DNA Microarray Technique For Detection

And Identification Of Viruses Causing

Encephalitis And Hemorrhagic Fever

Akash Mali , India.

1. A major goal of This work is to develop microarray-based methods for

detection and identification of viral nucleic acid from these viruses.

2. The main advantage the technique provides is an ability to screen a

sample for nucleic acid from several different viruses in one test.

Principle of Applying Microarray Technology for Virus Detection and

Identification

Depending on the design of the method, the DNA

can be labeled with fluorescent dyes directly during

amplifiCation or in an additional step .The labeled

nucleic acid is purified and hybridized to the

microarray.

On the microarray slides, virus-specific

DNA probes are attached. The

hybridization is performed in a

hybridization station that allows both

mixing of the sample during incubation as

well as a controlled stringency in terms of

temperature, incubation times and amount

of wash buffers used.

Finally, the slide is scanned in a laser

scanner and hybridization signals are

quantified from the produced image for

subsequent numerical analysis.

Viruses and the Importance of Rapid Diagnostics

The group of interesting viruses includes, from the Bunyaviridae family, hanta-

viruses such as Hantaan and Sin Nombre, Crimean-Congo hemorrhagic fever

virus, a nairovirus, and Rift Valley fever virus, a phlebovirus.

Due to the severity of disease and need for both supportive care and

patient isolation it is of utmost importance to make a rapid detection and

identification of the disease-causing pathogen. Nucleic acid-based

methods are suitable since often no antibodies have developed in early

disease.

Advantages and Drawbacks of Using the Microarray Technique

The microarray technique presents some potential advantages compared to the PCR-

based protocols.

Drawbacks with the technique are, primarily, the still rather undeveloped

and complicated format, and the non-quantitative result. As for most assays,

further verification by other methods is needed, but a microarray test can

efficiently function as a screening tool to assist in selecting a specific PCR.

Mainly, the high number of possible probes on the microarray provides a better

multiplexing capacity by allowing investigation of more DNA fragments. This

means that both more viruses and several parts of the genomes can be targeted in

one test. A broader test is valuable, saving time and effort, as well as sample, in

cases of an unclear clinical picture or for a broader screening of a set of samples.

Key Factors for Development of a Microarray-Based Test

Short or long probe strands on

microarrays modulate specificity and

ability to detect new strains. A new

strain of virus B might not be detected

based on mismatches with short

probes.

By applying random nucleic acid

amplification ,both a wide range of

viruses as well as new and

diverged strains could be amplified.

The hybridization to the microarray

extracts the viral sequence from the

randomly amplified mixture of

nucleic acid. Still, the drawback is

the amplification of parts of the

viral genome not targeted by the

microarray probes and non-viral

nucleic acid that consumes

reagents and makes the

amplification less efficient.. The

other factor that will in fluence the

ability of the method to detect and

discover new strains or to make a

specific identification of a certain

strain is the probe length .

To achieve a sufficient lower

limit of detection, signal

amplification involving more

amplification , and longer

incubations times for labeling

and hybridization are needed.

Hantavirus Microarray

A microarray was constructed containing overlapping 500 nucleotide PCR fragments covering

the S and M genome segments of a group of hantaviruses. Viral RNA was amplified from cell

culture and wild rodents using hantavirus universal primer sets before subsequent fluorescent

labeling and hybridization. There sults showed a distinction of Puumala virus strains up to 90%

similarinnucleicacidsequenceidentityinparallelwithanabilitytodetect new strains, differing up to

30%, by cross-hybridization.

Hantavirus microarray constructed with500-nucleotide fragment probes .

Closely related strains of Puumala virus could be identified and distinguished

The flavivirus microarray assay included seven mosquito-borne flaviviruses: West

Nile virus, Japanese encephalitis virus, Yellow fever virus and Dengue 1–4 viruses.

These are predominantly endemic in tropical and subtropical regions, causing

hundreds of millions of cases of disease every year, mainly Dengue virus infections.

Design of the flavivirus microarray assay . The amplicons generated from a sample

by the highly degenerated multiplex amplification are shown in yellow and the probe

fragments attached on the microarray slide surface are shown in red.

Strategy for amplification from sample

for the flavivirus microarray assay. The

tag on the 50 part of the primers is

shown in red.

A multiplex RT-PCR was designed,

targeting the same five positions of

all seven viruses for amplification

of viral RNA from a sample.Five

primer pairs were designed with a

highly degenerated 30 part targeting

the same position in all seven

viruses and with an artificial 50-tag

similar for all 10 primers

The method was demonstrated on

cell cultured virus and on clinical

samples from Dengue virus

infections. A lower limit of

detection of about 10 viral

genome copies was determined

on Dengue 3 virus and overall

the performance of the method

was comparable to the different

routinely used RT-PCR methods.

The method demonstrated its practical usefulness when a

sample take nearly from a patient with hemorrhagic fever

symptoms was tested. Based on the origin of the patient

from south-west India, RT-PCR stargeting Crimean-

Congo hemorrhagic fever virus and Dengue viruses were

selected and proved to be negative.

The flavivirus method is

currently under development

to improve the capacity to

target new strains, to get a

better distinction between

different West Nile virus

strains, and to simplify and

obtain a more rapidtest

Among the known West Nile virus

strains, there is both a significant

sequence variation, presently suggested

for division in five lineages

Generalized West Nile virus phylogeny with

representative strains. Lineages 3–5 have

been recently suggested. Lineage 3 includes

Rabensburg virus (RabV) recently

discovered in central Europe.

A microarray assay for a group of hemorrhagic fever viruses is under development. The

group includes some hantaviruses and flavi viruses, but also Marburg virus and Ebola

viruses, Crimean-Congo hemorrhagic fever virus, Lassa virus and Rift Valley fever virus. In

the first-generation microarray, 500 nucleotide probes were synthesized from the

glycoproteins. These viruses are not closely related, and a random amplification protocol

was applied and tested successfully for cell-cultured virus .

Conclusion

s1.The microarray technique offers some advantages compared to other nucleic-based

virus identification methods, both in terms of multiplexing capacity and ability to

find new strains.

2.The key factors for designing a microarray-based method are the amplification and

the probe length. These will decide how many different viruses the method should

target and the ability to distinguish virus strains as well as to detect new strains.

3. For setting up these methods, ‘standard’microarray equipment was used, including

hybridization machines for mixing during hybridization and controlled stringency.

4. The development is focused on gradual simplification and shortening down of the

protocols.

5. The hantavirus project demonstrated the usefulness of long 500mer probes on the

microarray for distinction of different viruses and detection of new strains.

6. The flavivirus method was tested and evaluated on Dengue clinical samples, and

performed with a lower limit of detection compared with the routinely used RT-

PCRs.

References

1. Tanja Kostic, Patrick Butaye, Jacques Schrenzel ,Detection of

Highly Dangerous Pathogens: Microarray Methods for the

Detection of BSL 3 and BSL 4 Agents Page No. 113-123