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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet

Short communication

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Detection of avian influenza A/H7N9/2013 virus by real-time reverse transcription-polymerase chain reaction

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Xiaoping Kang a , Weili Wu b,∗ , Chuntao Zhang c , Licheng Liu a,d , Huahua Feng d , Lizhi Xu d , Xin Zheng d , Honglei Yang d , Yongqiang Jiang a , Bianli Xu e , Jin Xu e , Yinhui Yang a , Weijun Chen b,d a State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Dongdajie Road 20, Beijing 100071, China b CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, No.1 Beichen West Road, Chaoyang District, Beijing 100101, China c National Institutes for Food and Drug Control, Beijing 100050, China d Beijing BGI-GBI Biotech Co., Ltd, Beijing 101300, China e Center for Disease Control and Prevention of Henan, Zhenzhou, China

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a b s t r a c t

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Article history: Received 12 May 2013 Received in revised form 12 December 2013 Accepted 9 January 2014 Available online xxx

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Keywords: Avian influenza A/H7N9 virus Real-time reverse transcription polymerase chain reaction (rRT-PCR)

The first case of avian influenza A/H7N9 infection was reported in Shanghai in mid-February 2013; by May 1, 2013, it had infected 127 people and caused 26 deaths in 10 provinces in China. Therefore, it is important to obtain reliable epidemiological data on the spread of this new infectious agent, a need that may be best met by the development of novel molecular methods. Here, a new method was described for the detection of avian influenza A/H7N9 using real-time reverse transcription-polymerase chain reaction (rRT-PCR). Using serial dilutions of avian influenza A H7N9 cultures, the detection limit of the assay was determined to be approximately 3.2 × 10−4 HAUs (hemagglutination units) for the H7 gene and 6.4 × 10−4 HAUs for N9 gene. In tests of serial dilutions of in vitro-transcribed avian influenza A H7 and N9 gene RNA, positive results were obtained for target RNA containing at least three copies of the H7 gene and six copies of the N9 gene. Thirteen throat swabs from H7N9 patients were tested; all tested positive in the assay. Specificity was evaluated by testing 18 other subtypes of influenza viruses; all tested negative. A total of 180 throat swabs from patients infected with influenza virus, including 60 from patients infected with seasonal influenza A/H1N1 virus, 60 from patients infected with pandemic influenza A/H1N1/2009 virus, 30 from patients infected with seasonal influenza A/H3N2 virus and 30 from patients infected with influenza B virus, were also tested; all tested negative. © 2014 Elsevier B.V. All rights reserved.

Sporadic human infections with avian influenza A viruses, which usually occur after recent exposure to poultry, have been reported, including infections with low pathogenic avian influenza (LPAI) A (H7N2, H7N3, H9N2, or H10N7) virus (Nguyen-Van-Tam et al., 2006; Eames et al., 2010; Butt et al., 2005; Arzey et al., 2012) and highly pathogenic avian influenza (HPAI) A (H5N1) virus (Yuen et al., 1998). These are classified as highly pathogenic for birds if the haemagglutinin protein contains a polybasic site that can be cleaved by avian enzymes to cause systemic illness. This designation thus relates to pathogenicity in avians rather than in humans. These infections cause a wide spectrum of illness, ranging from

∗ Corresponding author. Tel.: +86 1084097876. E-mail address: [email protected] (W. Wu).

conjunctivitis and upper respiratory tract disease to pneumonia and multiorgan failure. In February and March 2013, a large number of patients were reported in China with severe lower respiratory tract disease; these cases were associated with a novel, avian-origin reassortative influenza A/H7N9 virus (Gao et al., 2013). This virus had infected 77 people by April 16, 2013, causing 16 deaths (MOH, 2013a,b). Symptoms caused by avian influenza A/H7N9, seasonal influenza A and B viruses or other avian influenza A virus infections, such as fever, coughing and sneezing, appear very similar, making definitive clinical diagnosis difficult. Therefore, developing a rapid diagnostic method for detecting avian influenza A/H7N9 is an urgent priority for controlling the spread of the virus. In this study, a sensitive and specific real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay was developed for the detection of avian influenza A/H7N9. The data

http://dx.doi.org/10.1016/j.jviromet.2014.01.026 0166-0934/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Kang, X., et al., Detection of avian influenza A/H7N9/2013 virus by real-time reverse transcriptionpolymerase chain reaction. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.01.026

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Fig. 1. Sensitivity and dynamic range of rRT-PCR in detection influenza A virus H7N9 RNA. Serial dilutions of in vitro-transcribed H7 and N9 gene RNA were tested. A wide linear range (beginning at three copies (H7) or six copies (N9) and extending through 3 × 105 copies of the control RNA per reaction) was established in this assay. The intercept of the magnitude of the fluorescence signal (PCR Base line subtracted CF RFU) with the horizontal threshold line represents the Ct value for a given sample. (A) H7 assay; (B) N9 assay. 54 55 56 57 58 59 60 61 62 63 64 65

showed that the new method is sensitive and allows the rapid and accurate detection of avian influenza A/H7N9 RNA in patient samples. PCR primers and probes were designed using Primer Express Software (Applied Biosystems, Foster City, CA, USA) based on an alignment of all previously published avian influenza A/Zhejiang/DTID-ZJU01/2013(H7N9) hemagglutinin (HA) and neuraminidase (NA) gene sequences (published in April 10, 2013 on NCBI). The specific primers and probe sets for NA and HA genes were as follows: HA449F (forward primer), 5 -TCA GGA TCT TCA TTC TAT GCA GAA AT-3 , HA557R (reverse primer), 5 -ATT AGA GCT GGG CTT TTT CTT GTA TT-3 and HA479 (probe), 5 -FAM-TGG CTC CTG TCA

AAC ACA GAT AAT GCT GC-BQH1-3 ; NA416F (forward primer), 5 ACA CTC AAA CGG AAC AAT ACA CGA-3 , NA496R (reverse primer), 5 -GCA TTC CAC CCT GCT GTT GT-3 and NA455 (probe), 5 -FAMCGC CCT GAT AAG CTG GCC ACT ATC ATC A-BQH1-3 . The probe was labeled with the reporter FAM (6-carboxyfluorescein) and the quencher BQH1. Primers and probes were synthesized (Bgi-Gbi Biotech, Beijing, China). The amplification efficiency and detection limits of the assay were evaluated by generating and testing dilutions of RNA for avian influenza A H7N9 H7 and N9 genes. The H7 and N9 genes were amplified based on the aligned sequence of HA and NA genes of avian influenza A/H7N9 using the reference method (Zhu

Please cite this article in press as: Kang, X., et al., Detection of avian influenza A/H7N9/2013 virus by real-time reverse transcriptionpolymerase chain reaction. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.01.026

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et al., 2010). The resulting products were cloned into a pGEMT Easy vector (Promega, Shanghai, China) and linearized using a specific DNA restriction enzyme. RNA was generated by in vitro 80 transcription of the linearized plasmid DNA using the RiboMax 81 Express Large-Scale RNA Production System, according to the man82 ufacturer’s instructions (Promega, Madison, Wisconsin, USA). After 83 digestion of the template DNA with RNase-free DNase I, the tran84 scribed RNA was purified with an RNeasy kit (Qiagen, Hilden, 85 Germany). The purified RNA was quantified spectrophotometri86 cally at 260 nm, divided into aliquots, and stored at −80 ◦ C for 87 future use. Ten-fold serial dilutions of transcribed H7 RNA (6 × 107 88 to 5 copies/␮l) and two-fold serial dilutions of transcribed N9 89 RNA (6–0.75 copies/␮l) were subjected to rRT-PCR analyses. rRT90 PCR was performed in 20-␮l reaction volumes containing 4 ␮l of 91 the RNA dilution, 10 ␮l 2× Taqman One-Step RT-PCR Master Mix 92 Reagents (Applied Biosystems, Foster City, CA, USA), 0.5 ␮l 40× 93 MultiScribe and RNase inhibitor mixture, 0.25 ␮M forward primer, 94 0.25 ␮M reverse primer, and 0.125 ␮M probe using a fluorometric 95 PCR instrument (Applied Biosystems, Foster City, CA, USA). Ther96 mal cycling parameters were 30 min at 42 ◦ C followed by 10 min at 97 95 ◦ C and a 40 cycles of amplification (95 ◦ C for 15 s and 55 ◦ C for 98 30 s); fluorescence was collected during the 55 ◦ C step. 99 Detection of live virus was assessed using allantoic fluid from 100 virus-infected chicken embryos. Ten-fold serial dilutions (from 101 approximately 128 HAUs to 1.28 × 10−3 HAUs) and two-fold serial 102 dilutions (from 1.28 × 10−3 HAU to 8 × 10−5 HAU) of virus were 103 prepared in phosphate-buffered saline (PBS). RNA from 140-␮l 104 aliquots of each dilution was extracted using TRIzol (Invitrogen, 105 Carlsbad, CA, USA) and resuspended in 60 ␮l of DEPC (diethyl 106 pyrocarbonate)-treated water. rRT-PCR was performed in 20107 ␮l reaction volumes according to the method described above. 108 Influenza A was also detected by rRT-PCR using a commercial kit, 109 as described by the manufacturer (Bgi-Gbi Biotech, Beijing, China). 110 This procedure has been published previously (Fang et al., 2010) 111 Q2 and is approved by the State Food and Drug Administration of China 112 (Fig. 1). 113 Detection of 12 other influenza viruses was assessed using 114 allantoic fluid from virus-infected chicken embryos. All virus 115 concentrations were determined using the commercial kit after 116 diluting samples to approximately 106 copies/ml (Fang et al., 117 2010). The following 18 viruses were kindly provided by the 118 Academy of Military Medical Science and National Academy for 119 the Control of Pharmaceutical and Biological Products: influenza 120 virus A/Swine/Guangdong/2/2001 (H1N1); influenza virus 121 A/Beijing/16/2009 (H1N1); influenza virus A/duck/ST/1734/03 122 (H1N1); influenza virus A/duck/ST/992/00 (H2N8); influenza virus 123 A/Beijing/30/95 (H3N2); influenza virus A/duck/ST/708/00 (H3N3); 124 influenza virus A/duck/Siberia/378/01 (H4N6); influenza virus 125 A/Beijing/01/2003 (H5N1); influenza virus A/teal/HK/w312/97 126 (H6N1); influenza virus A/duck/Taiwan/4201/99 (H7N7); 127 influenza virus A/turkey/Ontario/6118/68 (H8N4); influenza 128 virus A/Swine/Shandong/nb/2003 (H9N2); influenza virus 129 A/Oa/HA/g1/97 (H9N2); influenza virus A/duck/ST/1796/01 130 (H10N4); influenza virus A/duck/ST/834/01 (H11N8); 131 influenza virus A/duck/HK/838/80 (H12N5); influenza virus 132 A/Gull/MD/704/77 (H13N5); influenza B virus (Hongkong/5/72). 133 RNA from 140-␮l aliquots of each dilution was extracted and resus134 pended in 60 ␮l of DEPC-treated water. rRT-PCR was performed in 135 20-␮l reaction volumes according to the method described above. 136 Standard curves of diluted serially RNA versus threshold cycle 137 were generated to determine both the efficiency of the rRT-PCR 138 and the limit of detection. The assay exhibited a wide linear range, 139 beginning at 12 copies of target RNA per reaction and extend140 ing through 1.2 × 107 copies/reaction (R2 = 0.9918) for the H7 gene 141 assay. For the N9 gene assay, the linear range extended from 142 24 copies/reaction to 2.4 × 107 copies/reaction (R2 = 0.9949). The 143 78 79

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detection limit for avian influenza A/H7N9 RNA was