Development And Application Of Real-time Rt-PCRfor Detection Of H1and H3Subtype Influenza Virus And ArmoredRNAstandards For Influenza A Virus

Influenza A virus (IAV) is an important causative agent responsible for devastating influenza occurring in both human and animal populations. The subtypes H1and H3are the most prevalent subtypes of IAV, infecting a wide range of host species. Real-time RT-PCR (rRT-PCR) has been widely used in IAV detection and subtyping worldwide, and has been recommended by World Health Organization (WHO) for detection of2009H1N1and2013H7N9influenza viruses. In China, universal rRT-PCR assay has successfully been implemented for molecular subtyping of H5, H7, and H9of IAV, serving as a national standard (GB). Nevertheless, so far there has no rRT-PCR assay available as GB for subtyping of H1and H3IAVs in China. In this study, we sought to develop rRT-PCR assays for detection of H1and H3IAVs. Additionally, we intended to prepare recombinant bacteriophage MS2particles containing armored RNA, which were used as a novel material standard for quality control in testing of IAV nucleic acid.1. Development of two rRT-PCR assays for detection of H1-and H3-subtype IAVsPrimers and TaqMan probes (LNA or MGB) were designed, respectively, based on the nucleotide sequences of HA gene of H1-and H3-subtype IAVs. rRT-PCR reaction systems have been successfully optimized. The specificities of two rRT-PCR assays were evaluated in this study. Our results indicated that all HI subtype IAVs, including human seasonal H1IAV, classical swine H1N1IAV, avian-like swine H1N1IAV and pandemic A (HIN1)2009virus could be accurately detected by the rRT-PCR assay for H1IAV we developed. As for rRT-PCR for H3IAV, all H3IAVs originated from different species could be detected, including H3N8equine IAVs, H3avian IAVs and swine IAVs. All other viruses as well as all other subtype IAVs, excepting H1and H3, were detected negative employing these two methods. The sensitivity of the two assays were evaluated and the results showed that46copies of H1cRNA and69copies of H3cRNA in each reaction could be detected respectively, indicating high sensitivity of the approach. Reproducibility test showed that coefficient variability of both assays was lower than5%. To evaluate the applicability of the methods,612clinical specimens were tested using the two assays, and the results were compared with conventional RT-PCR from GB/T27521-2011. The consistency is still greater than95%between rRT-PCR and RT-PCR, and rRT-PCR is10-fold and100-fold more sensitive than conventional RT-PCR. Our results suggested that rRT-PCR were suitable for detection of IAVs in practice.2. Validation of the rRT-PCR assays as GB for detection of animal IAV H1and H3subtypesBased on the validations of the two assays as described above, two drafts, titled "Real-time RT-PCR Assay for the Detection of H1Subtype Influenza Virus" and "Real-time RT-PCR Assay for the Detection of H3Subtype Influenza Virus" have been completed for standardization of H1and H3AIV detection. Detailed and specific information regarding these two assays were indicated in the drafts, including sampling, sample pretreatments, extraction of nucleic acids, formulating of reaction system, sample loading, setting of reaction parameters, result analysis, sequences of primers and probes, etc. A total of1519clinical samples were analyzed in13independent domestic laboratories using the two assays to evaluate these two GB methods. In addition, we consulted a number of experts from13scientific institutions before we submitted the drafts to the official authorities. 3. Expression of virus like particles (VLPs) of bacteriophage MS2containing cRNA of influenza virus M (Armored RNA)To develop a novel reference material for detection of IAV RNA, the cDNA fragment of maturation protein (A-protein), coat protein, packaging sites of MS2, and the matrix (M) gene of IAV was cloned into vector pET32a to prokaryotically express fusion proteins. The armored RNA (AR-M), encapsidated with M gene in the presence of bacteriophage coat proteins, was purified by cellufine sulfate mini-column and subsequently identified by RT-PCR and rRT-PCR. The stability test indicated that AR-M particles were resistant to RNase degradation and could be reproducibly used in rRT-PCR for90days when stored at-20℃or4℃. AR-M resembled authentic IAV, noninfectious, stable, RNase-resistant. Accordingly, AR-M was an idea reference control for testing of IAV nucleic acids and monitoring the entire detection process.4. Armored RNA served as standard material for nucleic acid testing of IAVBased on the quantitation of AR-M using rRT-PCR for IAV, a new RNA standard for influenza virus was prepared using AR-M. The homogeneity test indicates that the prepared recombinant RNA materials were homogenously distributed, confirmed by a vial-viral variation which was lower than5%when10randomly selected vials were examined. Our data demonstrated that AR-M particles were stable, evidenced by the results that AR-M could be stored for at least14days at room temperature,3months at2-8℃or6months at-20℃without any degradation. The quantitation standard of AR-M was (3.54±0.34)×106copies/μL and the averaged uncertainty value was0.34×106copies/μL, which were verified by our collaborative laboratories. This suggested that the AR-M particles could be used for quantitative assay of positive samples.In summary, in the present study two rRT-PCR assays have successfully been developed for detection of H1or H3subtype IAVs. Consequently, the corresponding GB drafts have been completed and submitted. Additionally, a phage packaging systems, containing armored RNA and genome of IAV was established. The GB drafts and nucleic acid standards obtained in this study would enhance capability of quality control for nucleic-acid testing of IAV.