Transcript Introduction (1)

Overview of Molecular Epidemiological
Methods for the Subtyping and
Comparison of Viruses
Derek Wong
http://virology-online.com
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.
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.
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.
Principles behind Restriction Enzyme
Analysis and Hybridization Probes
REA
EcoRI (GAATTC)
0
Target
32
32
Hybridization
Probes
100
68
GAATTC
Target
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.
Nature of Restriction Enzymes

4-cutter Enzymes (frequency of cutting = 1/256)
taq 1
Hae III
Sau 96I

6-cutter Enzymes (frequency of cutting = 1/4096)
Eco RI
Hind III

TCGA
GGCC
GGNCC
GAATTC
AAGCTT
8-cutter Enzymes (frequency of cutting = 1/65536)
Not I
GCGGCCGC
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. RocheAmplicor.
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.
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.
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.
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