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Characterization of neuraminidase inhibitor‐resistant influenza A(H1N1)pdm09 viruses isolated in four seasons during pandemic and post‐pandemic periods in Japan

Background/Objectives Japan has the highest frequency of neuraminidase (NA) inhibitor use against influenza in the world. Therefore, Japan could be at high risk of the emergence and spread of NA inhibitor‐resistant viruses. The aim of this study was to monitor the emergence of NA inhibitor‐resistant viruses and the possibility of human‐to‐human transmission during four influenza seasons in Japan. Methods To monitor antiviral‐resistant A(H1N1)pdm09 viruses, we examined viruses isolated in four seasons from the 2008–2009 season through the 2011–2012 season in Japan by allelic discrimination, NA gene sequencing, and NA inhibitor susceptibility. Results We found that 157 (1·3%) of 12 026 A(H1N1)pdm09 isolates possessed an H275Y substitution in the NA protein that confers about 400‐ and 140‐fold decreased susceptibility to oseltamivir and peramivir, respectively, compared with 275H wild‐type viruses. The detection rate of resistant viruses increased from 1·0% during the pandemic period to 2·0% during the post‐pandemic period. The highest detection rate of the resistant viruses was found in patients who were 0–9 years old. Furthermore, among the cases with resistant viruses, the percentage of no known exposure to antiviral drugs increased from 16% during the pandemic period to 44% during the post‐pandemic period, implying that suspected human‐to‐human transmission of the resistant viruses gradually increased in the post‐pandemic period. Conclusions A(H1N1)pdm09 viruses resistant to oseltamivir and peramivir were sporadically detected in Japan, but they did not spread throughout the community. No viruses resistant to zanamivir and laninamivir were detected.


Introduction
During the 2007-2008 influenza season, oseltamivir-resistant former seasonal A(H1N1) viruses emerged in Europe 1 and became the majority of A(H1N1) viruses within a year. These oseltamivir-resistant A(H1N1) viruses possessed a mutation causing a histidine-to-tyrosine substitution at amino acid position 275 (N1 numbering, H275Y) in the neuraminidase (NA) protein. The NA of these viruses had a slightly higher activity and affinity for the substrate than that from previously circulating sensitive viruses. 2 Furthermore, some potentially permissive substitutions in the NA and the hemagglutinin (HA) protein in addition to the H275Y substitution were reported, 3,4 so that oseltamivir-resistant A (H1N1) viruses may acquire the capacity for efficient human-to-human transmission. The global spread of oseltamivir-resistant A(H1N1) viruses was obliged to change the chemotherapeutic strategy and limited the therapeutic options for influenza. Consequently, the worldwide monitoring of antiviral-resistant viruses is important for therapeutic and public health measures. In particular, in Japan, four NA inhibitors, oseltamivir, peramivir, zanamivir, and laninamivir, are approved for chemotherapy against influenza and are prescribed with the highest frequency in the world. 5 Therefore, Japan is at high risk of the emergence of NA inhibitor-resistant viruses. Because updated surveillance information on antiviral-resistant viruses from Japan is desired, year-round surveillance for influenza antiviral resistance has been conducted through the National Institute of Infectious Diseases, in cooperation with 74 local public health institutes in Japan.
We have reported oseltamivir-resistant A(H1N1)pdm09 viruses through a preliminary surveillance of NA inhibitorresistant viruses during 2009-2010. 6 In the present study, we screened more than 12 000 A(H1N1)pdm09 viruses isolated during four influenza seasons, from the 2008-2009 season through the 2011-2012 season, to monitor NA inhibitorresistant viruses, and we detected 157 (1Á3%) H275Y mutant viruses exhibiting cross-resistance to oseltamivir and peramivir. We describe here the virological characteristics of the oseltamivir-and peramivir-resistant A(H1N1)pdm09 viruses, the prescription history of patients infected with these resistant viruses and clusters of resistant virus infection.

Viruses
Clinical specimens and the corresponding patient records were collected in 500 sentinel hospitals consisting of pediatrics and internal medicine between May 2009 and August 2012 as part of the National Epidemiological Surveillance of Infectious Diseases in Japan. Most specimens were collected at the first medical examination. Influenza A(H1N1)pdm09 viruses were isolated from the specimens in 74 local public health institutes using Madin-Darby canine kidney (MDCK) or human colon carcinoma (Caco-2) cells. About three isolates per week were used for allelic discrimination assay and/or partial NA gene sequencing in the local public health institutes. Approximately 5-10% of total isolates were propagated in MDCK cells and used for NA inhibition assay and/or full-length NA and HA gene sequencing in the National Institute of Infectious Diseases.

Allelic discrimination assay
To detect the H275Y substitution in NA, the allelic discrimination assay was performed as previously described. 7 Briefly, supernatants of A(H1N1)pdm09 virus-infected cells were directly added to the one-step duplex RT-PCR mixture without RNA purification, and the reaction was performed using the QuantiTect Virus kit (Qiagen, Dusseldorf, Germany). Fluorescent signals were collected during the annealing and extension steps, and amplification data and endpoint data were analyzed. Detection sensitivity of the allelic discrimination assay is 7Á5 copies/reaction 7 , and the assay detects a mixed population of H275Y mutant viruses and 275H wild-type viruses. We considered all mixed population as H275Y.

Sequence analysis
Viral RNA was prepared using the QIAamp Viral RNA kit (Qiagen). To screen for the H275Y substitution in NA, the partial NA gene between 676 and 1,111 (from the start codon) was amplified from the viral RNA as previously described. 7 The full-length NA gene was amplified from viral RNA using a plus-sense primer N1-F1 and a minus-sense primer N1-R1 (Table 1). The HA1 domain of the HA gene was amplified using a plus-sense primer H1HA1-BEGIN and a minus-sense primer H1-1106-1087R (Table 1). Nucleotide sequences were determined using the BigDye Terminator v3Á1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) with Applied Biosystems 3730 DNA Analyzer. The sequencing primers are shown in Table 1. DNA sequence assembly and analysis were performed using Sequencher 4Á9 software (Gene Codes Corporation, Ann Arbor, MI, USA). Amino acids are described with the N1 numbering.

NA inhibition assay
An NA inhibition assay was performed to determine the susceptibility of viruses to NA inhibitors. Four NA inhibitors, oseltamivir carboxylate (F. Hoffmann-La Roche Ltd., Basel, Switzerland), zanamivir (GlaxoSmithKline, Stevenage, UK), peramivir (Shionogi & Co., Ltd., Osaka, Japan), and laninamivir (Daiichi Sankyo Co., Ltd., Tokyo, Japan) were kindly provided by the manufacturers. The susceptibility of viruses to NA inhibitors was determined by chemiluminescent  6 The WHO expert working group on surveillance of influenza antiviral susceptibility has defined criteria of NA inhibitor susceptibility based on the fold change of IC 50 values compared with reference IC 50 values. 8 For influenza A viruses, use of normal (<10-fold increase), reduced (10-to 100-fold increase), and highly reduced (>100-fold increase) inhibition, and for influenza B viruses, the same criteria but using <5fold, 5-to 50-fold and >50-fold increases, is recommended.

Statistical analysis
Box-and-whisker plots were used to identify extreme IC 50 values as previously described. 9 The box contains 50% of the results, representing the middle two quartiles (25-75%). The length of the box represents the interquartile range (IQR). The whiskers extend to the largest and smallest values in the central part of the distribution before the region containing outliers is reached. The mild (between 1Á5 and 3Á0 IQR from the 25th and 75th percentiles) and the extreme (>3Á0 IQR from the 25th and 75th percentiles) outliers are defined. Differences between groups were evaluated by chi-square test. P values less than 0Á05 were considered statistically significant.

Phylogenetic analysis
Nucleotide sequences of the full-length NA gene and the HA1 domain of the HA gene were subjected to phylogenetic analysis. Multiple alignments and phylogenetic trees were constructed using the MEGA 5Á0 software. 10 The phylogenetic trees were obtained by the neighbor-joining method with bootstrap analysis of 1000 replicates. Accession numbers for the sequences used in the trees are shown in Table 2.

Detection of A(H1N1)pdm09 viruses with the H275Y substitution
During the 2008-2009 through the 2011-2012 influenza seasons, 38 292 cases of A(H1N1)pdm09 virus isolation/ detection were reported in Japan ( Figure 1A). We screened 12,026 isolates by allelic discrimination and/or partial NA gene sequencing to detect the H275Y substitution (

Prescription history of patients with oseltamivirand peramivir-resistant A(H1N1)pdm09 viruses
The prescription history of patients with H275Y mutant viruses is shown in Among the cases of H275Y mutant virus infections, the percentage of no known exposure to antiviral drugs significantly increased from 16% during the pandemic period to 44% during the post-pandemic period (P < 0Á05), suggesting that human-to-human transmission cases of the resistant viruses had gradually increased. In fact, two clusters of resistant virus infection with suspected human-to-human transmission were independently observed. The first cluster occurred in a facility for handicapped individuals in eastern Japan in January-February 2011. In this cluster, resistant viruses with the H275Y mutation were detected in three patients who received prophylaxis with oseltamivir; 1 week later, the resistant viruses were detected in three patients who were not exposed to antivirals. The HA, NA, and matrix (M) genes of the six resistant viruses were sequenced and confirmed to possess the same amino acid sequences, implying that a single H275Y mutant virus circulated among the patients in the facility. The second cluster of cases occurred in a hospital in western Japan in February-March 2011. In this cluster, resistant viruses with the H275Y mutation were detected in three immunocompromised inpatients who had received prophylaxis with oseltamivir and was then later detected in a nurse. These four viruses possessed the same HA and NA amino acid sequences. However, there is no evidence of widespread outbreaks of the resistant viruses in the community, although the number of suspected cases of human-to-human transmission of drugresistant viruses increased.

Discussion
The highest frequency of antiviral drug use throughout the world has been recorded in Japan, ever since the NA inhibitors zanamivir and oseltamivir were licensed and covered by national health insurance in Japan in 2000 and 2001, respectively. 5 Consequently, surveillance information on antiviral-resistant viruses from Japan is valued by clinical and public health sectors throughout the world. We  related to longer periods of viral shedding and higher levels of viral titers. 15 Based on clinical history information, resistant virus infections were predominantly associated with the therapeutic use of oseltamivir or peramivir. However, 12-40% of the total resistant viruses were detected from patients under prophylaxis of oseltamivir with a subtherapeutic dose (75 mg once daily), but not a therapeutic dose (75 mg twice daily) as observed in our previous study. 6 Most resistant viruses from the prophylactic use emerged sporadically, although a suspected human-to-human transmission case from a patient under treatment to a patient under prophylaxis was observed. The emergence of resistant viruses in patients who received prophylaxis cannot be disregarded, so that the prophylactic use of antivirals is generally not recommended and should be followed by monitoring for emergence of resistant virus. In Japan, the Japanese Association for Infectious Diseases has recommended post-exposure prophylaxis in hospitals and nursing homes, following the recommendations of the Advisory Committee on Immunization Practices, USA 16 , and the Health Protection Agency, UK. 17 Consequently, the emergence of antiviral-resistant viruses from the prophylactic use should be paid particular attention.
Among the cases with resistant viruses, the percentage of no known exposure to antiviral drugs significantly increased from 16% during the pandemic period to 44% during the post-pandemic period. A similar increase was observed in other continents. [18][19][20][21][22] These observations may suggest that limited human-to-human transmission with H275Y mutant viruses has gradually increased in the post-pandemic periods. We observed suspected human-to-human transmission in two independent in-facility infection clusters that occurred in eastern and western Japan in 2011. The former cluster occurred in a facility for handicapped individuals. H275Y mutant viruses with the same HA, NA, and M amino acid  sequences were initially detected in patients who received prophylaxis with oseltamivir; 1 week after these initial infections, infections were detected in individuals who did not receive treatment or prophylaxis with antiviral drugs. In this cluster of cases, it appears that a single H275Y mutant circulated inside the facility and was transmitted among the patients. The second cluster of cases occurred in a hospital. The H275Y mutant viruses were initially detected in immunocompromised inpatients who had received prophylaxis with oseltamivir and then in a nurse. These H275Y mutant viruses possessed the same HA and NA amino acid sequences, suggesting human-to-human transmission of the mutant virus. These two suspected clusters of human-tohuman transmission did not spread beyond the residential facility or hospital to the outside community. However, the increasing number of H275Y mutant infections among those with no known exposure to antivirals is a warning that continued surveillance to monitor human-to-human transmission of NA inhibitor-resistant viruses is necessary worldwide; this is especially true as we had seen that oseltamivirresistant former seasonal A(H1N1) viruses with the H275Y mutation, which emerged in Europe in the 2007-2008 season, spread globally within a year. 1 Some potentially permissive substitutions together with the H275Y mutation in the NA protein were reported for A (H1N1) viruses 3,4 and A(H1N1)pdm09 viruses. 20,22 In the case of A(H1N1) viruses, R222Q and V234M (N2 numbering) substitutions in NA restored the surface-expressed NA activity, 3 and these substitutions occurred before the global emergence of H275Y mutant viruses. Three substitutions (T82K, K141E, and R189K) in the HA protein promoted virus replication in the presence of the H275Y substitution in NA. 4 In the case of A(H1N1)pdm09 viruses, three substitutions (V241I, N369K, and N386S) in the NA protein may offset the destabilizing effect of the H275Y substitution. 20 In our study, the H275Y mutant viruses did not possess these permissive substitutions and did not form any certain clade in the NA and the HA1 phylogenetic trees ( Figure 4A,B). However, oseltamivir-resistant A(H1N1)pdm09 viruses detected in a cluster of community cases in Australia contained the potentially permissive mutations in NA. 20 Therefore, sustained monitoring of the NA and HA gene sequences together with antiviral susceptibility assays is important to explore the possibility of human-to-human transmission of NA inhibitor-resistant viruses.