DNA micro Array Technique is still under development which is very useful for identification and detection of highly dangerous viruses and pathogen.

1. DNA Microarray Technique For Detection
And Identification Of Viruses Causing
Encephalitis And Hemorrhagic Fever
Akash Mali , India.

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

7. 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

8. 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.

9. 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.

10. 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.

11. 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 .

12. Conclusion
s
1.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.

13. 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