Ikuyo Takayama

Rapid discrimination of oseltamivir-resistant 275Y and -susceptible 275H substitutions in the neuraminidase gene of pandemic influenza A/H1N1 2009 virus by duplex one-step RT-PCR assay

Pandemic influenza A/H1N1 2009 (A/H1N1pdm) virus caused significant outbreaks worldwide last year (2009). A number of oseltamivir‐resistant A/H1N1pdm viruses possessing an H275Y substitution in the neuraminidase (NA) protein were reported sporadically in several countries, including Japan, but they were sensitive to zanamivir and did not spread in the community. In this study, to monitor rapidly and simply oseltamivir‐resistant A/H1N1pdm viruses possessing H275Y, a duplex one‐step RT‐PCR assay (H275Y RT‐PCR assay) was developed based on an endpoint genotyping analysis method. H275Y RT‐PCR assay evaluated using several subtypes/types of influenza A and B viruses and other respiratory pathogenic viruses and shown to have high sensitivity and high specificity. Forty‐four clinical specimens were tested after RNA purification using the H275Y RT‐PCR assay, resulting in one clinical specimen being found to contain a virus possessing the H275Y mutation. Seventy‐three clinical isolates were then tested with the H275Y assay by using clinical isolates in the cultured supernatants of cells directly, without RNA purification, and the results were consistent with the NA sequencing. Since the H275Y RT‐PCR assay could detect the H275Y mutation in clinical isolates without RNA purification, as well as a H275Y mutated virus in clinical specimens after RNA purification, the assay was considered a powerful tool for surveillance screening of oseltamivir‐resistant A/H1N1pdm virus activity. J. Med. Virol. 83:1121–1127, 2011.

Development of a reverse transcription loop-mediated isothermal amplification assay for the rapid diagnosis of avian influenza A (H7N9) virus infection

A genetic diagnosis system for detecting avian influenza A (H7N9) virus infection using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) technology was developed. The RT-LAMP assay showed no cross-reactivity with seasonal influenza A (H3N2 and H1N1pdm09) or influenza B viruses circulating in humans or with avian influenza A (H5N1) viruses. The sensitivity of the RT-LAMP assay was 42.47 copies/reaction. Considering the high specificity and sensitivity of the assay for detecting the avian influenza A (H7N9) virus and that the reaction was completed within 30 min, the RT-LAMP assay developed in this study is a promising rapid diagnostic tool for avian influenza A (H7N9) virus infection.

Real-time RT-PCR assays for discriminating influenza B virus Yamagata and Victoria lineages

Abstract Since the late 1980s, two genetically and antigenically distinct lineages of influenza B virus, namely, B/Victoria/2/87-like (B/Victoria) and B/Yamagata/16/88-like (B/Yamagata), have co-circulated. In this study, one-step real-time reverse transcription-PCR (rRT-PCR) assays were developed to differentiate B/Victoria and B/Yamagata lineages. The assays were evaluated using in vitro transcribed control RNA, isolated viruses, and other respiratory pathogenic viruses, and were shown to have high sensitivity, good linearity (R 2 =0.99), and high specificity. Using the developed rRT-PCR assays, 169 clinical specimens collected between 2010 and 2013 were then tested, resulting in the identification of 20 clinical specimens as positive for influenza B virus. Of these, 14 and 6 samples were identified as positive for the B/Victoria and B/Yamagata lineages, respectively, whereas 149 samples were negative for the influenza B virus. The rRT-PCR assays were also examined using 20 clinical isolates from 20 influenza B virus-positive specimens, revealing that there was no discrepancy between the results from the rRT-PCR assays and the hemagglutination inhibition (HI) test, with the exception that one clinical isolate with different antigenicity could not be discriminated by the HI test. The present results suggest that these highly sensitive and specific assays are useful not only for diagnosing influenza viruses but also for their surveillance.

Rapid detection of the S247N neuraminidase mutation in influenza A(H1N1)pdm09 virus by one-step duplex RT-PCR assay

Neuraminidase inhibitors, especially oseltamivir, have been used worldwide for the prophylaxis or treatment of pandemic influenza H1N1 2009 (A(H1N1)pdm09) viruses. The most commonly detected oseltamivir-resistant A(H1N1)pdm09 viruses possess the H275Y mutation in neuraminidase (NA). Recently, a novel A(H1N1)pdm09 variant containing the S247N mutation in NA was found mainly in the Asia-Pacific area. This mutation reduces sensitivity to oseltamivir and confers extremely high resistance to oseltamivir in association with the H275Y mutation compared with the resistance caused by the H275Y mutation alone. In this study, a rapid and simple one-step duplex RT-PCR assay for identifying A(H1N1)pdm09 viruses possessing the S247N mutation was developed. This assay is based on an endpoint genotyping analysis method and can use isolates from cell culture supernatants without RNA extraction, similar to the H275Y RT-PCR assay reported previously. The combination of the S247N and H275Y RT-PCR assays is a powerful surveillance method for determining whether A(H1N1)pdm09 viruses with the S247N mutation acquire the H275Y mutation.

Novel Reassortant Influenza A(H1N2) Virus Derived from A(H1N1)pdm09 Virus Isolated from Swine, Japan, 2012

We isolated a novel influenza virus A(H1N2) strain from a pig on January 13, 2012, in Gunma Prefecture, Japan. Phylogenetic analysis showed that the strain was a novel type of double-reassortant virus derived from the swine influenza virus strains H1N1pdm09 and H1N2, which were prevalent in Gunma at that time.

TMPRSS2 Independency for Haemagglutinin Cleavage In Vivo Differentiates Influenza B Virus from Influenza A Virus

Influenza A and B viruses show clear differences in their host specificity and pandemic potential. Recent studies have revealed that the host protease TMPRSS2 plays an essential role for proteolytic activation of H1, H3, and H7 subtype strains of influenza A virus (IAV) in vivo. IAV possessing a monobasic cleavage site in the haemagglutinin (HA) protein replicates poorly in TMPRSS2 knockout mice owing to insufficient HA cleavage. In the present study, human isolates of influenza B virus (IBV) strains and a mouse-adapted IBV strain were analysed. The data showed that IBV successfully underwent HA cleavage in TMPRSS2 knockout mice, and that the mouse-adapted strain was fully pathogenic to these mice. The present data demonstrate a clear difference between IAV and IBV in their molecular mechanisms for spreading in vivo.

Development of a Diagnostic System for Novel Influenza A(H7N9) Virus Using a Real-Time RT-PCR Assay in Japan

The first human cases of infection with avian influenza A(H7N9) virus were reported in March 2013 in China. The number of confirmed cases continues to increase, although almost all the cases are limited to China. In this study, a one-step real-time RT-PCR assay was developed for detecting the novel A(H7N9) virus. This assay was shown to have high specificity, good linearity, and high sensitivity to a broad range of Eurasian H7 viruses. The assay is useful both for diagnostic purposes in cases of suspected human infection with the influenza A(H7N9) virus and in the surveillance of both avian and human influenza viruses. A diagnostic system using this assay was prepared at 74 prefectural and municipal public health institutes and 16 quarantine stations in Japan early into the human H7N9 infection outbreaks, enabling potential diagnoses of H7N9 infection across Japan.

Novel Reassortant Avian Influenza A(H5N1) Virus in Human, Southern Vietnam, 2014

To the Editor: The first case of human infection with highly pathogenic avian influenza A(H5N1) virus in Vietnam was reported in December 2003 (1), and >120 human cases were confirmed through 2013, with a high case-fatality rate (2). In 2013, clade 2.3.2.1a/c H5N1 viruses circulated widely in poultry across the country, although clade 1.1.1/1.1.2 H5N1 viruses predominated in poultry from the Mekong Delta region to central Vietnam (3,4). In 2014, two cases of human infection with A(H5N1) virus were identified in southern Vietnam. One case was associated with a clade 1.1.2 reassortant virus, A/Vietnam/14012902/2014 (Global Initiative on Sharing All Influenza Data [GISAID; http://www.gisaid.org] accession nos. EPI624919–EPI624926), which had been previously detected in Cambodia and Vietnam (5,6). We isolated the virus from the other case, performed phylogenetic analysis to identify the clade of this virus, and identified a novel virus that had undergone gene reassortment. The case-patient was a 52-year-old man who lived in Binh Phuoc Province (140 km northeast of Ho Chi Minh City). On January 11, 2014, he experienced mild fever and general fatigue; high fever developed on January 13. He was hospitalized with dyspnea on January 16 and died 2 days later. He was not given antiviral drug treatment. Dead poultry infected with H5N1 viruses were found scattered near his house during January 1–16, and he buried his 2 dead chickens on January 5. H5N1 virus infection was detected in the patient’s throat swab specimen by real-time reverse transcription PCR at the Pasteur Institute in Ho Chi Minh City. Virus was isolated by inoculating the throat swab specimens into 10-day-old embryonated chicken eggs; the resulting isolate, A/Vietnam/14011801/2014 (GISAID accession nos. EPI624911–EPI624918), then underwent gene sequencing. The 8 viral genes were amplified with SuperScript III Reverse Transcriptase Kit (Fisher Scientific, Pittsburg, PA, USA) and Phusion High-Fidelity DNA Polymerase (New England BioLabs, Ipswich, MA, USA) with specific paired primers, according to the manufacturer’s instructions, and sequenced on an ABI 3730 automated sequencer with Big-Dye Terminator Cycle Sequencing reagents (Applied Biosystems, Foster City, CA, USA). Whole genome sequence was determined. By gene sequencing analysis, A/Vietnam/14011801/2014 was found to have the multibasic cleavage site of hemagglutinin (HA) protein, which indicates highly pathogenic avian influenza A(H5N1) viruses, and was shown to predict binding specificity to an avian α2,3 sialic acid receptor. The neuraminidase gene possessed no amino acid substitutions associated with decreased antiviral activity, nor did the virus have amino acid substitutions associated with increased adaptation, virulence, infectivity, or transmissibility in mammalian hosts, including the E627K and D701N mutations in polymerase basic protein 2 (7). Phylogenetic analyses of the 8 viral genes of A/Vietnam/14011801/2014 were performed by using databases (GISAID and the Influenza Virus Resource, National Center for Biotechnology Information, Bethesda, MD, USA; http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html) that contained complete sequences of viral genomes belonging to clades 1.1.1, 1.1.2, and 2.3.2.1 a/b/c, most of which were collected in Vietnam, particularly after 2012. Neighbor-joining and Kimura 2-parameter methods were implemented by using MEGA version 5.0 software (http://www.megasoftware.net). Reliability of the phylogenetic analysis was tested by using 1,000 bootstrap replications. Lineages of the HA gene were defined by using previously described criteria (8). Lineages of the other 7 genes were defined by using criteria and nomenclature of Nguyen et al. (9). The HA of A/Vietnam/14011801/2014 belonged to clade 2.3.2.1c (online Technical Appendix Figure, panel A, http://wwwnc.cdc.gov/EID/article/23/3/15-1360-Techapp.pdf). The neuraminidase, polymerase basic proteins 1 and 2, and polymerase acid protein genes of this virus were also derived from respective lineages of ancestor clade 2.3.2.1c (Technical Appendix Figure, panels B–E). However, nucleoprotein, matrix, and nonstructural genes were classified as lineages of ancestor clade 2.3.2.1a (online Technical Appendix Figure, panels F–H) and differed from the gene lineages of almost all clade 2.3.2.1c viruses isolated from poultry in Vietnam. As reported in the Influenza Virus Resource, 2 viruses collected in Vietnam in December 2013 (A/muscovy duck/Long An/43/2013 and A/muscovy duck/Long An/46/2013) were similar reassortant viruses of clade 2.3.2.1c (Figure). However, the ancestor of the nonstructural gene lineage of these 2 viruses is clade 2.3.2.1c, which differs from A/Vietnam/14011801/2014. The differences indicate that A/Vietnam/14011801/2014 is a novel reassortant virus between clades 2.3.2.1a and 2.3.2.1c, between clades 1.1.2 and 2.3.2.1c, or both (Figure). This novel reassortant virus has not been reported in poultry in Vietnam, although novel reassortants between clade 1.1.2 and clade 2.3.2.1a viruses have been detected in Vietnam since 2013 (i.e., A/Vietnam/VP13-28H/2013, GISAID accession nos. EPI624927–EPI624934; and A/Vietnam/14012902/2014) (6). These novel reassortment viruses were first identified in human, animal, and environmental samples in Cambodia in 2013 (5). Other novel gene reassortments in clade 2.3.2.1 viruses have been previously reported (10), and new clade 2.3.4.4 viruses have been observed in Vietnam since 2014. Figure Novel reassortant virus (A/Vietnam/14011801/2014) identified in a human case of influenza A(H5N1) virus infection in Vietnam, 2014. Ancestry of genes is denoted in the hemagglutinin clades. Arrows indicate genes that differ from the gene lineages of original ... As multiple clade viruses co-circulate, reassortment events occur frequently in Vietnam. Continuous surveillance of avian influenza A(H5N1) viruses, not only in humans but also in poultry and wild birds, is needed for infection control measures during epidemics of these viruses. Technical Appendix. Phylogenetic analyses of the genes of the avian influenza A(H5N1) virus identified in a human in Vietnam, 2014. Click here to view.(1.0M, pdf)

Characterization of influenza A(H1N1)pdm09 viruses isolated from Nepalese and Indian outbreak patients in early 2015

We characterized influenza A(H1N1)pdm09 isolates from large‐scale outbreaks that occurred in Nepal and India in early 2015. Although no specific viral features, which may have caused the outbreaks, were identified, an S84N substitution in hemagglutinin was frequently observed. Chronological phylogenetic analysis revealed that these Nepalese and Indian viruses possessing the S84N substitution constitute potential ancestors of the novel genetic subclade 6B.1 virus that spread globally in the following (2015/16) influenza season. Thus, active surveillance of circulating influenza viruses in the Southern Asia region, including Nepal and India, would be beneficial for detecting novel variant viruses prior to their worldwide spread.

Development of fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) using quenching probes for the detection of the Middle East respiratory syndrome coronavirus

Abstract Clinical detection of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) in patients is achieved using genetic diagnostic methods, such as real-time RT-PCR assay. Previously, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of MERS-CoV [Virol J. 2014. 11:139]. Generally, amplification of RT-LAMP is monitored by the turbidity induced by precipitation of magnesium pyrophosphate with newly synthesized DNA. However, this mechanism cannot completely exclude the possibility of unexpected reactions. Therefore, in this study, fluorescent RT-LAMP assays using quenching probes (QProbes) were developed specifically to monitor only primer-derived signals. Two primer sets (targeting nucleocapsid and ORF1a sequences) were constructed to confirm MERS cases by RT-LAMP assay only. Our data indicate that both primer sets were capable of detecting MERS-CoV RNA to the same level as existing genetic diagnostic methods, and that both were highly specific with no cross-reactivity observed with other respiratory viruses. These primer sets were highly efficient in amplifying target sequences derived from different MERS-CoV strains, including camel MERS-CoV. In addition, the detection efficacy of QProbe RT-LAMP was comparable to that of real-time RT-PCR assay using clinical specimens from patients in Saudi Arabia. Altogether, these results indicate that QProbe RT-LAMP assays described here can be used as powerful diagnostic tools for rapid detection and surveillance of MERS-CoV infections.