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Journal of Clinical Microbiology, January 2009, p. 86-92, Vol. 47, No. 1
0095-1137/09/$08.00+0     doi:10.1128/JCM.01090-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Design of Multiplexed Detection Assays for Identification of Avian Influenza A Virus Subtypes Pathogenic to Humans by SmartCycler Real-Time Reverse Transcription-PCR {triangledown}

Wei Wang,1 Peijun Ren,1 Sek Mardi,2 Lili Hou,1 Cheguo Tsai,1 Kwok Hung Chan,3 Peter Cheng,4 Jun Sheng,5 Philippe Buchy,2 Bing Sun,1 Tetsuya Toyoda,1 Wilina Lim,4 J. S. Malik Peiris,3,6 Paul Zhou,1 and Vincent Deubel1*

Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai Institute of Biological Sciences, Shanghai, China,1 Institut Pasteur du Cambodge, Phnom Penh, Cambodia,2 Department of Microbiology, the University of Hong Kong, Hong Kong,3 Public Health Laboratory Services Branch, Centre for Health Protection, Department of Health, Hong Kong,4 Shanghai Nanxiang Hospital, Shanghai, China,5 HKU-Pasteur Research Centre, Hong Kong SAR6

Received 9 June 2008/ Returned for modification 13 August 2008/ Accepted 13 October 2008


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ABSTRACT
 
Influenza A virus (IAV) epidemics are the result of human-to-human or poultry-to-human transmission. Tracking seasonal outbreaks of IAV and other avian influenza virus (AIV) subtypes that can infect humans, aquatic and migratory birds, poultry, and pigs is essential for epidemiological surveillance and outbreak alerts. In this study, we performed four real-time reverse transcription-PCR (rRT-PCR) assays for identification of the IAV M and hemagglutinin (HA) genes from six known AIVs infecting pigs, birds, and humans. IAV M1 gene-positive samples tested by single-step rRT-PCR and a fluorogenic Sybr green I detection system were further processed for H5 subtype identification by using two-primer-set multiplex and Sybr green I rRT-PCR assays. H5 subtype-negative samples were then tested with either a TaqMan assay for subtypes H1 and H3 or a TaqMan assay for subtypes H2, H7, and H9 and a beacon multiplex rRT-PCR identification assay. The four-tube strategy was able to detect 10 RNA copies of the HA genes of subtypes H1, H2, H3, H5, and H7 and 100 RNA copies of the HA gene of subtype H9. At least six H5 clades of H5N1 viruses isolated in Southeast Asia and China were detected by that test. Using rRT-PCR assays for the M1 and HA genes in 202 nasopharyngeal swab specimens from children with acute respiratory infections, we identified a total of 39 samples positive for the IAV M1 gene and subtypes H1 and H3. When performed with a portable SmartCycler instrument, the assays offer an efficient, flexible, and reliable platform for investigations of IAV and AIV in remote hospitals and in the field.


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INTRODUCTION
 
Influenza A viruses (IAV) form a large group in the orthomyxoviruses, causing diseases in human and other mammalian and avian species, including wild birds, poultry, pigs, rodents, and sea mammals (53). Orthomyxoviruses contain an eight-segment negative-stranded RNA genome encoding 10 proteins, including the envelope-forming proteins hemagglutinin (HA), neuraminidase (NA), membrane/ion channel protein M2, and matrix protein M1. HA protein exposed at the surface of the viral envelope is responsible for target cell tropism and is associated with pathogenesis (2). HA antigen induces neutralizing antibodies and, consequently, is subjected to selective pressures leading to genetic evolution (7) and possibly increasing virulence and an expanding host range (17, 24, 31).

A major natural reservoir of avian influenza viruses (AIV) is feral aquatic birds, which contain many viruses defined by 16 known HA and 9 known NA subtypes. Only two viruses, the H5 and H7 subtypes containing HA genes, are highly pathogenic AIV (HPAIV) for birds. AIV containing the H1, H2, H3, H5, H7, and H9 subtypes have shown their capacity to cross the species barrier and cause human diseases (26, 40). H1N1 and H3N2 viruses are the most commonly recovered strains from among the classical influenza viruses that have been cocirculating for more than 30 years (30). The H2N2 subtype was the causative agent of the Asian influenza pandemic in 1957 (33). H9N2 caused influenza in Hong Kong and China (24, 32). H7N3 and H7N7 subtypes caused conjunctivitis on several continents (11, 14, 20, 44). HPAIV H5N1 emerged in 1997 in Hong Kong and currently poses a serious health threat worldwide, and a mutant or reassortant virus capable of efficient human-to-human transmission could trigger another influenza pandemic (17, 52; see also http://www.who.int/csr/disease/avian_influenza/en/index.html). All these viruses originate from avian and pig reservoirs and have a potency sufficient to cause a pandemic with substantial economic losses and dramatic public health issues. Thus, monitoring AIV in wild or domestic birds and pigs and diagnosing AIV infections in humans should be implemented in urban and rural settings to rapidly initiate appropriate medical, veterinarian, and epidemiological measures.

Standard viral diagnostic methods for AIV infections applied to active surveillance programs or disease investigations are time-consuming (1). Sample transportation to a reference laboratory is constraining, and inoculation of chicken embryos and cell cultures under high-containment conditions is a laborious process which may take more than 1 week (50). Many different molecular techniques allow IAV M and HA gene identification within a few hours (1, 5, 42) but require trained personnel working in well-equipped laboratories. Because of its rapidity, improved sensitivity over that of cell culture, relatively low cost, and reliability in reducing the cross-contamination possible with reverse transcription-PCR (RT-PCR), the one-step multiplexed real-time RT-PCR (rRT-PCR) has been developed for AIV surveillance (6, 8, 10, 13, 15, 22, 27-29, 36-39, 41, 46-48, 52, 54-56). However, the genetic diversity of subtypes and variants causing diseases in avian species and in humans has been a challenging issue for multiplex rRT-PCR, often requiring primer and probe mixtures incompatible with the specificity and sensitivity of the method.

In this study, we performed an algorithm of four rRT-PCR assays using a SmartCycler instrument for identification of IAV M1 and HA sequences from six AIV known to infect humans and birds. This method, which was performed with a portable machine, can be used for rapid clinical diagnosis of out- or inpatients in a hospital and by mobile investigation teams in a field laboratory to overcome problems of sample transportation and to implement rapid response strategies to control virus outbreaks.


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MATERIALS AND METHODS
 
Clinical samples, viruses, virus HA and M1 genes, and bacteria. Clinical specimens from nasopharyngeal swabs (NPS) were collected from pediatric patients, under 15 years old, experiencing acute respiratory infections (ARI) and who consulted the pediatric department of Shanghai Nanxiang Hospital between October 2006 and January 2008. The swabs were kept in 2 ml viral transport medium (2.95% tryptose phosphate broth, 0.5% gelatin, 500 mg/liter amphotericin B, 4 mg/liter gentamicin, and 0.5 x 10–6 U/liter penicillin G and streptomycin), transported at 4°C to the Institut Pasteur of Shanghai laboratory, divided into aliquots, and stored at –80°C.

Different IAV and AIV from the collection of the Department of Microbiology, the University of Hong Kong, isolated from embryonating chicken egg allantoic fluids are shown in Table 1. Viruses were not titrated, and RNA was extracted from undiluted fluids.


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  		TABLE 1. Influenza virus and other respiratory viruses used in this study

HK/948/06 (H1N1) and HK/2268/06 (H3N2) viruses from the collection of the Hong Kong Public Health Laboratory were cultivated in MDCK cells and titrated by determining the 50% tissue culture infective dose (TCID50), and RNA was extracted from viruses in cell supernatants (Table 1).

A collection of viruses responsible for ARI was isolated and cultivated in MDCK cells at Institut Pasteur of Shanghai (Table 1).

DNA clones of the subtype H5 HA genes from different H5N1 clades were prepared at Institut Pasteur of Shanghai, either from viral RNA or by gene synthesis (Table 2).


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  		TABLE 2. IAV M and HA gene cDNA clones

The M1 gene of PR/8/34 (H1N1) virus was amplified by RT-PCR and cloned into the pET14b-M1 plasmid (45), whereas the H1 HA gene of the same virus was cloned into the pPSP-H1 plasmid (16).

Streptococcus hemolyticus (Streptococcus pyogenes) Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Legionella pneumoniae, Chlamydia pneumoniae, and Mycoplasma pneumoniae were isolated from patients with ARI at Institut Pasteur of Cambodia.

RNA extraction and transcript preparation. Total RNA from NPS aliquots obtained from children with ARI in Shanghai Nanxiang Hospital or from virus-infected chicken egg allantoic fluids, virus-infected cell supernatants, or bacterial lysates was extracted using a QIAamp viral RNA minikit (Qiagen, Hilden, Germany) in accordance with the manufacturer's protocol. Purified RNA was frozen at –80°C in aliquots.

In vitro-transcribed RNAs from different HA genes from IAV subtypes and H5 clades was used for internal controls and for the study of sensitivity and specificity of the rRT-PCR (Table 2). The HA genes of IAV were used to construct plasmid DNAs by inserting the full-length genes into the pGEM-T Easy vector (Promega, Madison, WI) or the pCMV vector (Invitrogen, Carlsbad, CA). The plasmidic T7 RNA polymerase transcription initiation site and promoter sequence located upstream of the IAV cloned sequences were utilized to synthesize RNA transcripts in vitro, using a T7 RiboMAX large-scale RNA production system (Promega) in accordance with the manufacturer's instructions. The concentration of RNA transcripts was calculated using a UV spectrophotometer (Bio-Rad, Hercules, CA).

To perform sensitivity tests, the RNAs were serially 10-fold diluted, ranging from 105 RNA copies/reaction to 1 RNA copy/reaction. To compare the detection limits obtained by rRT-PCR with titrated virus particles, RNAs extracted from the H1N1, H3N2, and H5N1 viruses in supernatant fluids (Table 1) were serially diluted 10-fold from 106 to 0.01 TCID50/reaction.

rRT-PCR. Highly conserved regions of each M1 and HA gene were identified using Clustal W sequence alignment algorithms. The primer and probe sequences were designed with highly conserved sequences of IAV genes by using Primer Express (ABI, Foster, CA) software and Beacon Designer (Premier Biosoft) software with selection criteria by default (Table 3). The sequences of the M1 or HA genes of virus subtypes from human, bird, and pig isolates used for alignment are from GenBank (http://www.ncbi.nlm.nih.gov/GenBank/index.html) and are available by request from the authors. After synthesis (Takara, Dalian, China), primers and probes were divided into aliquots and conserved undiluted at –80°C. H1 and H9 virus probes were labeled at their 5' ends with Texas Red reporter dye and at their 3' ends with Black Hole 2 quencher (BHQ-2) dye. The H3 probe was labeled at its 5' end with TET (6-carboxytetrachlorofluorescein) reporter dye and at its 3' end with BHQ-1 quencher dye. H2 and H7 virus probes were labeled with 6-carboxyfluorescein reporter dye at their 5' ends and at their 3' ends with BHQ-1 quencher dye (Table 3).


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  		TABLE 3. Sequences of primers and probes used in rRT-PCR

We adapted the HA genotyping assay for application to the SmartCycler II system (Cepheid, Sunnyvale, CA), which has four channels and can detect at most four different fluorescent dyes. Reactions were based either on a Sybr technique or on a TaqMan probe technique. Sybr reactions were done with a Sybr green I dye one-step RT-PCR kit (Takara), and the mixtures contained 5 U Takara Ex Taq HS, 5 U Primer Script II RT enzyme mixture, 12 U RNasin (Promega), and 200 pmol of each primer in a 25-µl final volume. TaqMan reactions were performed using a QuantiTect Probe RT-PCR kit (Qiagen). The reaction mixture contained 2.5 U enzyme mixture, 12 U RNasin, and 200 pmol of each primer and probe in a 25-µl final volume. Five microliters of RNA was added to each reaction mixture. All reactions used the same program: reverse transcription at 50°C for 30 min, transcriptase denaturation at 95°C for 5 min and then DNA polymerization for 45 cycles of 94°C for 15 s, 53°C for 30 s, and 72°C for 15 s, ending with (Sybr) or without the melting step. Results were considered positive if the cycle threshold (CT) values were less than 35 cycles and the {Delta}S values (representing changes in the magnitude of the fluorescent signal) were higher than 100.

The WHO H5 HA rRT-PCR TaqMan protocol developed by the laboratory at the National Institute of Infectious Diseases (NIID), Tokyo, Japan, the WHO Collaborating Centre for Reference and Research on Influenza, and the WHO H5 Reference Laboratory was used as a reference technique for determining the sensitivity and specificity of H5 HA gene detection assay (51).

rRT-PCR and M1 RT-PCR assays of viruses from NPS responsible for ARI. A total of 202 NPS specimens were prepared for IAV detection using the rRT-PCR described above and for IAV M1 gene detection using the RT-PCR protocol previously described (4). Briefly, 2.5 µl of RNA extracted from NPS specimens was added to 22.5 µl of a master mixture containing PCR buffer (OneStep RT-PCR kit; Qiagen), 0.2 mM of each deoxynucleoside triphosphate, 0.5 µM of each of the M1 primers (5'-CAG AGA CTT GAA GAT GTC TTT GCT AG-3' and 5'-GCT CTG TCC ATG TTA TTT GGA ATC-3') previously described (9), and enzymes. Samples were subjected to RT-PCR using the cycle program as follows: 30 min at 50°C, 15 min at 94°C, followed by 40 cycles of 30 s of denaturation at 94°C, 30 s of annealing at 55°C, and 1 min of extension at 72°C. A final extension was programmed for 10 min at 72°C. The amplification products were analyzed in a 0.5 µg/ml ethidium bromide-2% agarose gel. All RT-PCR-positive samples were analyzed by using rRT-PCR for the HA genes of subtypes H1, H3, and H5. Statistical analysis was performed by using SAS software (SAS Institute, Inc., Cary, NC).


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RESULTS
 
rRT-PCR. We have developed a sequential rRT-PCR that contains four reactions (Fig. 1). The AIV M gene-positive samples were further processed for H5 HA gene identification, and negative samples were tested subsequently with either the TaqMan assay for H1 and H3 HA genes or the TaqMan assay for H2, H7, and H9 HA genes and the beacon probe rRT-PCR identification assay. All reactions were programmed with the same PCR program, so they could be run not only with SmartCycler but also with other real-time PCR instruments in case of a large sample quantity.


Figure 1
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  		FIG. 1. Algorithm of the four-tube rRT-PCR assay for the detection of AIV.

Sensitivity. In order to optimize and compare the sensitivities of the four rRT-PCRs, RNA transcripts of each full-length HA gene were prepared and tested by using either monoplex or multiplex reactions (Table 4). The limit of detection was calculated using 10-fold dilutions of the transcripts. The limit of detection of the A/Henan/04 HA RNA transcript found by reaction 2 (Fig. 1) corresponded to 10 RNA copies, and these results were similar to those of the standard rRT-PCR technique recommended by WHO (see Materials and Methods) (data not shown). The limit of detection of reaction 3 for the H1 or H3 HA gene corresponded to 1 RNA copy, whereas the limit was 10 RNA copies of each gene in reaction 3. The limit of detection in reaction 4 was about 10 RNA copies for the H2 and H7 HA genes and 100 copies for the H9 HA gene.


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  		TABLE 4. Comparison of CT values of monoplex-specific and multiplex real-time PCR for HA RNA transcripts

To determine the sensitivity of the detection of live virus particles, serial dilutions of RNAs extracted from titrated H1N1, H3N2, and H5N1 virus stocks (TCID50) were tested with reactions 1, 2, and 3. The fluorescent signals observed for the M1 genes of H1N1 and H3N2 viruses in reaction 1 corresponded to 1 TCID50, and those for the H1 and H3 HA genes in reaction 3 corresponded to 0.1 TCID50 (Table 5). Signals observed for H5N1 virus were 0.1 TCID50 and 0.01 TCID50 for reactions 1 and 2, respectively.


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  		TABLE 5. Comparison of monoplex RT-PCR and multiplex rRT-PCR sensitivitiesa

Specificity. The specificity of the H5 rRT-PCR for different clades of viruses, including low-pathogenicity, high-pathogenicity, and avian and human H5N1 viruses, was tested. Eight HA clones of H5N1 virus corresponding to clades 0, 1, 2.1.3, 2.3.1, 2.3.2, and 8 (21) (Table 2) were used in reaction 2 and showed positive results with a melting temperature of 77°C to 78°C for the amplicons (data not shown). All genes also showed positive results when tested using the technique provided by WHO (data not shown).

To assess the integrity of the primers and probes of the six HA subtypes, reactions 1 to 4 were tested for cross-reactivity among H1 to H11 subtypes of AIV (Table 1). The primer set corresponding to the M1 gene of IAV was positive for all subtypes, and only the HA primer-probe set for the homologous virus subtypes showed positive results. The H4, H6, H8, H10, and H11 virus subtypes remained undetected by reactions 2 to 4. None of the several unrelated viruses causing ARI (Table 1) showed any positive results by reactions 1 to 4 (data not shown).

The cross-reactivities of the primers used in the four rRT-PCRs (Table 3) with RNA molecules extracted from seven bacteria commonly found in the respiratory tract were assessed by RT-PCR (see Materials and Methods). None of the samples showed any visible amplified product after agarose gel electrophoresis (data not shown).

Comparative study of RT-PCR and rRT-PCR using NSP samples. As reaction 1 is essential in the detection of IAV, it is important to address its specificity and sensitivity with samples from natural specimens. The specificity of reaction 1 for the IAV M gene was addressed by testing a collection of 202 NPS specimens from children with ARI (Table 6). Calculation of the melting temperature of the amplified products (80°C to 81°C) in reaction 1 helped to discriminate false positives due to internal primer dimers (data not shown). The reaction identified 39 positive samples (P = 19.3%; n = 202; S = 0.028; 95% confidence limits 0.778 to 0.978 [{alpha}, <0.05]). To confirm the results obtained by rRT-PCR, samples were tested by RT-PCR (see Materials and Methods). Thirty-eight samples (18.8%) were found positive by RT-PCR, of which two (1%) had previously been found negative by rRT-PCR (Table 6). The specificity between reaction 1 and RT-PCR for detection of the IAV M gene was 99.5%. The sensitivity of reaction 1 relative to that of RT-PCR for the detection of IAV was 94.9%. Statistical analysis showed that the two methods have no significant differences in performance (chi-square = 0.90; continuity adjustment chi-square = 1.0; kappa coefficient = 0.92, with 95% confidence limits of 0.85 to 0.99[{alpha}, <0.05]).


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  		TABLE 6. Comparison of detection of rRT-PCR and RT-PCR assaysa

Reactions 2 and 3 were performed subsequently with all 202 samples, of which 2 were found positive for H1, 37 were found positive for H3, and all samples were negative for subtype H5. However, two samples found negative by rRT-PCR but positive by RT-PCR were confirmed positive and corresponded to the H3 subtype in reaction 3. These two samples had CT values of 28 and 29, respectively, values close to the limit of sensitivity for the detection of H3 virus sequences (Table 5), whereas all other positive samples had CT values between 15 and 19 (data not shown). One sample positive by rRT-PCR but negative by RT-PCR for the M gene was found positive for subtype H3 (Table 6) but with high CT values of 30 and 34 for the M1 gene and H3 subtype, respectively, indicating a low concentration of viral RNA. Results for two other samples found positive for the M gene in reaction 1 (CT values of 20.6 and 27.7, respectively) but negative in reactions 2, 3, and 4 should be considered false positives, since the samples were also negative by RT-PCR. The reasons for the positive results in duplicated reaction 1 for the two samples were not evident. Concordance of reaction 3 with reaction 1 was 94.9%.


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DISCUSSION
 
Two subtypes of IAV containing the H1 and H3 HA genes cause recurrent seasonal outbreaks due to a high mutation rate, with high morbidity and mortality. Treatment of infected patients remains efficient if the virus is detected early, and therefore, rapid identification of IAV would benefit the patient. AIV subtypes H5 and H7 are highly pathogenic in domestic birds and have important economic impacts and alarming rates of expansion and transmission to humans. Massive vaccination of domestic birds against the zoonotic H5N1 virus remains difficult and is not policed in many countries. Except for H5N1, subtypes H2, H7, and H9 are the most likely candidates for the next pandemic (11, 33, 49). Therefore, tracking HPAIV and other virus subtypes that can infect humans in aquatic and migratory birds, poultry, and pigs is essential for epidemiological surveillance and outbreak alerts and can contribute to rapid implementation of infection control measures at the national and international levels.

Several molecular methods have been developed to identify different subtypes of AIV pathogenic for humans and for poultry (1, 5, 39, 42); these methods include RT-PCR, nested RT-PCR, rRT-PCR, nucleic acid sequence-based amplification (21), loop-mediated isothermal amplification (18, 19), and DNA microarrays (23, 43). In the present study, a new rRT-PCR assay was established for simultaneous or sequential identification of six AIV subtypes pathogenic for humans, based on M1 gene identification and H5, H1, and H3 or H2, H7, and H9 subtype identification, respectively. This assay used different algorithms according to medical or ecological situations. The four-tube assay also took into consideration the specificity and sensitivity of the tests. The first tube contained primers for IAV M1 gene identification to screen for the presence of any IAV subtype. The Sybr green I dye detection system was chosen for this test because it shows high sensitivity and offers the capacity to screen large numbers of specimens at low cost. Usually, rRT-PCR has proven to be more sensitive than cell culture for IAV identification. The limit of virus particle detection by the Sybr M1 gene rRT-PCR was similar to that previously published by others using a similar test and a SmartCycler instrument (12).

In our protocol, the M1 gene-positive specimens were subsequently processed, first for H5 subtype identification, since this virus subtype is currently endemic in poultry and widely spread, with high risk for the human population in direct contact with avian species (41). The large genetic variability due to virus evolution and cocirculation of different clades of subtype H5N1 subtypes increases the difficulty of elaborating an rRT-PCR containing an ad hoc primer mixture that hybridizes with all H5 HA genes identified to date in avian, porcine, and human populations (7) without affecting the specificity and sensitivity of the technique. Our strategy was to use a one-tube assay for the detection of different H5 clades that is easy to update when new variants emerge and sequences are made available. Reaction 2, containing only two sets of primers, recognized the six different clades tested (52). However, this assay as performed with RNA transcripts, plasmidic DNAs, or egg and cell cultures needs to be validated with H5 virus-positive biological samples and all 10 existing clades (52). Recent techniques have developed a one-step TaqMan rRT-PCR specific for the detection of less than 1 PFU of H5 AIV from clades 0, 1, 2, and 3 (10, 27). The limit of detection in reaction 2 of 10 copies of RNA molecule and 0.01 50% lethal dose of virus was similar to limits obtained using one of the WHO-recommended protocols and to those previously published (13, 22, 55, 56).

Two H1 and 37 H3 virus subtypes positive for the M gene in rRT-PCR or RT-PCR (Table 6) were subtyped using reaction 3. These results validate the reaction which could be implemented in hospitals for screening patients suspected of having seasonal influenza. However, two samples were found negative in reaction 1 but positive in reaction 3, probably reflecting the small amount of viral material in those samples and a difference in sensitivity of 1 log between reaction 1 and reaction 3 (Table 5). Thus, reactions 2 and 3 may be used concomitantly with, rather than subsequently to, reaction 1. This rapid test has considerable value by contributing to the choice of anti-influenza treatment for AIV-infected patients. If the result is positive, rRT-PCR 2 targeting the H5-specific sequence should be controlled using WHO-recommended assays (51), and similar tests should be carried out at a reference center for influenza virus.

The fourth reaction of the rRT-PCR test contains three sets of specific primers and fluorogenic probes that match the HA sequences of all H2, H7, and H9 subtypes with avian, pig, and human origins available in GenBank. The three virus subtypes are found in infected feral and aquatic birds and fowl and have the ability to cause diseases in human hosts. Avian H2N2 was the cause of the 1957 pandemic and the source of laboratory contamination (33-35). Avian H7N7 transmission to humans was the origin of an epidemic of conjunctivitis in Canada and in Europe (11). AIV H9N2 has infected several children, and H9 HA has receptors for efficient human host cell infectivity (24, 32). Several multiplex rRT-PCR assays have been developed for H5 and H7 or for H5, H7, and H9 subtype detection (6, 22, 37-39, 54). In this study, we present the first one-tube rRT-PCR for three subtypes, H2, H7, and H9. Reaction 4 could be used subsequently for patients who are AIV M1 gene positive but negative for subtypes H5, H1, and H3. However, this reaction may be used concomitantly with reactions 2 and 3 for surveillance programs and for monitoring large bird flocks and pig herds. Samples positive for the M gene but negative for the six HA genes may be investigated further by other available techniques targeting all HA or NA genes of AIV (3, 25, 31a).

The present set of rRT-PCR assays, which were set up as a high priority for rapid detection of causative agents in humans, is being further validated with different sample types obtained from human biological specimens, including the lower respiratory tract and serum or fecal samples, as well as specimens from other species. This study is a starting point for further tests of a large set of specimens and is ongoing in China and in Cambodia. Our assays performed with a portable SmartCycler offer an efficient, flexible, and reliable platform for investigations in the field and overcome the need to transport samples from remote stations lacking the means for sampling and shipping in safety and under appropriate conditions to central facilities for analysis. The feasibility of this mobile system has already been described for rapid detection of Ebola virus by mobile investigation teams in Africa (47). The availability of cartridge-based sample preparation adapted to SmartCycler equipment would provide reproducible performance and also limit extra pipetting steps and multiple reagent mixtures that would speed up analysis and reduce contamination risk.


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ACKNOWLEDGMENTS
 
The study was supported by the French Ministry of Health, the Li Kha Shing Foundation, the French Agency for Development (SISEA project), and the European Union (RIVERS project).

W.W. is a recipient of AREVA from AREVA-Pasteur partnership. J.S.M.P. is supported by the university grants committee of the Hong Kong SAR (project AoE/M-12/06).

We thank A. Vabret and F. Freymuth (Laboratory of Virology, Centre Hospitalier Regional de Caen, France) for providing 229E-CoV and OC43-CoV; C. Y. H. Leung for providing viruses tested in the Department of Microbiology, the University of Hong Kong; K. Nakajima of Nagoya City University for pME18SA (H3 HA of A/Aichi/2/68, Hong Kong influenza virus); and M. Ohuchi of Kawasaki Medical College for pUC-H2HA (H2 HA of A/Adachi/2/57, Asian influenza virus) and pUC-H7 (H7 HA, A/chicken/Rostock/8/34). We also thank Y. M. Zheng for manuscript preparation.


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FOOTNOTES
 
* Corresponding author. Mailing address: Unit of Emerging Viruses, Chinese Academy of Sciences, Institut Pasteur of Shanghai, 411 Hefei Road, 200025 Shanghai, People's Republic of China. Phone: 86 21 6384 5146. Fax: 86 21 6384 3571. E-mail: vdeubel{at}sibs.ac.cn Back

{triangledown} Published ahead of print on 29 October 2008. Back


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Journal of Clinical Microbiology, January 2009, p. 86-92, Vol. 47, No. 1
0095-1137/09/$08.00+0     doi:10.1128/JCM.01090-08
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