seek it with care; You may hunt it with forks and hope; You may threaten its life with a railway-share; You may charm it with smiles and soap--' " from "The Hunting of the Snark" (Lewis Carroll 1872 )

A novel coronavirus Aligned coronavirus orf1ab amino acid sequences: First available sequences were about equally distant from all known coronaviruses! SARS MHV (1) TGEV (2) IBV (3)

What is known about SARS CoV? •An until now entirely unknown virus: •Prototype virus defining an entirely new group within the coronaviridae virus family GROUP 4 ?

SARS CoV - A completely novel coronavirus

Coronavirus

SARS CoV - genetic organisation Marra et al., Science 1 May 2003 •All "standard" coronavirus genes (replicase polyprotein; S, E, M, N proteins) plus a number of open reading frames of unknown function •Per. 11 June: Sequence information on >15 isolates, some information on genetic variation

Predicted membrane proteins of SARS CoV

Genetic variation All characterised viruses are >99% similar at RNA level mid-May: 15 long sequences: differences in 127 positions (out of 29727); 16 of them occur twice or more Are there differences in virulence, transmissibility, etc? 2 main clades: "Hotel Metropol" and "mainland China", can be discriminated by five positions + TW1 Recent zoonotic event?

M M M? M M Genetic variation •All characterised viruses are >99% similar at RNA level •Are there differences in virulence, transmissibility, etc? 2 main clades: "Hotel Metropol" and "mainland China", can be discriminated by five positions: 9404; ; ; ; A

SARS and the novel coronavirus: - the evidence •Virus found in high proportion of probable SARS patients (PCR, culture; EM) •Antibody against SARS virus i high proportion of probable SARS patients • " not found in healthy controls (U.S., HK) •Serum from reconvalescents neutralise virus in culture •Serologocal conversion / titre rise from acute to convalescent sera •Primates inoculated with cultured virus developed SARS-like symptoms; virus could be reisolated •HOWEVER: The possible role of other bacteria/viruses as co-factors modulating e.g. severity of symptoms has not been ruled out

Hvor kommer viruset fra? •Zoonose mest sannsynlig •Hittil ukjent virus og ukjent sykdom •Mange av de tidlige tilfellene hadde kontakt med eksotiske dyr •Funn av virus hos eksotiske dyr på viltmarkeder i sør-Kina: Masked palm civet; Racoon dog; slanger, ville svin •Virus hos dyr nesten - men ikke helt - identisk

Predicted membrane proteins of ”wild” SARS CoV C 122 Did the virus need to get rid of this gene to work well in humans?? C 39 Orf10 The virus found in animals (and in GZ01) had a functional orf10 membrane protein gene

Recent zoonotic event? 29 bases lost, disabling gene for a membrane protein

HCoV229E=blue SARS CoV=magenta TGEV=green inhibitor bound to TGEV=red Anand et al.:Coronavirus Main Proteinase (3CLpro) Structure: Basis for Design of Anti-SARS Drugs Science : Molecular modeling suggests that available experimental rhinovirus 3C pro inhibitors may be modified to make them useful for SARS therapy. One such compound tested in anti SARS- CoV screening : moderately active in vitro A SARS Designer Drug? The virus enzyme protease 3C is essential to make active virus replication enzymes

Virus persistence and shedding Clinical and epidemiological significance of prolonged virus shedding is unknown Sputum: 10 4 (day3) (day 9) Throat swab (day9) <800 BAL (day11) 4 x 10 5 (Drosten et al. NEJM) data from: Peiris et al; Prospective study of the clinical progression and viral load of SARS-associated coronavirus pneumonia in a community outbreak Lancet

Stability / inactivation •stable in feces/urine, room temp; 1-2 days, increased stability at elev. pH (diarrhea) and on certain surfaces •Stable at 4°C for weeks; room temp: 1 log loss per 48h; inactivated rapidly at 56 °C •inactivated in: acetone, formaldehyde (10%), chlorox, 75% ethanol, phenol (2%) (source: WHO 5&16 May)

Sars coronavirus: Diagnostikk •Å påvise infeksjon med SARS CoV •Å påvise infeksjon med andre agens som forklarer sykdom - sjekke ut de mange som ikke har SARS MEN: Dobbeltinfeksjoner er beskrevet! •Metoder: Påvise virus Påvise antistoff –Dyrke virus- Immunfluorescens –RT-PCR - ELISA –Se partikler i EM- Virusnøytralisasjon

Testenes sterke og svake sider •Viruspåvisning: virus er lett å dyrke; PCR er sensitiv for påvisning av virus, •MEN: Prøver tatt tidlig i sykdommen inneholder lite virus; netto sensitivitet blir derfor dårlig •Antistoffpåvisning: Til slutt utvikler de aller fleste antistoff som kan påvises, •MEN det tar lang tid (opptil 4 uker)

SARS-diagnostikk i Norge •30 mars: Første PCR-protokoll offentliggjort (Bernhard Nocht-Institut, Hamburg) –Primere, reagenser og positiv kontroll-RNA bestilt og prøvd ut på FHI - klar ca 10 d senere –Flere sykehuslab har nå operativ RT-PCR diagnostikk for SARS-CoV - 1 lab gjør dyrking på SARS-prøver •FHI utpekt av HD som nasjonalt referanselaboratorium 6 mai •Etter vurdering kan prøver videresendes til internasjonale lab for tester vi (ennå) ikke har i Norge •Antistoff-testreagenser (immunfluorescens) nå kommersielt tilgjengelig

Konklusjoner •Store framskritt på kort tid - dette vil fortsette •Kjennskap til viruset åpner for: –Identifisering av smitte –Utvikling av behandling –Utvikling av vaksine –Forstå sykdom •Sars-virus i naturen? : –Er det "ville" viruset klart til å lage nye utbrudd? –Et det like smittsomt? Like farlig? –Hvor fins det og hvor vanlig er det? •Betydning av mikrobiologisk diagnostikk vil øke Det er viruset vi må hindre i å bli endemisk •Test-resultat og kasusdefinisjon –hvordan forholde seg til viruspositive som ikke oppfyller andre krav i overvåkningsdefinisjon?