Molecular Epidemiological Methods for Virus Subtyping

1. Overview of Molecular Epidemiological Methods for the Subtyping and Comparison of Viruses An Overview

2. Uses of molecular epidemiological methods • Subtyping - in some viruses, different subtypes are associated with different clinical manifestations e.g. enteroviruses, adenoviruses, and human papillomaviruses. • General Epidemiology - by identifying the viral subtypes at different times and geographical locations, one can detect major changes in the epidemiological patterns of infection e.g. HIV and HCV. • Investigation of Outbreaks - to support or disprove a link between the donor and recipient viruses e.g. HIV, HBV, HCV, Norwalk virus.

3. Methods Used - Complete or Partial genome? • For greatest degree of accuracy, the complete genome should be used for the purpose of comparison. • However, since viral genomes ranges from 3500 bp to over 200,000 bp, it would be highly impractical to sequence the whole genome. • Certain simple methods are still used for the comparison of complete genomes e.g. RFLP for CMV, HSV, and Adenoviruses. • Nowadays in practice, a small part of the genome is amplified first by PCR and the product investigated by sequencing or other methods.

4. Strategies for identification of the PCR Product (Commonly used methods) • Sequencing of the PCR product • the gold standard but expensive and not widely available. • PCR product may be sequenced directly or cloned before sequencing. • However, it is the test of choice in outbreak situations where there are serious public health and/or medical-legal implications. • Sequencing can be used to confirm results of other molecular epidemiological assays. As a matter of fact, all other assays can be considered as simpler screening assays. • Restriction Fragment Length Polymorphism (RFLP) - very simple, rapid and economical technique but the result may be difficult to read. • Hybridization with a specific oligonucleotide probe - A wide variety of formats is available e.g. dot-blot, Southern blot, reverse hybridization, DNA enzyme immunoassay etc.

5. Principles behind Restriction Enzyme Analysis and Hybridization Probes REA EcoRI (GAATTC) Target 0 32 100 32 68 Hybridization Probes GAATTC Target

6. PCR-RFLP (PRA) • The gene target must be present in all viral strains. • It is amplified with primers directed against conserved areas in the target gene so that all subtypes can be amplified. • The PCR product is then digested with one or more restriction enzymes and on an agarose or polyacrylamide gel. • The species or genotype is identified from the restriction patterns seen. • Therefore PRA can be considered as probably the simplest DNA fingerprinting technique. • The principle of PRA is similar to that of RFLP of whole viral genomes and pulse field gel electrophoresis. • It is quick, simple and cheap and this is why it is preferred by many molecular biologists. • Examples include HCV genotyping and identification of mycobacteria.

7. Nature of Restriction Enzymes • 4-cutter Enzymes (frequency of cutting = 1/256) taq 1 TCGA Hae III GGCC Sau 96I GGNCC • 6-cutter Enzymes (frequency of cutting = 1/4096) Eco RI GAATTC Hind III AAGCTT • 8-cutter Enzymes (frequency of cutting = 1/65536) Not I GCGGCCGC

8. Specific Oligonucleotide Probe • Simple to carry out, particularly suitable for large scale testing • Results are usually easier to read than REA and requires less skill to interpret • Preferred strategy by commercial companies e.g. INNO-LIPA HCV, Sorin DEIA, Roche Amplicor and Taqman. • Can be made into a highly automated closed system e.g. Roche-Amplicor. • Therefore more attractive than PRA for the routine laboratory but the costs could be prohibitive. • Specific nucleic acid probe assays are available where the specimen is tested directly without amplification. However the sensitivity is much lower.

9. Choice of Genomic Region • The choice of genomic region to use for analysis is critical and could affect the outcome of results. • Too conserved – will not be able to demonstrate any differences between subtypes. • Too variable – may not be able to demonstrate a link between source and recipient viruses in outbreak studies because of the high mutation rate. • In general, RFLP is not suitable to highly conserved regions while nucleic acid probes are not suitable for highly variable regions. • It is often advisable to use more than one gene region, especially where there are serious medical-legal implications.

10. DNA Sequencing • DNA Sequencing is a gold standard of molecular virological investigation. • Automated Sequencing facilities are now used in many routine diagnostic laboratories. • The PCR product is directly sequenced without cloning. • A Blast search of the sequence is then carried out in GENBANK. This is normally good enough for identification. • For epidemiological investigations, a phylogentic analysis should be carried out.

11. GENBANK • GENBANK is the most important bioinformatics resource – may be accessed through Entrez or Blast searches. • However, there are a number of problems. • Sequences not refereed • Many sequences are very old and obtained by manual sequencing techniques that were not very accurate. • Many sequences are from strains of microorganisms that had not been well characterized. • A lot of the sequences deposited in Genbank are contaminated with plasmid vector sequences or PCR primer sequences. • Therefore there is a need for other well validated databases.

12. Phylogenetic Analysis • DNA sequences of outbreak strains are compared to those of the suspected source strains and reference strains. • A phylogenetic tree is drawn up with bootstrap resampling analysis. • Where the outbreak strains and source strains are similar, they should be close together on the tree with a high bootstrap value. • The actual gene that is used and the length that is sequenced is critical. • Too conserved – will not be able to demonstrate any differences between subtypes. • Too variable – may not be able to demonstrate a link between source and recipient strains in outbreak studies because of the high mutation rate. • Most useful for RNA viruses such as HIV, HCV and Norwalk where there is a lot of variation, but less useful for DNA viruses, bacteria, fungi and protozoa.

13. Investigation of Norovirus Outbreak Patients 1, 2, 3.

14. Summary • A wide variety of molecular epidemiological methods are available, of which DNA sequencing is the gold standard. • It is now usual to analyze a small part of the genome rather than the complete genome. The target fragment is first amplfied by PCR before analysis. • The most widely used screening methods involve either restriction enzyme analysis or hybridization with specific nucleic acid probes, or a combination of the two. • Other screening methods such as SSCP, dHPLC and other heteroduplex analysis techniques are rarely used outside a research setting because they often suffer from poor inter-laboratory reproducibility. • The choice of the genomic region to use is critical: it is often advisable to use more than one genomic region. • It is important to remember that all molecular epidemiological methods available for viruses can be applied to bacteria but not vice-versa.

15. Points to Consider • Molecular epidemiology techniques are can be used to good effect to disprove a link between donor and recipient strains of a particular infectious agent but they cannot prove a definitive link. • Therefore a negative result is much greater predictive value than the positive result. • The probability of a link depends on many factors including the prevalence of that particular genotype and the methods used. • Where the outbreak carries huge medical-legal implications e.g. HIV transmitted through blood factors, the case would have to be argued on an individual basis in court, preferably with the help of a statistician. • It is important to remember that molecular epidemiological investigation does not replace a good basic epidemiological investigation