Influenza A viruses contain an eight-segmented, single-stranded, and negative-sense RNA genome and can be differentiated from type B and C influenza viruses based on the internal antigens, the nucleoprotein (NP) and matrix (M1) proteins (Palese P, et al., 2007; Wright P F, et al., 2007). Among influenza A viruses, 16 hemagglutinin (HA) (H1-H16) and 9 neuraminidase (NA) (N1-N9) subtypes have been identified based on major differences within the HA and NA surface glycoproteins(Palese P, et al., 2007; Wright P F, et al., 2007). An influenza pandemic is an epidemic of a possibly emerging virus strain that spreads on a worldwide scale and infects a large proportion of the human population in which individuals might have encountered previous viral strains but have no immunity against these emerging strains(Nicholls H, 2006). Thus, pandemics can cause substantial morbidity and mortality, and pose a significant public health concern(Palese P, et al., 2007; Wright P F, et al., 2007).
Developing better methods for detection of influenza viruses is important to carry out surveillance of virus infection and outbreak in both humans and animals. While the traditional "gold standard" methods include culturing viruses and immunoassays (Amano Y, et al., 2005), the molecular diagnostic tests include reverse transcription-PCR (RT-PCR), microarray, molecular nucleic acid hybridization, quantitative real time RT-PCR (qRT-PCR), mismatch amplification mutation assay (MAMA) PCR, and multiplex RT-PCR assay (Burns N, et al., 1994; Dawson E D, et al., 2007; Hata M, et al., 2007; Storch G A, 2000; Wu C, et al., 2008). As viral strains change and evolve due to constant mutating of the virus genome sequence, this has presented a challenge to develop a universal system or assay that can detect every strain, and poses a need for developing better detection tools in order to monitor the distribution and outbreaks of influenza virus.
In 2009, infection associated with a new swine-origin strain of H1N1 influenza A virus (called pandemic H1N1/2009 strain herein) was declared to be a global pandemic (Stage 6) by the World Health Organization (WHO) after evidence of spreading in the world(Garten R J, et al., 2009; Vijaykrishna D, et al., 2010). By the end of 2009, more than 200 countries had officially reported over more than 500, 000 laboratory confirmed cases of the influenza pandemic H1N1/2009 infection, including more than 6, 250 deaths(World Health Organization, 2009). In China, there were more than 123, 000 laboratory confirmed cases, including at least 700 deaths by the end of 2009(Cao B, et al., 2009). The incidents associated with the H1N1/2009 influenza virus had declined significantly since early 2010, leading to the WHO announcement of the end of the epidemic in late 2010 (World Health Organization, 2010). However, it is unclear whether outbreaks associated with this novel strain will occur again, and sporadic cases of infections associated with this strain and its derivative strains have been found in China in 2011 (Yang, Z., Mao, G., Liu, Y., Liu, W., Luo, Y., Wu, J., and Liu, F., unpublished results). Today, it is still important to continue developing better and improved assays that can rapidly and specifically detect the pandemic H1N1/2009 influenza virus as well as its derivative strains in order to monitor the infection of these viruses in the human and animal populations.
As a rapid response to the outbreak of the pandemic H1N1/2009 virus, the WHO provided a quantitative real time RT-PCR (qRT-PCR)-based assay for detection of this virus in October 2009 (World Health Organization, 2009). The WHO-recommended method included a panel of oligonucleotide primers and dual-labeled hydrolysis (Taqman®) probes to be used in qRT-PCR assays for the in vitro detection and characterization of the novel H1N1/2009 strain in respiratory specimens and viral cultures. The primers and probes targeted the conserved regions of the HA genes of the pandemic viruses. In addition, many assays that were based on the qRT-PCR technology or the WHO-recommended systems have been developed and studied (Bennett S, et al., 2010; Gunson R, et al., 2010; Jiang T, et al., 2010; Klungthong C, et al., 2010; Lorusso A, et al., 2010; Pabbaraju K, et al., 2009; Schulze M, et al., 2010; Yang Y, et al., 2011). These assays using the oligonucleotides have greatly facilitated the detection and monitoring of the infections associated with the pandemic H1N1/2009 virus.
As influenza viruses spread and replicate, mutations are constantly introduced into the viral genome(Palese P, et al., 2007; Wright P F, et al., 2007). Thus, potential mismatch of the sequences in the emerging strains with the primers and probes of the WHO-recommended assay arises, and may reduce the sensitivity and effectiveness of the WHO assay for detecting the pandemic H1N1/2009 strain (Klungthong C, et al., 2010; Pabbaraju K, et al., 2009; World Health Organization, 2009). It is important to continue developing better and improved assays, including new sets of primers and probes with optimal match of virus sequences that can be used in the qRT-PCR assays, for detection and surveillance of the infections associated with the pandemic H1N1/2009 strains.
In this study, we have developed a novel set of primers that can be used in a qRT-PCR assay for detection of the pandemic H1N1/2009 influenza virus. A primer-probe set recommended by WHO was used as a reference method. The newly designed primers target three regions that are highly conserved among the HA genes of the pandemic H1N1/2009 viruses and are different from those targeted by the WHO-recommended primers. The qRT-PCR assays with the newly designed primers are highly specific in detecting pandemic H1N1/2009 viruses. Furthermore, the qRT-PCR assays with our designed primers appeared to be 10-fold more sensitive than those with the WHO-recommended primers, with a detection limit of about 2.5 copies of target RNA per reaction. Of 83 human clinical nasopharyngeal swabs tested, 32 were detected to be positive using the qRT-PCR assays with the newly designed primers, while only 25 were positive in the assays with the WHO-recommended primers. These results suggest that the qRT-PCR assay with the newly designed primers is highly sensitive for detecting the pandemic H1N1/2009 virus infection. Furthermore, our study demonstrates the feasibility of developing highly sensitive detection assays for early diagnosis of emerging influenza virus strains.
All research involving human participants was approved by the Institutional Review Boards of the Taizhou Institute of Virology and College of Life Sciences, Wuhan University, in accordance with the guidelines for the protection of human subjects. Written informed consent was obtained from each participant. No research involving human participants was carried out at the University of California-Berkeley.
A total of 83 clinical samples of nasopharyngeal swab were collected at the People's Hospital of Taizhou, China from December 5, 2009 to January 10, 2010. The H1N1/2009 reference strain (A/Taizhou/09/2009 (H1N1)) was isolated from a clinical sample of nasopharyngeal swabs obtained from the People's Hospital of Taizhou. The genomic segment coding for the HA gene was cloned and its sequence was determined(accession number JN863077). The other virus strains used in this study (Table 1) have been described previously (Gu H, et al., 2010; Li X, et al., 2009; Qi X, et al., 2009). All the virus isolates were grown in chicken eggs and the infectious allantoic fluids were collected and used as the virus stocks and samples. The titers of the virus samples were determined by plaque assays in Madin-Darby canine kidney (MDCK) cells using a standard protocol (Salomon N, et al., 1999). Total RNA samples were isolated from 140 μL samples of the allantoic fluid or nasopharyngeal swabs (suspended in Dulbecco's modified Eagle medium, DMEM) using the QIAamp viral RNA mini extraction kit (QIAGEN, Shanghai, China) in accordance with the manufacturer's protocol, and were stored at -70℃ until used.
Table 1. Virus isolates used in this study
The HA genomic segment of the reference strain (A/Taizhou/09/2009 (H1N1)) was cloned following the procedures described previously (Gu H, et al., 2010; Li X, et al., 2009; Qi X, et al., 2009). Briefly, RNA was extracted from the reference strain using Trizol reagent (Invitrogen, Shanghai, China). The HA sequence was reverse transcribed into cDNA and amplified by PCR with the uni12 primer and specific primer sets described previously (Hoffmann E, et al., 2001). The PCR-amplified products were cloned into vector pMD-18T (TAKARA, Dalian, China) and sequenced to verify its accuracy (Invitrogen, Shanghai, China). The resulting plasmid, designated as pT-HA (H1N1/2009/Taizhou), was used as the template for the subsequent studies.
The qRT-PCR primers and probes were designed based on the sequence information obtained from the Influenza Sequence Database (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html). Each of the available pandemic H1N1/ 2009 virus HA sequences from the database were aligned, and primers and probes were designed specifically to target the highly conserved regions of these sequences using the Beacon Designer (version7.6) program (Premier Biosoft). The primers and probes that were used in the study and yielded the best qRT-PCR results in detecting the pandemic H1N1/2009 virus were listed in Table 2.
Table 2. Primers and probes used in our developed qRT-PCR assays for the detection of the pandemic H1N1/2009 virus. A primer-probe set recommended by WHO, which was used as a reference method in our study, is also included
The RNA transcript corresponding to the HA gene of a reference strain (A/Taizhou/09/2009 (H1N1)) was in vitro transcribed from construct pT-HA (H1N1/2009/Taizhou) and used as the template to optimize qRT-PCR conditions. The qRT-PCR reactions were optimized at different temperatures and times, using the reagents and procedures as described in the WHO-recommended qRT-PCR assays (World Health Organization, 2009). The qRT-PCR reactions were performed in a reaction mixture containing 1 μL in vitro transcribed RNA of HA (H1N1/2009/Taizhou), 12.5μL 2X reaction mix and 0.5μL SuperScriptTMⅢ RT/Platimun® Taq Mix (SuperScriptTMⅢ Platinum® One-Step Quantitative Kit (Invitrogen, Ct.no.11732-020), Invitrogen, Shanghai, China), and 800 nmol/L of the forward and reverse primers and 200 nmol/L of Taqman probe that were either recommended by the WHO or generated based on our primer design. The reaction was performed for 30 min at 50℃ followed by 2 min at 95℃ with subsequent 45 cycles of amplification (95℃ for 15 s and 55℃ for 30 s), and the fluorescence was recorded at 55℃. For determination of the optimal melting temperature, the melting temperature was set at 55.0, 56.1 57.0, 58.3, 59.2, and 60.0℃. Regardless of the WHO-recommended or the newly designed primers to be used, the qRT-PCR reactions consistently yielded the lowest threshold cycle (Ct) values at a melting temperature of 55℃. Accordingly, the qRT-PCR assays described in the study were carried out under these conditions unless specified.
RNAs were in vitro synthesized by T7 polymerase from constructs containing different gene segment (HA, NA, PA, PB1, PB2, NP, M, NS) of a human seasonal H1N1 influenza virus reference isolate (A/Nanchang/ 14/1996) and the HA sequences of other viruses listed in Table 1. These constructs have been either constructed in our laboratory or described previously (Gu H, et al., 2010; Li X, et al., 2009; Qi X, et al., 2009). The in vitro transcribed RNAs were used as the templates to assess the specificity of the qRT-PCR assays with the designed primers. All the experiments were performed in triplicate and repeated three times.
To study the sensitivity of the qRT-PCR assays, total RNA was extracted from infectious allantoic fluid containing the reference virus strain (A/Taizhou/09/2009 (H1N1)) using QIAamp viral RNA mini kit (QIAGEN, Shanghai, China). Serially diluted RNAs ranging from 10-1 to 10-6 were used in the sensitivity test and the detection limit of the qRT-PCR assay was determined.
To further determine the sensitivity of the assays, RNA was in vitro transcribed from construct pT-HA (H1N1/ 2009/Taizhou) using the RiboMAXTM Large Scale RNA Production System-T7 (Promega, Beijing, China). Serial dilutions of the in vitro transcribed RNAs, which range from 2.5 to 5×106 copies per reaction, were used as the template for the sensitivity test.
The qRT-PCR reaction was performed following the WHO recommendations (World Health Organization, 2009). The assays were carried out, using the SuperScriptTMⅢ Platinum® One-Step Quantitative Kit (Invitrogen), in a 25 μL mixture containing 5 μL of serial diluted RNA samples, 12.5 μL 2X Reaction mix, 0.5μL SuperScriptTMⅢ RT/Platimun® Taq Mix (Invitrogen), and 800 nmol/L of forward and reverse primers and 200 nmol/L of Taqman probe that were either recommended by the WHO or generated based on our primer design. Amplification and detection were performed on a Bio-Rad iCycler iQ5TM real time PCR system (Hercules, CA) for 30 min at 50℃ followed by 2 min at 95℃ with subsequent 45 cycles of amplification (95℃ for 15 s and 55℃ for 30 s), and the fluorescence was recorded at 55℃. All the experiments were done in triplicate and repeated three times.