The validation of a real-time RT-PCR assay which detects influenza A and types simultaneously for influenza A H1N1 (2009) and oseltamivir-resistant (H275Y) influenza A H1N1 (2009)

Influenza A H1N1 (2009) was declared by the World Health Organisation (WHO) as the first influenza pandemic of the 21st century. Rapid detection of influenza A and differentiation of influenza A H1N1 (2009) and seasonal influenza A is beneficial. In addition the rapid detection of antiviral resistant strains of influenza A H1N1 (2009) would be useful for clinicians to allow for change to an effective treatment at a much earlier stage if resistance is found. It was the aim of this study to develop a real-time RT-PCR that can detect all influenza A viruses and type simultaneously for influenza A H1N1 (2009) and oseltamivir resistant (H275Y) influenza A H1N1 (2009). This multiplex assay will allow laboratories to screen respiratory samples for all types of influenza A, influenza A H1N1 (2009) virus and oseltamivir resistant (H275Y) influenza A H1N1 (2009) virus in a rapid and cost effective format, ensuring that typing methods for seasonal and avian viruses are used on a smaller subset of samples. Since most virology laboratories already offer a molecular service for influenza A this assay could easily be implemented into most areas at little cost therefore increasing local access to resistance testing.     

Ultrasensitive detection of drug-resistant pandemic 2009 (H1N1) influenza A virus by rare-variant-sensitive high-resolution melting-curve analysis

Oseltamivir (Tamiflu), an oral neuraminidase inhibitor, has been widely used to treat pandemic 2009 (H1N1) influenza A. Although a majority of 2009 (H1N1) influenza A virus remains oseltamivir susceptible, the threat of resistance due to the His275Tyr mutation is highlighted by the limitations of alternative therapies and the potential for rapid, global fixation of this mutation in the circulating influenza A virus population. In order to better understand the emergence of resistance, we developed a rare-variant-sensitive high-resolution melting-curve analysis method (RVS-HRM) that is able to detect the His275Tyr oseltamivir resistance mutation to 0.5% in a background of susceptible virus. We applied RVS-HRM to clinical specimens from patients who developed oseltamivir resistance and demonstrated the ultrasensitive detection of influenza A virus N1 neuraminidase quasispecies. Interestingly, we were unable to detect the oseltamivir resistance mutation in pretreatment samples, suggesting that resistant virus does not reach even this very low detection threshold until exposed to selective drug pressure. Thus, patients naive to oseltamivir are most likely to be susceptible when this drug is used as a first-line treatment modality.     

Influenza virus A(H1N1)pdm09 hemagglutinin polymorphism and associated disease in southern Germany during the 2010/11 influenza season

A novel influenza A virus emerged in early 2009 to cause the first influenza pandemic of the 21^st century. Understanding the evolution of influenza virus is crucial to determine pathogenesis, vaccine efficacy, and resistance to antiviral drugs. In this study, we investigated the molecular evolution of influenza virus A(H1N1)pdm09 in the 2010/11 influenza season in southern Germany by sequence analysis of the influenza virus hemagglutinin gene from 25 patients with mild, moderate, and severe disease. Phylogenetic analysis revealed co-circulation of different genetic groups. The D222G mutation, which had previously been observed in severe cases, was not detected. Immunocompromised patients were not affected more severely than non-immunocompromised patients (p>0.05), although longer shedding was observed in some of them. Interestingly, additional mutations and potential glycosylation sites were detected in samples from the lower respiratory tract in two patients, but not in the corresponding upper respiratory tract specimens. The H275Y mutation in the influenza virus neuraminidase gene, known to confer resistance to the neuraminidase inhibitor oseltamivir, was detected in one patient.     

一起甲型H1N1流感疫情病原检测及其HA与NA基因特性研究

目的 确认引起一起流感暴发疫情的病原,阐明该病原的血凝素基因(HA)和神经氨酸酶基因(NA)的特性.方法 疫情中最早出现流感样症状病例的咽拭子样本用real-time RT-PCR方法检测甲型H1N1流感病毒核酸,采用鸡胚分离法进行病毒培养,选取两病毒分离株进行HA和NA核苷酸序列测定,并进行基因特性分析.结果 此次流感疫情是由甲型H1N1流感病毒引起的,其HA和NA基因均与参比毒株的HA和NA基因高度同源,NA基因没有发生H274Y突变.结论 本研究的甲型H1N1流感病毒分离株为疫苗亲本株和中国分离株的类似株,对神经氨酸酶抑制剂类药物(如达菲)敏感.     

Development of a real-time RT-PCR assay for detection of resistance to oseltamivir in influenza A pandemic (H1N1) 2009 virus using single nucleotide polymorphism probes

Resistance to oseltamivir in pandemic (H1N1) 2009 influenza A virus is linked to an amino acid change from histidine (H) to tyrosine (Y) at position 275 in the neuraminidase protein (NA). A real-time one step RT-PCR assay using single nucleotide polymorphism (SNP) probes was developed to detect this mutation in respiratory specimens. The limit of detection was 47.6 copies/reaction for wild-type H275 RNA and 52.9 copies/reaction for the mutant H275Y RNA. The assay did not cross-react with other respiratory pathogens. The clinical sensitivity and specificity of the assay was compared to the gold standard Sanger sequencing method using 25 sensitive, 15 resistant and 20 negative samples. The sensitivity and specificity was 88.0% and 100% respectively with the SOIV_Osel_SEN probe designed to detect the H275 allele and 100% for the SOIV_Osel_RES probe detecting the 275Y allele. The sensitivity of the assay using nine admixtures of sensitive and resistant alleles was 88.9% and 77.8% with the SOIV_Osel_SEN probe and SOIV_Osel_RES probe respectively. The presence of mixed sensitive and resistant alleles in patient samples and mixtures of in vitro RNA were detected reproducibly. This assay can be used for screening of original samples for oseltamivir resistance without the need for culture and phenotypic testing.     

Characterization of oseltamivir‐resistant influenza A(H1N1)pdm09 viruses in Taiwan in 2009–2011

The early isolated swine‐origin influenza A(H1N1)pdm09 viruses were susceptible to oseltamivir; however, there is a concern about whether oseltamivir‐resistant influenza A(H1N1)pdm09 viruses will spread worldwide as did the oseltamivir‐resistant seasonal influenza A(H1N1) viruses in 2007–2008. In this study, the frequency of oseltamivir resistance in influenza A(H1N1)pdm09 viruses was determined in Taiwan. From May 2009 to April 2011, 1,335 A(H1N1)pdm09‐positive cases in Taiwan were tested for the H275Y mutation in the neuraminidase (NA) gene that confers resistance to oseltamivir. Among these, 15 patients (1.1%) were found to be infected with H275Y virus. All the resistant viruses were detected after the patients have received the oseltamivir. The overall monthly ratio of H275Y‐harboring viruses ranged between 0% and 2.88%, and the peak was correlated with influenza epidemics. The genetic analysis revealed that the oseltamivir‐resistant A(H1N1)pdm09 viruses can emerged from different variants with a great diversity under drug pressure. The ratio of NA/HA activities in different clades of oseltamivir‐resistant viruses was reduced compared to those in the wild‐type viruses, indicating that the balance of NA/HA in the current oseltamivir‐resistant influenza A(H1N1)pdm09 viruses was interfered. It is possible that H275Y‐bearing A(H1N1)pdm09 virus has not yet spread globally because it lacks the essential permissive mutations that can compensate for the negative impact on fitness by the H275Y amino acid substitution in NA. Continuous monitoring the evolution patterns of sensitive and resistant viruses is required to respond to possible emergence of resistant viruses with permissive genetic background which enable the wide spread of resistance. J. Med. Virol. 85:379–387, 2013. © 2012 Wiley Periodicals, Inc.     

Identification of oseltamivir resistance among pandemic and seasonal influenza A (H1N1) viruses by an His275Tyr genotyping assay using the cycling probe method

Neuraminidase inhibitors are agents used against influenza viruses; however, the emergence of drug-resistant strains is a major concern. Recently, the prevalence of oseltamivir-resistant seasonal influenza A (H1N1) virus increased globally and the emergence of oseltamivir-resistant pandemic influenza A (H1N1) 2009 viruses was reported. In this study, we developed a cycling probe real-time PCR method for the detection of oseltamivir-resistant seasonal influenza A (H1N1) and pandemic influenza A (H1N1) 2009 viruses. We designed two sets of primers and probes that were labeled with 6-carboxyfluorescein or 6-carboxy-X-rhodamine to identify single nucleotide polymorphisms (SNPs) that correspond to a histidine and a tyrosine at position 275 in the neuraminidase protein, respectively. These SNPs confer susceptibility and resistance to oseltamivir, respectively. In the 2007-2008 season, the prevalence of oseltamivir-resistant H1N1 viruses was 0% (0/72), but in the 2008-2009 season, it increased to 100% (282/282). In the 2009-2010 season, all of the pandemic influenza A (H1N1) 2009 viruses were susceptible to oseltamivir (0/73, 0%). This method is sensitive and specific for the screening of oseltamivir-resistant influenza A (H1N1) viruses. This method is applicable to routine laboratory-based monitoring of drug resistance and patient management during antiviral therapy.     

Monitoring the susceptibility to oseltamivir of Influenza A(H1N1) 2009 virus by nested-PCR and pyrosequencing during the pandemic and in the season 2010-2011

For the early detection of the H275Y mutation as a marker of oseltamivir resistance in A(H1N1) pandemic strains, a sensitive and specific pyrosequencing assay was developed. This assay analyses a region 99nts long, encompassing the H275Y site, amplified by a nested PCR. Seventy-five respiratory specimens, obtained from 62 patients during the pandemic and in the 2010-2011 influenza season, in Tuscany, were tested. Resistant strains were demonstrated in 10 patients. In three other patients, resistant and sensitive variants were found. This pyrosequencing assay may be a useful method for monitoring the spread of resistant influenza H1N1 2009 strains.     

Rapid identification of neuraminidase inhibitor resistance mutations in seasonal influenza virus A(H1N1), A(H1N1)2009, and A(H3N2) subtypes by melting point analysis

The high mutation rate of influenza virus, combined with the increasing worldwide use of influenza virus-specific drugs, allows the selection of viruses that are resistant to the currently available antiviral medications. Therefore, reliable tests for the rapid detection of drug-resistant influenza virus strains are required. We evaluated the use of a procedure involving real-time polymerase chain reaction (PCR) followed by melting point analysis (MPA) of hybrids formed between the PCR product and a specific oligonucleotide probe for the identification of point mutations in the influenza A virus neuraminidase gene (NA) that are associated with oseltamivir resistance [resulting in the amino acid change H275Y for seasonal and pandemic influenza A(H1N1) viruses and E119V for A(H3N2) viruses]. Therefore, 54 seasonal A(H1N1) (12 oseltamivir-resistant and 42 sensitive strains), 222 A(H1N1)2009 (5 resistant, 217 sensitive), and 51 A(H3N2) viruses (2 resistant, 49 sensitive) were tested by MPA, and the results were compared to those obtained by sequencing the NA gene. The results clearly indicate that the identification of drug resistance mutations by MPA is as accurate as sequencing, irrespective of whether MPA is performed using clinical material or the corresponding isolate. MPA enables a clear identification of mutations associated with antiviral resistance.     

Recovery of influenza B virus with the H273Y point mutation in the neuraminidase active site from a human patient

The H275Y oseltamivir resistance mutation confers high-level resistance to oseltamivir in isolates of human A(H1N1) influenza. We report the recovery and identification of an influenza B virus with the H273Y neuraminidase point mutation directly from a human patient. The H273Y influenza B isolate is resistant to oseltamivir and peramivir but sensitive to zanamivir.     

深圳市2008-2009年A型H1N1季节性流感病毒神经氨酸酶抑制剂耐药性监测

目的 对深圳市2008-2009年分离到的H1N1季节性流感病毒神经氨酸酶(NA)抑制剂的耐药性进行监测.方法 根据原始临床样本的采集时间,按周抽取了55株2008-2009年分离到的H1N1季节性流感病毒,对其NA片段进行全长测序,选取WHO推荐的疫苗株和部分国内外分离到的H1N1季节性流感病毒作为参考株,运用Mega3.1软件进行种系发生树的构建、耐药相关位点及糖基化位点的分析.结果 对NA片段的序列分析发现2008年有2株(7.1%)出现了H275Y突变,但是2009年则有25株(92.6%)出现了该突变.提示H275Y达菲耐药突变株成为了2009年深圳市社区传播的优势株.同时还发现了一株Q136K变异株,显示对乐感清出现耐药.分子进化分析结果显示,H275Y变异成为了毒株在系统进化树上分布的主要依据.所有的深圳株NA片段上潜在的糖基化位点序列保守.结论 大量H275Y达菲耐药株的出现提示在今后的工作中应当密切关注流感病毒的耐药进展,进一步加强其耐药机制的研究.

Abstract：

Objective To analyze neuraminidase(NA) inhibitor resistance of seasonal H1N1 influenza A viruses isolated in Shenzhen during 2008 to 2009. Methods The NA gene of these viruses were sequenced. Phylogenetic analysis of the sequences was performed with Mega3. 1 software. Results In 2008, most isolates of the seasonal H1 N1 virus were susceptible to neuraminidase inhibitors, but the H275Y mutation in the neuraminidase gene region associated with high-level oseltamivir resistance had been detected in 92.6% of the strains isolated in 2009. Furthermore, a strain with Q136K was found, which showed the resistance to Zanamivir. Conclusion In the light of emerging resistance, close monitoring and understanding of the nature and dynamics of resistance mutations in influenza virus should be a priority.     

Investigation of causes of oseltamivir chemoprophylaxis failures during influenza A (H1N1-2009) outbreaks

BackgroundAntiviral post-exposure prophylaxis with oseltamivir has been used as a strategy in mitigating the Influenza A (H1N1-2009) pandemic. There have been few reports of well-documented prophylaxis failures and the reasons for failure.ObjectivesWe report herein a series of 10 cases of prophylaxis failures and explore the reasons behind their prophylaxis failure.Study designIn the early pandemic phase, the military employed oseltamivir post-exposure ring-prophylaxis of affected units. From June 22 to July 30, 2009, cases of laboratory-confirmed prophylaxis failures were identified. Nasopharyngeal swabs were collected and tested by PCR. Samples with sufficient RNA material were sent for whole genome sequencing, and screened for mutations that confer oseltamivir resistance, especially the H275Y mutation.ResultsTen cases of laboratory-confirmed prophylaxis failure were identified, with a mean age of 22.3 years. One case was asymptomatic; the remaining 9 had fever or cough but without severe complications. The mean duration of exposure before starting oseltamivir was 1.9 days (SD 0.9), while the mean duration of oseltamivir consumption before symptom onset was 1.9 days (SD 1.4). None of the samples had the H275Y mutation or other known mutations that confer resistance. From the whole genome sequencing, several mutations at the HA (T220S, E275V, T333A, D239G); PB2 (K660R, L607V, V292I); NS1 (F103S), and NP (W104G) gene segments were detected, but none of them were likely to result in anti-viral resistance.ConclusionsPrimary prophylaxis failures exhibited mild symptoms without complications; all did not have the H275Y mutation and were unlikely to result from other mutations.     

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.     

Molecular genetic studies of the susceptibility of epidemic influenza A(H1N1) virus strains isolated in the 2006-2009 seasons in Russia to oseltamivir (Tamiflu)

Oseltamivir (Tamiflu) is recommended by WHO experts as a drug to treat and prevent of influenza and to create stocks if its new pandemic variant occurs. The susceptibility of influenza viruses to oseltamivir was studied by polymerase chain reaction-based techniques detecting specific mutations in the neuraminidase gene. The increase in the number of oseltamivir-resistant influenza viruses, isolated from the Russian Federation, with type 1 neuraminidase H274Y mutation from 49% (2007-20008) to 92% (2008-2009) did not depend on the frequency of oseltamivir use. Full correlation of the results obtained by various techniques allows them to be used to monitor the susceptibility of influenza viruses to oseltamivir.     

Genetic variability of isolates of pandemic influenza virus A H1N1 isolated in Russia in 2009

In 14 isolates of pandemic influenza virus A H1N1 extracted from patients with influenza in 2009, complete nucleotide sequences of genome segments encoding surface proteins hemagglutinin and neuraminidase were determined. Phylogenetic analysis of these sequences showed their genetic similarity with the corresponding genes of other isolates of pandemic influenza virus A (H1N1) 2009 extracted in other countries, and the degree of homology for each gene was over 99%. Neuraminidase mutations known in the literature that lead to the stability of the virus to oseltamivir and other drugs of neuraminidase inhibitors were not recorded in the studied isolates. In four isolates from autopsy material, G155E mutation was found in hemagglutinin of the isolate A/Salekhard/01/2009 (H1N1). This mutation leads to increased viral replication in developing chick embryos. The frequency and nature of the nucleotide substitutions in the hemagglutinin and neuraminidase genes are determined.     

Singleplex real-time RT-PCR for detection of influenza A virus and simultaneous differentiation of A/H1N1v and evaluation of the RealStar influenza kit

BackgroundA novel influenza A virus, subtype A/H1N1v emerged in April 2009 and caused the first influenza pandemic of the 21st century. Reliable detection and differentiation from seasonal influenza viruses is mandatory for appropriate case management as well as public health.ObjectivesTo develop and technically validate a novel one-step real-time RT-PCR assay which can be used for influenza A virus screening and subtyping of A/H1N1v in a singleplex fashion. To assess the clinical performance of a novel commercial influenza RT-PCR kit based on the in-house version.Study designA real-time RT-PCR assay targeting the matrix gene of influenza A viruses was developed and validated using in vitro transcribed RNA derived from influenza A/H1N1v, A/H1N1 and A/H3N2 virus as well as plaque-quantified influenza A/H1N1v, A/H1N1 and A/H3N2 virus samples. After validation of the in-house version the commercial RealStar kit was used to assess the clinical performance and specificity on a panel of influenza viruses including A/H1N1v, A/H1N1, swine A/H1N1, A/H3N2, avian A/H5N1 as well as patient specimens.ResultsThe lower limit of detection of the in-house version was 2149, 1376 and 2994 RNA copies/ml for A/H1N1v, A/H1N1 and A/H3N2, respectively. The RealStar kit displayed 100% sensitivity and specificity and could reliably discriminate influenza A viruses from A/H1N1v. No cross reaction with swine A/H1N1 and A/H1N2 was observed with the RealStar A/H1N1v specific probe.ConclusionBoth assays demonstrated high sensitivity and specificity and might assist in the diagnosis of suspected influenza cases.