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Letter
Discrimination between Highly
Pathogenic and Low Pathogenic H5 Avian Influenza A Viruses
Sunchai Payungporn,* Salin Chutinimitkul,* Arunee Chaisingh,† Sudarat
Damrongwantanapokin,† Bandit Nuansrichay,† Wasana Pinyochon,† Alongkorn
Amonsin,* Ruben O. Donis,‡ Apiradee Theamboonlers,* and Yong Poovorawan*
*Chulalongkorn University, Bangkok, Thailand; †National Institute of Animal
Health, Bangkok, Thailand; and ‡Centers for Disease Control and Prevention,
Atlanta, Georgia, USA
Suggested
citation for this article
To the Editor: To thoroughly investigate avian influenza outbreaks,
identifying highly pathogenic avian influenza (HPAI) and low pathogenic
avian influenza (LPAI) is essential. Currently, determination of inserted
basic amino acids within the hemagglutinin cleavage site of HPAI relies
on nucleotide sequencing (1–3). Direct sequencing
is relatively time-consuming and laborious and thus is not suitable for
local and regional diagnostic laboratories that receive large numbers
of samples that may contain HPAI or LPAI subtype H5N1. Therefore, a rapid
diagnostic assay was developed to discriminate between HPAI and LPAI subtype
H5 viruses by 1-step real-time reverse transcription–polymerase chain
reaction (RT-PCR) with melting curve analysis.
H5 primers flanking the cleavage site were designed from conserved regions
among HPAI and LPAI strains by using nucleotide sequences obtained from
GenBank and our previous studies (4–6). The primers
consisted of a forward primer H5F3+ (nucleotides 1001–1021: 5´-AACAGATTAGTCCTTGCGACTG-3´)
and a reverse primer H5R2+ (nucleotides 1124–1103: 5´-CATCTACCATTCCCTGCCATCC-3´),
which yielded products of ≈124 bp and ≈112 bp, corresponding
to HPAI and LPAI, respectively. Consequently, the sizes of the amplicons
and percentage guanine-cytosine content were different, allowing discrimination
between HPAI and LPAI by melting curve analysis (7).
One-step, real-time RT-PCR with melting curve analysis was performed
in an ABI 7500 system (Applied Biosystems, Foster City, CA, USA). In each
reaction, 3.0 μL of RNA sample was combined with a reaction mixture
containing 10 μL 2× SYBR Green PCR Master Mix (Applied Biosystems),
0.5 μL 40× MultiScribe (Applied Biosystems) and RNase inhibitor,
each primer (at final concentration of 0.5 μmol/L), 1.5 mmol/L MgCl2,
and RNase-free water in a final volume of 20 μL. The thermal profile
began with incubation at 48°C for 45 min (reverse transcription),
then incubation at 95°C for 10 min (predenaturation), followed by
40 cycles of amplification alternating between 94°C for 15 s (denaturation)
and 68°C for 40 s (annealing/extension). The SYBR Green I fluorescent
signal was obtained once per cycle at the end of the extension step. After
amplification, melting curve analysis was performed by heating the sample
to 95°C for 15 s, then cooling it to 70°C for 1 min, followed
by a linear temperature increase to 95°C at a rate of 0.5°C/s,
while continuously monitoring the fluorescent signal. Data were analyzed
by the 7500 System SDS Software version 1.2 (Applied Biosystems).
To develop the assay, samples previously identified as influenza A/chicken/Nakorn-Patom/Thailand/CU-K2/04
(H5N1) served as a control for HPAI, and A/duck/Hong Kong/308/78 (H5N3)
served as a control for LPAI. The H5 genes (nucleotides 914–1728) of each
strain were inserted into pGEM-T Easy Vector and then transcribed in vitro
by using RiboMAX Large Scale RNA Production System-T7 (Promega, Madison,
WI, USA). Serial 10-fold dilutions of the standard H5 RNA were subjected
to a sensitivity test (8). The fluorescent signal
can be detected at RNA dilutions as low as 102 copies/μL.
To assess the specificity, viral RNA extracted from other subtypes of
influenza A viruses (H1–H4 and H6–H15) was tested. The assay was specific
for the H5 subtype, since no amplification was detected from other subtypes.
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enlarged image
Figure. Discrimination between highly pathogenic
avian influenza (HPAI) and low pathogenic avian influenza (LPAI)
by melting curve analysis...
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Three preliminary melting curve analyses showed that this assay was effective
in discriminating between the melting peaks of HPAI and LPAI (Figure).
The variations of melting temperature (Tm) between runs were
experimentally determined. The mean (standard deviation) of Tm
values for HPAI and LPAI were 77.43°C (0.21°C) and 79.57°C
(0.23°C), respectively. This assay provided high reliability and reproducibility,
since the coefficients of variation were <0.30.
Seventy-eight specimens of influenza A virus were used to validate the
assay. The 75 HPAI samples were isolated during the 2004 outbreak in Thailand;
3 LPAI samples, including A/avian/NY/01 (H5N2), A/Chicken/Mexico/31381-3/94
(H5N2), and A/shoveler/Egypt/03 (H5N2), were provided by the Centers for
Disease Control and Prevention. The viruses were isolated in embryonated
chicken eggs as described previously (9). RNA
was extracted from 140 μL of allantoic fluid, and RT-PCR was performed
as described above. Melting curve analysis showed that all H5N1 isolates
from the 2004 outbreak in Thailand were interpreted as HPAI, whereas the
3 samples of H5N2 subtype were classified as LPAI. The Tm varied
from 77.2°C to 78.1°C for HPAI and 78.75°C to 79.5°C for
LPAI. The melting curve analysis results were completely concordant with
the results of direct sequence analysis of the H5 gene for all samples
tested.
The melting curve analysis described here provides a rapid, accurate,
and high-throughput approach to discriminate between HPAI and LPAI. This
assay is particularly attractive for large-scale screening of suspected
subtype H5 influenza A virus during outbreaks to identify candidate LPAI
that could be used as vaccine strains.
Acknowledgments
We thank K.F. Shortridge,
S. Yingst, E. Dubovi, and A. Klimov for providing specimens.
This research was
supported by Thailand Research Fund.
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Suggested citation
for this article:
Payungporn S, Chutinimitkul
S, Chaisingh A, Damrongwantanapokin S, Nuansrichay B, Pinyochon W, et
al. Discrimination between highly pathogenic and low pathogenic H5 avian
influenza A viruses. Emerg Infect Dis [serial on the Internet]. 2006 Apr
[date cited]. Available from http://www.cdc.gov/ncidod/EID/vol12no04/05-1427.htm
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