Journal of Medical Virology 87:18–24 (2015)

Evaluation and Verification of the Nanosphere Verigene RVþ Assay for Detection of Influenza A/B and H1/H3 Subtyping Han Jin Cho,1 Jin Woo Jang,2 Sun Young Ko,2 Sung Hyuk Choi,1 Chae Seung Lim,2* and Seong Soo A. An3 1

Department of Emergency Medicine, Korea University, Seoul, Republic of Korea Department of Laboratory Medicine, College of Medicine, Korea University, Seoul, Republic of Korea 3 College of Bionanotechnology & Gachon Medical Research Institute, Gachon University & Gil Medical Center, Seongnam-si, Gyeonggi Do, Republic of Korea 2

With the emerging risks of drug-resistant viruses and pandemic influenza, rapid and accurate detection of influenza viruses and determination of their subtypes is a crucial component of patient management. This study evaluated the performance of the Verigene respiratory virus plus nucleic acid (Verigene RVþ) test for the detection of influenza A/B and subtype determination compared it with conventional molecular methods. Nasopharyngeal swabs were collected from 228 patients with influenza-like illness (influenza A (n ¼ 67), 2009-H1N1 (n ¼ 21), influenza B (n ¼ 80), mixed A & B (n ¼ 3), mixed RSV A and influenza (n ¼ 3), and influenza-negative (n ¼ 54)). Patient samples were analyzed by Influenza A/B one-step typing (Seegene, Seoul, Korea), Seeplex RV15 ACE Detection (Seegene), Nanosphere Verigene RVþ assay (Nanosphere, Northbrrook, IL) and virus culture. Out of 228 samples, 109 (47.8%) were positive by culture, and an additional 65 (28.5%) were positive by Seeplex RV15 ACE Detection, Influenza A/B one-step typing or Nanosphere Verigene RVþ assay. In comparison tests with Seeplex RV15 ACE Detection RT-PCR, the sensitivity of the Verigene RVþ kit for detection of the influenza A, 2009H1N1, influenza B, and mixed A & B was 97.1%, 100%, 100%, and 100%, respectively. The specificity of the Verigene RVþ was 100% for all types. The concordance between Verigene RVþ and Influenza A/B one-step typing for H1, H3, H1/H3 mixed, and 2009-H1N1 was 100% (26/26), 100% (35/35), 100% (4/4), and 100% (21/21), respectively. The Verigene RVþ assay showed acceptable sensitivity and specificity for detection and subtyping of influenza viruses compared with the convenC 2014 WILEY PERIODICALS, INC. 

tional RT-PCR method. J. Med. Virol. 87:18–24, 2015. # 2014 Wiley Periodicals, Inc. KEY WORDS:

molecular; diagnostic; influenza

INTRODUCTION Despite remarkable developments in the diagnosis and prevention of influenza, timely detection and appropriate management remain challenging [Kehl and Kumar, 2009]. Influenza infection is a major cause of morbidity and mortality in both old and young age groups, especially in immuno-compromised subjects [Vallieres and Renaud, 2013; van der Vries et al., 2013]. Therefore, continuous efforts toward rapid and accurate diagnosis are necessary. Among the many types of infectious viruses, influenza viruses affect people of all ages through personto-person transmission, resulting in hundreds of thousands of hospitalizations every year [Thompson et al., 2004]. Efficient and sensitive diagnosis of influenza among hospital in-patients, emergency

Grant sponsor: Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea; Grant number: A103001. Competing interests: None declared.  Correspondence to: Chae Seung Lim, PhD, Department of Laboratory Medicine, College of Medicine, Korea University Guro Hospital, Guro 2 Dong, Guro Gu, Seoul, 152-703, Republic of Korea. E-mail: [email protected] Accepted 7 April 2014 DOI 10.1002/jmv.23970 Published online 5 May 2014 in Wiley Online Library (wileyonlinelibrary.com).

Evaluation and Verification of the Nanosphere Verigene RVþ Assay

room patients, or outpatients is important for the control and management of infection. In particular, fast and accurate tests that can be performed at reasonable cost are critical for emergency room patients in order to differentiate respiratory viruses and allow physicians to start an appropriate antiviral therapy and implement patient quarantine strategies [Ganzenmueller et al., 2010]. Furthermore, since the emergence of pandemic A/H1N1 and drug-resistant influenza viruses, changes in the paradigm of influenza management have become important [Dawood et al., 2009; Vinikoor et al., 2009]. Clinical confirmatory methods for influenza viruses include viral culture, reverse transcription polymerase chain reaction (RT-PCR), and nucleic acid tests [Kehl and Kumar, 2009; Selvaraju and Selvarangan, 2010; Cao et al., 2012]. As each method has characteristic strengths and weaknesses, the choice of the appropriate confirmatory test at each hospital depends on its clinical laboratory situation. The RT-PCR test was chosen as a reference test for this study because of its high sensitivity and specificity, but it has the drawbacks of high cost, the requirement for trained personnel, and a relatively long turnaround time [Fouchier et al., 2000; Jindal et al., 2009]. After the A/H1N1 influenza pandemic in 2009, interest in fast and inexpensive molecular detection methods has increased. As a result, several tests based on nanoparticle detection have been developed, including the semi-automated respiratory virus nucleic acid test and the fully automated respiratory virus nucleic acid test SP, Verigene respiratory virus plus nucleic acid (Verigene RVþ; Nanosphere, Northbrook, IL) [Jannetto et al., 2010; Zhao et al., 2010]. Designs based on nanoparticle detection overcome the multiplexing limitations associated with spectral overlaps between fluorescent probes in liquid phase detection systems. These tests also have the advantages of short turnaround time and enhanced sensitivity [Storhoff et al., 2004]. Among currently available tests, the Verigene respiratory virus plus nucleic acid test, Verigene RVþ (Nanosphere) detects the influenza A virus, A subtype (H1, H3 and 2009H1N1), the influenza B virus, and respiratory syncytial virus (RSV) subtype (A and B) using a special instrument for nucleic acid amplification and array hybridization. In this study, the efficiency of the Verigene RVþ test for detecting influenza A/B and their subtypes was evaluated in comparison with the Seeplex RV15 ACE detection kit and Seeplex Influenza A/B OneStep Typing set (Seegene, Seoul, South Korea). MATERIALS AND METHODS Clinical Sample Collection and Preparation To examine the performance of the Verigene RVþ test, 228 nasopharyngeal swabs were obtained from patients with influenza-like illness at the Korea University Guro Hospital, Seoul, Korea between

19

January 2008 and March 2011. All of the samples (flocked nasopharyngeal swabs) were transported in 3 ml of universal transport medium (UTM; COPAN, Murrieta, CA). Influenza-like illness was defined as the acute onset of fever and cough. Specimens were transferred within 24 hr to the laboratory, where they were cryopreserved at 70˚C prior to use. The stored samples were thawed once and tested immediately by RT-PCR. The study protocol was approved by the Human Use Ethical Committee at Korea University Guro Hospital. Informed consent was obtained from all subjects. Influenza Virus Culture All of the 228 clinical samples were cultured for influenza viruses. Virus culture was performed with Cryopreserved R-Mix culture R-Mix ReadyCells (Diagnostic HYBRIDS, Athens, OH) as previously reported [Kim et al., 2008]. Seeplex RV15 ACE Detection RT-PCR Initially, the commercial multiplex RT-PCR assay, Seeplex RV15 ACE Detection (Seegene), was used to confirm the presence or absence of influenza A and B viruses. Viral RNA was extracted with the NucliSENS easyMAG apparatus (bioMerieux, Durham, Netherlands), and reverse transcription was performed with a RevertAid First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania), according to the respective manufacturer’s instructions. PCR was performed using the Seeplex RV15 ACE detection kit as instructed by the manufacturer. The completed reaction was analyzed with the Tape Station platform (Lab901, Edinburgh, UK) through a software interpretation module. The results were analyzed qualitatively and semi-quantitatively according to the migration rate and band intensity of each species. A peak intensity greater than 40 was considered a positive identification. Influenza A/B OneStep Typing Another multiplex RT-PCR was conducted using the Seeplex Influenza A/B One Step Typing set (Seegene). The Seeplex Influenza A/B One Step Typing set is capable of detecting influenza A, influenza B, and three subtypes of influenza A (H1, H3, and H1N1-2009). Briefly, RT and PCR amplifications were performed on the GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA) in a total volume of 50 ml containing RNA (10 ml), 5 Flu A/B OneStep Typing PM (10 ml), 5 OneStep RT-PCR Buffer (10 ml), 8-Mop solution (16 ml), dNTP Mix (2 ml), and OneStep RT-PCR Enzyme Mix (2 ml), in accordance with the manufacturer’s instructions. The PCR protocol included the following steps: initial incubation at 50˚C for 30 min and 95˚C for 15 min, followed by 45 cycles at 94˚C for 30 sec, 60˚C for 90 sec, and 72˚C for 60 sec. The completed reaction J. Med. Virol. DOI 10.1002/jmv

Cho et al.

J. Med. Virol. DOI 10.1002/jmv

3 17.0  12.4 years 1–34 years 2:1 3 8.3  5.4 years 5–16 years 1:2 80 27.1  22.7 years 6 months–72 years 49:31 21 20.7  10.8 years 3–40 years 12:9

RSVA & Influenza A/B Influenza A & B Influenza B

228 36.3  38.2 years 4 months–87 years 146:82

For the samples positive for seasonal influenza A, the sensitivity of the Verigene RVþ test and Influenza A/B OneStep Typing was 97.1% (n ¼ 65/67, 95% CI 88.7–99.5%) and 98.5% (n ¼ 66/67, 95% CI 90.9– 99.9%), respectively. For samples positive for 2009H1N1 only, the sensitivity of both the Verigene RVþ test and Influenza A/B OneStep Typing was 100% (n ¼ 21/21, 95% CI 80.8–100%). For samples positive for influenza B, the sensitivity of the Verigene RVþ test and Influenza A/B OneStep Typing was 100%

No. of subjects Age (mean  SD) Age range Male/female

Sensitivities and Specificities of the Influenza RT-PCR Assays for Clinical Specimens

2009-H1N1

The distributions of the age and gender of patients included in this study are shown in Table I. The age of the patients ranged from 4 months to 87 years (mean  SD 36.3  38.2 years). Of the 228 nasopharyngeal samples examined for respiratory viruses, 174 (76.3%) were positive by any of the influenza virus assays: 38.6% (88/228) were positive for influenza A virus and 35.1% (80/228) were positive for influenza B virus. Three cases (1.3%, 3/228) were coinfected with influenza A and B viruses or with RSV A and influenza, and 54 (23.7%) cases were negative. Of the 93 samples positive for influenza A, 90 (96.8%), 88 (94.6%), and 52 (55.3%) were positive by the Verigene RVþ test, Influenza A/B OneStep Typing, and viral culture, respectively. Of the 84 samples positive for influenza B, 83(98.8%), 79 (94.0%), and 57 (67.8%) were positive by the Verigene RVþ test, Influenza A/B OneStep Typing, and viral culture, respectively.

Seasonal

RESULTS

Total

The main purpose of this study was to evaluate the sensitivity and specificity of the Verigene RVþ test, compared with the Seeplex RV15 ACE Detection kit and Seeplex Influenza A/B One Step Typing set as reference standards. The performance parameters are expressed as 95% confidence intervals (CI). All analyses were performed using SPSS (version 20.0; SPSS, Chicago, IL).

Characteristics

Data Analysis

Influenza A

The Verigene RVþ test (Nanosphere) was developed as a fully automated system for detecting influenza A (H1, H3), B and A/H1N1 (2009). Nucleic acid extraction, multiplex RT-PCR amplification, and microarray hybridization were performed using a Verigene Processor SP with 50 ml of nasopharyngeal swab specimen. Microarrays were then transferred to a Verigene Reader for data analysis and reporting results. All procedures were carried out according to the manufacturer’s instructions.

TABLE I. Characteristics of Subjects With Influenza-Like Symptoms Confirmed By Seeplex RV15 ACE Detection

Verigene RVþ Test

67 28.8  26.8 years 6 months–72 years 38:29

Negative

was analyzed using the Tape Station platform (Lab901).

54 38.1  30.0 years 4 months–87 years 30:24

20

100% (1/1) 50.0% (1/2) 68.8% (55/80) 47.6% (10/21)

33.3% (1/3)

100% (1/1) Not available 50.0% (1/2) Not available 100% (3/3) 33.3% (1/3) 100% (80/80) 95.5% (78/80) 100% (21/21) 100% (21/21)

Seeplex RV15 detection Verigene RVþ 97.1% (65/67) Influenza A/B 98.5% (66/67) OneStep Typing Virus culture 59.7% (40/67)

RSV Aþ Influenza B (n ¼ 1) RSV Aþ Influenza A (n ¼ 2) Mixed A & B (n ¼ 3) Influenza B (n ¼ 80) 2009-H1N1 (n ¼ 21) Seasonal (n ¼ 67)

Given that it can be difficult to differentiate viral and bacterial infections, early and accurate detection of the influenza virus would reduce unnecessary administration of antibiotics. In addition, hospital stays of affected patients could be shortened, and early isolation could also prevent nosocomial transmission of the influenza virus [Garbino et al., 2004; Lee et al., 2006; Mahony, 2008]. In outpatient and emergency departments it is especially important to obtain results within minutes using rapid detection methods. Several products that satisfy such criteria are available, but they demonstrate a wide range of sensitivities [Hurt et al., 2007; Glezen, 2008; Cho et al., 2013]. When the novel 2009H1N1 strain was spreading at pandemic levels, the rapid antigen test did not function well as a screening test in clinical settings because of its low sensitivity. As a result, many hospitals experienced an extremely high laboratory workload [CDC, 2009; Ginocchio et al., 2009; Crawford et al., 2010]. During the 2009 influenza pandemic, over-administration of antiviral agents to patients with suspected infections resulted in a shortage of stocked drugs worldwide, and occasionally caused side effects in some patients. As a result, interest in fast, accurate, and inexpensive methods for detecting the highly infective influenza virus has increased. In this study, the Seeplex RV15 ACE Detection kit, Verigene RVþ test, and viral culture on the same specimens were used. The Seeplex RV15 ACE Detection test and Verigene RVþ test showed good sensitivity (96.8–100%) for the detection of influenza A or B, whereas viral culture showed low sensitivity (55.3–67.8%). The two molecular assays detected influenza pathogens from 65 (28.5%) samples that

Test (n ¼ 228)

DISCUSSION

Influenza A (n ¼ 88)

Overall concordance rates between the Verigene RVþ and Influenza A/B OneStep Typing for influenza A subtypes were calculated using 86 influenza Apositive samples (Table III). For the H1, 2009-H1N1, H3, and mixed H1 & H3 subtypes, the concordance rate between Influenza A/B OneStep Typing and Verigene RVþ was 100% (n ¼ 26/26, 95% CI 84.0– 100%), 100% (n ¼ 21/21, 95% CI 80.7–100%), 100% (n ¼ 35/35, 95% CI 87.7–100%), and 100% (n ¼ 4/4, 95% CI 51.0–100%), respectively.

TABLE II. Evaluation of the Verigene RVþ Test and RT-PCR in Comparison With the Reference Method

Comparison Between the Verigene RVþ and Influenza A/B OneStep Typing for Influenza A Subtyping

Negative (n ¼ 54)

(n ¼ 80/80, 95% CI 95.4–100%) and 95.5% (n ¼ 78/80, 95% CI 91.3–99.3%), respectively. The specificity of both the Verigene RVþ and Influenza A/B OneStep Typing was 100% (n ¼ 54/54, 95% CI 91.7–100%). Therefore, in comparison with conventional PCR and Influenza A/B OneStep typing, the Verigene RVþ test demonstrated acceptable sensitivity and specificity (Table II).

100% (54/54)

21 100% (54/54) 100% (54/54)

Evaluation and Verification of the Nanosphere Verigene RVþ Assay

J. Med. Virol. DOI 10.1002/jmv

22

Cho et al.

TABLE III. Agreement of Influenza A (n ¼ 86) Subtyping Between Verigene RVþ Test and Influenza A/B OneStep Typing Influenza A/B OneStep Typing

Verigene RVþ H1 2009-H1N1 H3 H1 & H3 mixed Total

H1

2009-H1N1

H3

H1 & H3 mixed

Total

26 0 0 0 26

0 21 0 0 21

0 0 35 0 35

0 0 0 4 4

26 21 35 4 86

were negative for virus culture. The virus cultures therefore showed lower detection rates for influenza than the RT-PCR. Because the influenza virus is an RNA virus, the nucleic acid testing methods included RT-PCR, realtime RT-PCR, and nucleic acid sequence-based amplification. These techniques are gold-standard methods in many laboratories because of their high sensitivity and specificity [Spackman et al., 2002]. Nevertheless, they are expensive, require trained personnel for nucleic acid extraction, and take 6–8 hr to obtain results. The Verigene RVþ test includes Food and Drug Administration (FDA)-cleared random-access molecular test equipment, which is based on nanoparticle technology without the requirement for preextraction of samples [Alby et al., 2013]. In this study, the Verigene RVþ test was directly compared with conventional PCR and the Influenza A/B OneStep Typing set. The Verigene RVþ test equipment revealed excellent concordance with the reference test, Seeplex RV15 ACE Detection. As reported in the study by Alby et al. [2013], the Verigene RVþ test produced results with approximately 5 min of handson time for each sample and took about 2.5 hr in total to produce the final results [Spackman et al., 2002]. Considering the relatively short period needed to obtain accurate results, the Verigene RVþ test can be used in both the emergency and outpatient departments. However, one drawback is that only one sample could be tested in each module. In addition, this method was too expensive to be used as a single test: each test costs $70 in the USA and its cost in Republic of Korea has not been determined. Recently, several molecular sample-to-result systems that are almost fully automated and produce results relatively rapidly (1–2.5 hr) have been introduced [Jenny et al., 2010; Loeffelholz et al., 2011; Salez et al., 2012; CDC, 2013]. Molecular sample-toresult tests such as the Cepheid Xpert Flu Assay (Cepheid, Sunnyvale, CA), Idaho Technology FilmArray Assay (Idaho Technology, Salt Lake City, UT) and Simplexa Flu A/B & RSV kit assay (Focus Diagnostics, Cypress, CA) have demonstrated high sensitivities and specificities for the detection of influenza (over 90% for each test) compared with real-time RT-PCR [Sambol et al., 2010; Popowitch et al., 2011; Pierce et al., 2012]. In addition, the Simplexa Flu A/B & RSV kit demonstrated potential J. Med. Virol. DOI 10.1002/jmv

for a higher throughput and the FilmArray Respiratory Panel was able to identify 20 respiratory viruses. Using the Cepheid Xpert Flu Assay, the time to the final result was approximately 60 min with less than 5 min of hands-on time. The Verigene RVþ test has certain advantages such as decreased turnaround time, ability to determine the subtypes of RSV and influenza A viruses, and detection of oseltamivir resistance in seasonal influenza H1N1 and influenza H1N1 (2009) [Van Wesenbeeck et al., 2013]. Although, the Luminex xTAG respiratory viral panel (Luminex, Houston, TX) determines influenza A subtypes (H1, H3), it has a relatively long turnaround time and low sensitivity for detecting the influenza B virus (41.3%) [Pabbaraju et al., 2008]. The dominant influenza A virus subtype during the 2009–2010 influenza season was novel 2009-H1N1; A/H1N1 and A/H3N2 dominated the 2011 and 2012 influenza seasons. The co-circulation of the pandemic influenza A/H1N1 virus with seasonal influenza A/H1N1, A/H3N2, and B viruses, made subtyping particularly important because antiviral susceptibilities differ according to influenza virus subtype. Most seasonal influenza A/H1N1 viruses were resistant to oseltamivir, and susceptible to zanamivir (100%) and the adamantanes (99.4%) [Fiore et al., 2011], whereas influenza A/H3N2 and novel 2009-H1N1 viruses were susceptible to neuraminidase inhibitors (100%) and resistant to adamantanes (100%). Therefore, the USA Centers for Disease Control (Atlanta, GA) recommended empirical treatment with either zanamivir alone or a combination therapy of oseltamivir and adamantine unless results for the influenza A virus subtypes were available [Fiore et al., 2011]. From 2009 to 2010, the Verigene RVþ test subtyped 21 of 21 specimens as novel 2009-H1N1 with a sensitivity of 100%. In our study, 1 influenza A/H1N1 specimen was reported as negative for A/H1 by the Verigene RVþ test and all 35 influenza A/H3N2 virus specimens were detected accurately by the Verigene RVþ test. The overall sensitivity of Verigene RVþ for subtyping the influenza A/H3N2 virus was 98.9%. This result suggests that the Verigene RVþ test could be used as the primary assay for subtyping the influenza virus. In a recent report comparing FDA-cleared molecular multiplex platforms, the Verigene RVþ test

Evaluation and Verification of the Nanosphere Verigene RVþ Assay

demonstrated a high percentage of invalid results (15.8%, 27/171) [Van Wesenbeeck et al., 2013]. In addition, Verigene RVþ showed relatively poor performance in influenza virus detection and subtyping compared with Prodesses ProFLUþ, Prodesses ProFASTþ (GeneProbe, San Diego, CA), and FilmArray respiratory panel (BioFire Diagnostics, Salt Lake City, UT) [Van Wesenbeeck et al., 2013]. However, the current study showed a very low rate of invalid results (2.3%) for Verigene RVþ, and showed good performance for both influenza virus detection and subtyping, compared with Seeplex RV15 ACE Detection. These discrepant results could be caused by differences in the distribution of influenza subtypes and criteria used for sample collection; in the previous report most of the samples constituted influenza A virus, subtype H3 [Van Wesenbeeck et al., 2013]. In summary, the Verigene RVþ test demonstrated similar sensitivity and specificity to the Seeplex RV15 ACE Detection test for the detection of influenza viruses. The Verigene RVþ test also showed good agreement with the Influenza A/B OneStep Typing for subtyping influenza viruses. Our data indicate that the Verigene RVþ test would be suitable for the detection and subtyping of influenza viruses. ACKNOWLEDGMENTS We thank Chi Hyun Cho, Jong Han Lee, Young Duck Cho, and Woo Joo Kim for their helpful comments in analysis of data and editing the manuscript. REFERENCES Alby K, Popowitch EB, Miller MB. 2013. Comparative evaluation of the Verigene RVþ assay and the Simplexa Flu A/B & RSV kit for detection of influenza and respiratory syncytial viruses. J Clin Microbiol 51:352–353. Cao Q, Mahalanabis M, Chang J, Carey B, Hsieh C, Stanley A, Odell CA, Mitchell P, Feldman J, Pollock NR, Klapperich CM. 2012. Microfluidic chip for molecular amplification of influenza A RNA in human respiratory specimens. PLoS ONE 7:e33176. Centers for Disease Control and Prevention (CDC). 2009. Evaluation of rapid influenza diagnostic tests for detection of novel influenza A (H1N1) Virus—United States, 2009. MMWR Morb Mortal Wkly Rep 58:826–829. Centers for Disease Control and Prevention (CDC). 2013. Rapid diagnostic testing for influenza. http://www.cdc.gov/flu/professionals/diagnosis/rapidlab.htm [Accessed 10 September 2013]. Cho CH, Woo MK, Kim JY, Cheong S, Lee CK, An SA, Lim CS, Kim WJ. 2013. Evaluation of five rapid diagnostic kits for influenza A/B virus. J Virol Methods 187:51–56. Crawford JM, Stallone R, Zhang F, Gerolimatos M, Korologos DD, Sweetapple C, de Geronimo M, Dlugacz Y, Armellino DM, Ginocchio CC. 2010. Laboratory surge response to pandemic (H1N1) 2009 outbreak, New York City metropolitan area, USA. Emerg Infect Dis 16:8–13. Dawood FS, Jain S, Finelli L, Shaw MW, Lindstrom S, Garten RJ, Gubareva LV, Xu X, Bridges CB, Uyeki TM. 2009. Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N Engl J Med 360:2605–2615. Fiore AE, Fry A, Shay D, Gubareva L, Bresee JS, Uyeki TM. 2011. Antiviral agents for the treatment and chemoprophylaxis of influenza recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 60:1–24. Fouchier RA, Bestebroer TM, Herfst S, Van Der Kemp L, Rimmelzwaan GF, Osterhaus AD. 2000. Detection of influenza A viruses from different species by PCR amplification of conserved sequences in the matrix gene. J Clin Microbiol 38:4096–4101.

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H3 subtyping.

With the emerging risks of drug-resistant viruses and pandemic influenza, rapid and accurate detection of influenza viruses and determination of their...
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