THE SARS VIRUS GENOME The Quick and the Dead?

SARS Severe Acute Respiratory Syndrome First identified in Guangdong Province, China Mortality 3-6% (45-63% in persons over-60) Associated Coronavirus SARS-HCoV

Coronavirues Enveloped Replicates in cytoplasm of animal cells Single-strand 30 kb RNA genome With 5’ cap & poly-A tail Respiratory, enteric, hepatic, neurological Cause 30% respiratory infections Can acquire genes by horizontal transfer and co-infection Three main classes

Application of Genomics to Pandemic Competing Interests: CDC & BCCA Draft Sequence: 12 April 2003 Complete Sequence: 1 May 2003

Look! I stay up all night studying genomics, too!

Genomics Strategy for SARS-HCoV Virus isolated from bronchioalveolar sample from fatal case from Toronto (Tor2 isolate) Virus particles purified & RNA extracted Converted to cDNA by oligo-dT priming RACE used to obtain far 5’end of genome Large cDNAs cloned & bidirectionally sequenced Sequence Alignment and Consensus Analyses

Surprising & Not-So-Surprising Features Sequence nearly identical to CDC isolate No evidence for mutability Major genes conserved among coronaviruses replicases 1a and 1b S protein the small envelope (E) protein s2m motif common to avian bronchitic and equine rhinitic viruses Suggests horizontal transfer 9 unique ORFs some overlap, some nested do not readily align with other coronaviruses suggests new coronavirus class Membrane (M) glycoprotein Nucleocapsid (N) protein

The Power of Analyzing Sequences for Consensus ORF 3 (Fig. 2; base pairs 25,268 to 26,092) encodes a predicted protein of 274 amino acids that lacks significant BLAST (24), FASTA (25), or PFAM (26) similarities to any known protein. Analysis of the N-terminal 70 amino acids with SignalP provides weak evidence for the existence of a signal peptide and a cleavage site (probability 0.540). Both TMpred (27) and TMHMM predict the existence of three transmembrane regions spanning approximately residues 34 to 56, 77 to 99, and 103 to 125. The most likely model from these analyses is that the C terminus and a large 149–amino acid N-terminal domain would be located inside the viral or cellular membrane. The C-terminal (interior) region of the protein may encode a protein domain with ATP-binding properties (ProDom ID PD037277).Fig

Conclusions and Summary from SARS-HcoV Genome Project SARS defines unique class of coronavirus. No evidence of intervirus recombination between known coronaviruses Besides s2m motif, no evidence of intervirus recombination from non-coronaviruses. Suggests recent mutation of existing animal (nonhuman) virus caused ability to infect humans. Demonstrates how fast technology and expertise has grown and can be applied CDC & BCCA have filed for patents

SARS History: SARS History: Guangdong to Genome in Six Months Virus to Genome in 38 Days November 2002 November 2002: An outbreak of a mysterious respiratory illness occurs in Guangdong Province, China, making hundreds seriously ill and killing dozens. Mid-February 2003 Mid-February 2003: Virus spreads to Vietnam and Hong Kong; international travel blamed. Mid-March Mid-March: Virus spreads to Singapore and Canada. March 15 March 15: A Singaporean doctor travels through New York on his way to Germany, becoming ill en route; he is diagnosed with SARS in Frankfurt. March 17 March 17: World Health Organization facilitates the collaboration of 11 laboratories in 10 countries to identify the cause of SARS. March 24 March 24: Centers for Disease Control and Prevention announces that a coronavirus strain causes SARS. March 29 March 29: Dr. Carlo Urbani, a WHO officer who treated the earliest cases in Hanoi, dies of SARS. May 1 May 1: CDC and the British Columbia Cancer Agency in Canada publish near-identical sequences of the SARS virus in Science.

What to do after getting sequence?

Knowledge of Sequence will… allow precision diagnosis of virus presence by specific DNA amplification of virus regions help consolidate association of virus with disease facilitate development of antiviral treatments (including neutralizing antibodies) and vaccines help understand epidemiology of rapid viral mutation and host changing

The Next Genome Project?

Antennalope

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