JCM Figure table search 04
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Other Versions of this Article:
JCM.00382-07v1
45/5/1637    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Auewarakul, P.
Right arrow Articles by Puthavathana, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Auewarakul, P.
Right arrow Articles by Puthavathana, P.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, May 2007, p. 1637-1639, Vol. 45, No. 5
0095-1137/07/$08.00+0     doi:10.1128/JCM.00382-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Surveillance for Reassortant Virus by Multiplex Reverse Transcription-PCR Specific for Eight Genomic Segments of Avian Influenza A H5N1 Viruses{dagger} ,{triangledown}

Prasert Auewarakul,1 Kantima Sangsiriwut,1 Kridsda Chaichoune,2 Arunee Thitithanyanont,3 Witthawat Wiriyarat,2 Taweesak Songserm,4 Rasameepen Ponak-nguen,2 Jarunee Prasertsopon,1 Phisanu Pooruk,1 Pathom Sawanpanyalert,5 Parntep Ratanakorn,2 and Pilaipan Puthavathana1*

Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Bangkok,1 Faculty of Veterinary Science, Nakhon Pathom,2 Faculty of Science, Bangkok,3 Mahidol University, Faculty of Veterinary Medicine, Kasetsart University, Nakhon Pathom,4 Department of Medical Sciences, Ministry of Public Health, Nonthaburi, Thailand5

Received 19 February 2007/ Returned for modification 23 February 2007/ Accepted 14 March 2007


   ABSTRACT
Top
 ABSTRACT
TEXT
 REFERENCES
 
Avian influenza H5N1 virus is a global threat. An emergence of a reassortant virus with a pandemic potential is a major concern. Here we describe a multiplex reverse transcription-PCR assay that is specific for the eight genomic segments of the currently circulating H5N1 viruses to facilitate surveillance for a virus resulting from reassortment between human influenza virus and the H5N1 virus.


   TEXT
Top
 ABSTRACT
 TEXT
REFERENCES
 
The genome of influenza A viruses is composed of eight gene segments, which encode polymerase (PB1, PB2, and PA), nucleocapsid (NP), hemagglutinin (HA), neuraminidase (NA), matrix (M) protein, and nonstructural (NS) protein. Since influenza viruses have segmented genomes, they are able to evolve through reassortment (1). Both the 1957 and 1968 pandemic strains are believed to be reassortants in which HA and PB1 genes, with or without the NA gene, were replaced by the corresponding genomic segments of avian influenza virus strains (5). The reassortments provided the viruses with new HA and NA antigenic profiles and the ability to spread efficiently from human to human. The ongoing outbreak of the highly virulent avian influenza virus H5N1 is concerning because of the possibility of a reassortant virus emerging with pandemic potential (3). Monitoring for a reassortant with mixed genomic segments from human and avian viruses is therefore very important. However, detection of a reassortant requires sequencing of all eight genomic segments, a process which is laborious and time consuming. Therefore, we developed a simple and high-throughput screening method using multiplex reverse transcription-PCR (RT-PCR) specific for eight genomic segments of recent H5N1 viruses to detect the probable reassortant viruses worthy of further characterization.

For the primer design, 100 full-length sequences arbitrarily selected from the Influenza Virus Resource Database (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/genomes/FLU/) were used to search for the sequence(s) that was conserved in H5N1 and that differed from those of other subtypes. The viruses used in this study comprised 28 human influenza virus isolates and 103 H5N1 isolates from various animal species (94 isolates from poultry and other birds, 6 isolates from tigers, 2 isolates from leopards, and 1 isolate from a cat). These viruses have been collected since the beginning of the epidemic in the country. In 2003, 2004, 2005, and 2006, 7, 34, 60, and 2 animal H5N1 virus isolates were collected, respectively. The human viruses comprised 6 H5N1, 3 H1N1, 8 H3N2, and 11 influenza B viruses. These viruses were propagated in MDCK cells or embryonated eggs. The H5N1 viruses were originally subtyped by RT-PCR. The subtyping was confirmed by sequencing for all human H5 and some animal H5 viruses (2). Available accession numbers of some of the H5N1 virus isolates used in this study are shown in Table S1 in the supplemental material and also in a report by Puthavathana et al. (4). The human H1, H3, and influenza B viruses were characterized and confirmed with respect to their subtypes by hemagglutination inhibition assay at the WHO Reference Laboratories, Melbourne, Australia, or the Centers for Disease Control and Prevention of the United States via the National Reference Laboratory on Influenza, NIH, Thailand.

RNA was extracted by using a QIAamp viral RNA Mini kit (QIAGEN, Valencia, CA). A 140-µl volume of virus suspension from the infected culture or embryonated egg yielded a volume of 60 µl of extracted RNA. Multiplex RT-PCR was carried out in three reaction tubes with combinations of the following primer sets: NA plus PB1 plus NP, HA plus PA plus PB2, or NS plus M. Sequences of these primers are shown in Table 1. The thermocycling was performed in a GeneAmp PCR system 2400 thermal cycler (Perkin Elmer) using a QIAGEN OneStep RT-PCR kit (QIAGEN, Valencia, CA) with standard reaction conditions (a 50 µl volume containing 1x PCR buffer, 5 to 10 µl of extracted RNA, 400 µM (each) deoxynucleoside triphosphate, 2.5 mM MgCl2, 2 µl enzyme mix, and 2 U RNase inhibitor). The thermocycling profile was 50°C for 30 min, 95°C for 15 min, 35 cycles of 95°C for 45 s, 55°C for 45 s, and 72°C for 45 s, and a final extension of 72°C for 10 min. The PCR products were visualized by ethidium bromide staining after electrophoresis in a 2% agarose gel.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Sequences, concentrations used in the reaction, nucleotide positions, and product sizes of PCR primers for the eight gene segments

 
In our multiplex RT-PCR assay, potential reassortants would show the lack of one or more of the eight amplicons in the case of H5N1 viruses and the presence of one or more amplicons in the case of H1N1 and H3N2 viruses. With the designed primer set, we could initially amplify all eight genomic segments from 105 out of 109 H5N1 viruses. Four viruses initially gave negative amplification results for NA. However, these four viruses yielded positive NA band results in separated single-gene amplifications, and multiplex reamplification using 1.5- to 2-fold-higher RNA input gave positive amplification results for all genes. Representative results obtained with the multiplex RT-PCR products are shown in Fig. 1. None of the human H3N2, H1N1, or influenza B virus isolates yielded any amplification products by this method (data not shown). In order to verify the lack of reassortment, all human H5N1 viruses and a subset of animal viruses (62 out of 103) were subjected to a full-genome sequencing protocol as previously described (4). Phylogenetic analyses of the sequence data revealed that all viruses contained their respective genomic segments and that no evidence of reassortment was found (data not shown). In addition to detecting human-avian virus reassortants, our assay may also be able to detect some reassortants that acquire genomic segments from some other unrelated avian viruses. However, the assay was not designed to detect reassortment among avian viruses, which are unlikely to play a role in viral adaptation to the human host. The absence of a band in the amplification of H5N1 viruses is not always due to reassortment. Full-genome sequencing is needed for result confirmation. Because the performance of this assay is directly affected by variation in the sequences of circulating H5N1 strains, the primer sequences should be frequently reevaluated and updated accordingly.


Figure 1
View larger version (79K):
[in this window]
[in a new window]

  		FIG. 1. A representative photograph of multiplex RT-PCR products from an H5N1 virus after electrophoresis in 2% agarose and ethidium bromide staining. Each lane shows products from each tube. The first lane is a 100-bp DNA ladder, and the target gene of each amplification product is labeled on the right.

 
Our multiplex RT-PCR was designed for egg- or MDCK-propagated viruses. Although the assay may be able to detect some of the genes in some clinical specimens, the level of sensitivity is probably not sufficient to reliably detect all the genomic segments simultaneously. This makes our assay less suitable for direct screening using clinical specimens. Nevertheless, the possibility of using a more sensitive version of this assay with clinical specimens should be further explored.


   ACKNOWLEDGMENTS
 
This study was supported by research grants from the National Center for Genetic Engineering and Biotechnology and the Thailand Research Fund for Advanced Research Scholar.

We thank Darat Lauhakirti and Sirawat Srichatrapimuk for their collaboration in the preparation of the manuscript.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, 2 Prannok Road, Bangkok Noi, Bangkok 10700, Thailand. Phone: 662 419 7059. Fax: 662 418 4148. E-mail: siput{at}mahidol.ac.th Back

{dagger} Supplemental material for this article may be found at http://jcm.asm.org/. Back

{triangledown} Published ahead of print on 21 March 2007. Back


   REFERENCES
Top
 ABSTRACT
 TEXT
 REFERENCES
 

  1. Guan, Y., J. S. Peiris, A. S. Lipatov, T. M. Ellis, K. C. Dyrting, S. Krauss, L. J. Zhang, R. G. Webster, and K. F. Shortridge. 2002. Emergence of multiple genotypes of H5N1 avian influenza viruses in Hong Kong SAR. Proc. Natl. Acad. Sci. USA 99:8950-8955.[Abstract/Free Full Text]
  2. Hoffmann, E., J. Stech, Y. Guan, and R. G. Webster. 2001. Universal primer set for the full-length amplification of all influenza viruses. Arch. Virol. 146:2275-2289.[CrossRef][Medline]
  3. Li, K. S., Y. Guan, J. Wang, G. J. Smith, K. M. Xu, L. Duan, A. P. Rahardjo, P. Puthavathana, C. Buranathai, T. D. Nguyen, A. T. Estoepangestie, A. Chaisingh, P. Auewarakul, H. T. Long, N. T. Hanh, R. J. Webby, L. L. Poon, H. Chen, K. F. Shortridge, K. Y. Yuen, R. G. Webster, and J. S. Peiris. 2004. Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia. Nature 430:209-213.[CrossRef][Medline]
  4. Puthavathana, P., P. Auewarakul, P. C. Charoenying, K. Sangsiriwut, P. Pooruk, K. Boonnak, R. Khanyok, P. Thawachsupa, R. Kijphati, and P. Sawanpanyalert. 2005. Molecular characterization of the complete genome of human influenza H5N1 virus isolates from Thailand. J. Gen. Virol. 86:423-433.[Abstract/Free Full Text]
  5. Webster, R. G., W. J. Bean, O. T. Gorman, T. M. Chambers, and Y. Kawaoka. 1992. Evolution and ecology of influenza A viruses. Microbiol. Rev. 56:152-179.[Abstract/Free Full Text]

Journal of Clinical Microbiology, May 2007, p. 1637-1639, Vol. 45, No. 5
0095-1137/07/$08.00+0     doi:10.1128/JCM.00382-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Other Versions of this Article:
JCM.00382-07v1
45/5/1637    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Auewarakul, P.
Right arrow Articles by Puthavathana, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Auewarakul, P.
Right arrow Articles by Puthavathana, P.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Antimicrob. Agents Chemother. Clin. Microbiol. Rev.
Clin. Vaccine Immunol. ALL ASM JOURNALS