Virus Evolution Molecular Epidemiology of Viral Infections Jen-Ren Wang, Ph. D. 王貞仁 Dept. of Medical Laboratory Science and Biotechnology National Cheng Kung University

Virus evolution Virus evolution ： constant change of a viral population in the face of selection pressures Virus populations display diversity. The sources of diversity ： mutation (genetic drift), recombination, natural selection (adaptation, fitness)

Replicating viruses produce large numbers of mutant genomes RNA virus ： Error rate- 1 in 10 4 or 10 5 DNA virus ： lower error rate – 1 in 10 7 or error-correcting DNA polymerase - latent infection

RNA viruses and quasispecies Viruses exist as dynamic distributions of non identical but related replicons. Polymorphism

Viruses exchange information Recombination - polymerase changes templates (copy choice) during replication (RNA virus) - nucleic acid segments are broken and rejoined (DNA virus) Reassortment

Molecular studies are useful in Epidemiological investigation Real-time surveillance Make predictions about future developments

Molecular epidemiology of viral infections Distinguish between related strains of viruses Deduce the relationships between viruses from different outbreaks or individual patients Dissemination and evolution of viruses can be followed locally and globally

Molecular epidemiology of viruses Determine the sources of imported viruses Monitor pathways of virus transmission Monitor the process of control activities Develop molecular reagents for rapid detection of viral infections

Implications of sequence information Maintain effective diagnostics, treatment, and prophylaxis –Strain-specific treatment: HIV, HCV, HBV, CMV –HCV ： indicator of susceptibility to specific treatments eg. Genotype 1 is resistant to interferon therapy –Detection of mutation that confer antiviral resistance –HIV ： monitor emergence of drug resistance Distinguish between more or less pathogenic strains –Avian influenza ： HAPI or LAPI –HCV ： differ in the in tendency to cause liver damage –Attenuated vaccine strains

Methods for molecular epidemiological analysis Oligonucleotide fingerprinting: Rnase T1 Recognize relationship between isolates separate from ancestral infection by no more than 3 to 5 years Monoclonal antibody characterization of viral epitopes Nucleic acid analysis Nucleic acid hybridization PCR-RFLP PCR-SSCP PCR-sequencing

Oligonucleotide fingerprint analysis Restriction enzyme- No Probe- No Basis for distinctions- Rnase T1 cleavage sites Level of resolution- subtypes, quasispecies Advantages- simple: directly applicable to RNA viruses. Can detect point mutations Disadvantages- complex electrophoresis procedure

Nucleotide sequencing Restriction enzyme- No Probe- No Basis for distinctions- nucleotide sequence Level of resolution- single genome (if cloned) Advantages- wide applicability, can identify single nucleotide mutation Disadvantages- Technically complex; produces large amounts of data; automated sequencing requires expensive equipment

Antigenic drift - Epidemics - increase in incidence of pneumonia and lower respiratory disease - excess rates of hospitalization or mortality Antigenic shift -Widespread and severe epidemics - Pandemic Influenza viruses

Viruses recommended in the influenza vaccines, 1968-

Each year a new flu vaccine is produced, and judging which strains to target is a tricky business. A new study evaluating viral evolution suggests a more systemic approach to predicting next year’s virus. Plotkin et al. PNAS 99: , 2002

They found that among the sequences within each of these large clusters, those sequences isolated in China or Hong Kong are found preferentially in the first half of the cluster’s lifetime. These results support the hypothesis that dominant viral swarms tend to originate in Asia and thereafter spread across the globe. Joshua B. Plotkin et.al., PNAS 99 ： , 2002

Predicting evolutionary change in the influenza A virus Neil M. Ferguson and Roy M. Anderson Nature Medicine 8: , 2002

Ferguson and Anderson. Nature Medicine 8:562, 2002 Unlike HIV, which is constantly growing in diversity, influenza change constantly but with limited diversity at any point in time- giving an unusual “conifer” tree shape (R. Bush).

Fitch et al. PNAS 94: 7712, 1997 The thick line running from the lower left to the upper right (open square) is called the trunk and represents the successful H3N2 lineage.

Bush et al. Science 286: 1921, 1999 The average life- span of a nontrunk lineage is 1.5 years, although one recent nontrunk lineage persisted for 5 years (*).

Bush et al. Science 286: 1921, 1999 Positive selection is defined as a significant excess of nucleotide substitutions that result in amino acid replacements.

Evolution isn’t just something that happened in the past; evolution can be observed in the present, and in some cases, used to predict the future. School boards and science educators need to understand this simple fact: If student don’t learn about evolution, they can’t possibly understand modern biology or medicine. David M. Hillis. Predictive Evolution. Science 286: 1866, 1999.