Jennifer Laplante

Deep Sequencing Reveals Mixed Infection with 2009 Pandemic Influenza A (H1N1) Virus Strains and the Emergence of Oseltamivir Resistance

Mixed infections with seasonal influenza A virus strains are a common occurrence and an important source of genetic diversity. Prolonged viral shedding, as observed in immunocompromised individuals, can lead to mutational accumulation over extended periods. Recently, drug resistance was reported in immunosuppressed patients infected with the 2009 pandemic influenza A (H1N1) virus within a few days after oseltamivir treatment was initiated. To better understand the evolution and emergence of drug resistance in these circumstances, we used a deep sequencing approach to survey the viral population from an immunosuppressed patient infected with H1N1/2009 influenza and treated with neuraminidase inhibitors. This patient harbored 3 genetic variants from 2 phylogenetically distinct viral clades of pandemic H1N1/2009, strongly suggestive of mixed infection. Strikingly, one of these variants also developed drug resistance de novo in response to oseltamivir treatment. Immunocompromised individuals may, therefore, constitute an important source of genetic and phenotypic diversity, both through mixed infection and de novo mutation.

Characteristics of Patients with Oseltamivir-Resistant Pandemic (H1N1) 2009, United States

During April 2009–June 2010, thirty-seven (0.5%) of 6,740 pandemic (H1N1) 2009 viruses submitted to a US surveillance system were oseltamivir resistant. Most patients with oseltamivir-resistant infections were severely immunocompromised (76%) and had received oseltamivir before specimen collection (89%). No evidence was found for community circulation of resistant viruses; only 4 (unlinked) patients had no oseltamivir exposure.

Influenza Antiviral Resistance Testing in New York and Wisconsin, 2006 to 2008: Methodology and Surveillance Data

ABSTRACT The need for effective influenza antiviral susceptibility surveillance methods has increased due to the emergence of near-universal adamantane resistance in influenza A/H3N2 viruses during the 2005-2006 season and the appearance of oseltamivir resistance in the influenza A/H1N1 virus subtype during the 2007-2008 season. The two classes of influenza antivirals, the neuraminidase inhibitors (NAIs) and the adamantanes, are well characterized, as are many mutations that can confer resistance to these drugs. Adamantane resistance is imparted mainly by a S31N mutation in the matrix gene, while NAI resistance can result from a number of mutations in the neuraminidase gene. During the 2007-2008 season, a neuraminidase mutation (H274Y) conferring resistance to the NAI oseltamivir emerged worldwide in the A/H1N1 virus subtype. Surveillance methodology and data from New York (NY) and Wisconsin (WI) for the 2006-2007 and 2007-2008 influenza seasons are presented. We used an existing pyrosequencing method (R. A. Bright et al., Lancet 366:1175-1181, 2005) and a modified version of this method for detection of adamantane resistance mutations. For NAI resistance mutation detection, we used a mutation-specific pyrosequencing technique and developed a neuraminidase gene dideoxy sequencing method. Adamantane resistance in the A/H3N2 virus samples was 100% for 2007-2008, similar to the 99.8% resistance nationwide as reported by the CDC. Adamantane resistance was found in only 1.2% of NY and WI A/H1N1 virus samples, compared to that found in 10.8% of samples tested nationwide as reported by the CDC. Influenza A/H1N1 virus H274Y mutants were found in 11.1% of NY samples for 2007-2008, a level comparable to the 10.9% nationwide level reported by the CDC; in contrast, mutants were found in 17.4% of WI samples. These results indicate the need for regional influenza antiviral surveillance.

Next generation sequencing for whole genome analysis and surveillance of influenza A viruses

BACKGROUND The Wadsworth Center, New York State Department of Health (NYSDOH), conducts routine diagnosis and surveillance of influenza viruses. Whole genome sequencing (WGS) with next generation sequencing (NGS) was initiated to provide more rapid, detailed, thorough, and accurate analysis. OBJECTIVES To optimize and implement a method for routine WGS of influenza A viruses. To use WGS to monitor influenza A viruses for reassortment, mutations associated with antiviral resistance and antigenicity changes, as well as those potentially affecting virulence and tropism. STUDY DESIGN Multiple extraction and amplification methods were investigated and optimized for the production of template to be used for NGS. Additionally, software options were considered for data analysis. Initial WGS influenza projects have included the comparison of mixed population sequence data obtained with NGS, Sanger dideoxy sequencing, and pyrosequencing, the comparison of sequences obtained from paired primary/cultured samples, the analysis of sequence changes over several influenza seasons, and phylogenetic analysis. RESULTS Procedures were optimized for extraction and amplification such that WGS could be successfully performed on both cultured isolates and primary specimens. Data is presented on 15 A/H1pdm09 and 44 A/H3N2 samples. Analysis of influenza A viruses identified and confirmed variant and mixed populations affecting antigenicity and antiviral susceptibility in both primary specimens and cultured isolates. CONCLUSIONS An influenza A whole genome PCR method has been optimized for the reliable production of template for NGS. The WGS method has been successfully implemented for enhanced comprehensive surveillance and the generation of detailed clinical data on drug resistance and virulence. Data obtained with this method will also aid in future vaccine selection.

Antiviral Resistance in Influenza Viruses: Laboratory Testing

Influenza continues to be a significant health care issue. Although vaccination is the major line of defense, antiviral drugs play an important role in prophylaxis and disease management. Approved drugs for influenza are currently limited to those that target the viral matrix protein or neuraminidase enzyme. Resistance-associated sequence changes in the genes encoding these proteins have been extensively studied. Available methods for genotypic and phenotypic antiviral susceptibility testing have expanded and are being further developed and improved. The sporadic emergence of drug-resistant variants and the global spread of resistant strains have demonstrated the ongoing need for vigilant testing and surveillance.

A Pyrosequencing-Based Approach to High-Throughput Identification of Influenza A(H3N2) Virus Clades Harboring Antigenic Drift Variants.

ABSTRACT The rapid evolution of influenza A(H3N2) viruses necessitates close monitoring of their antigenic properties so the emergence and spread of antigenic drift variants can be rapidly identified. Changes in hemagglutinin (HA) acquired by contemporary A(H3N2) viruses hinder antigenic characterization by traditional methods, thus complicating vaccine strain selection. Sequence-based approaches have been used to infer virus antigenicity; however, they are time consuming and mid-throughput. To facilitate virological surveillance and epidemiological studies, we developed and validated a pyrosequencing approach that enables identification of six HA clades of contemporary A(H3N2) viruses. The identification scheme of viruses of the H3 clades 3C.2, 3C.2a, 3C.2b, 3C.3, 3C.3a, and 3C.3b is based on the interrogation of five single nucleotide polymorphisms (SNPs) within three neighboring HA regions, namely 412 to 431, 465 to 481, and 559 to 571. Two bioinformatics tools, IdentiFire (Qiagen) and FireComb (developed in-house), were utilized to expedite pyrosequencing data analysis. The assays analytical sensitivity was 10 focus forming units, and respiratory specimens with threshold cycle (CT) values of <34 typically produced good quality pyrograms. When applied to 120 A(H3N2) virus isolates and 27 respiratory specimens, the assay displayed 100% agreement with clades determined by HA sequencing coupled with phylogenetics. The multi-SNP analysis described here was readily adopted by another laboratory with pyrosequencing capabilities. The implementation of this approach enhanced the findings from virological surveillance and epidemiological studies between 2013 and 2016, which examined more than 3,000 A(H3N2) viruses.

Standardizing the influenza neuraminidase inhibition assay among United States public health laboratories conducting virological surveillance.

BACKGROUND Monitoring influenza virus susceptibility to neuraminidase (NA) inhibitors (NAIs) is vital for detecting drug-resistant variants, and is primarily assessed using NA inhibition (NI) assays, supplemented by NA sequence analysis. However, differences in NI testing methodologies between surveillance laboratories results in variability of 50% inhibitory concentration (IC50) values, which impacts data sharing, reporting and interpretation. In 2011, the Centers for Disease Control and Prevention (CDC), in collaboration with the Association for Public Health Laboratories (APHL) spearheaded efforts to standardize fluorescence-based NI assay testing in the United States (U.S.), with the goal of achieving consistency of IC50 data. METHODS For the standardization process, three participating state public health laboratories (PHLs), designated as National Surveillance Reference Centers for Influenza (NSRC-Is), assessed the NAI susceptibility of the 2011-12 CDC reference virus panel using stepwise procedures, with support from the CDC reference laboratory. Next, the NSRC-Is assessed the NAI susceptibility of season 2011-12 U.S. influenza surveillance isolates (n = 940), with a large subset (n = 742) tested in parallel by CDC. Subsequently, U.S. influenza surveillance isolates (n = 9629) circulating during the next three influenza seasons (2012-15), were independently tested by the three NSRC-Is (n = 7331) and CDC (n = 2298). RESULTS The NI assay IC50s generated by respective NSRC-Is using viruses and drugs prepared by CDC were similar to those obtained with viruses and drugs prepared in-house, and were uniform between laboratories. IC50s for U.S. surveillance isolates tested during four consecutive influenza seasons (2011-15) were consistent from season to season, within and between laboratories. CONCLUSION These results show that the NI assay is robust enough to be standardized, marking the first time IC50 data have been normalized across multiple laboratories, and used for U.S. national NAI susceptibility surveillance.

Detection of a transient R292K mutation in Influenza A/H3N2 viruses shed for several weeks by an immunocompromised patient

ABSTRACT We describe the case of an immunocompromised patient, positive for influenza A virus (H3N2), in whom the neuraminidase R292K mutation was transiently detected during oseltamivir treatment. The R292K mutation was identified by direct testing in 3 of 11 respiratory specimens collected throughout the patients illness but in none of the cultures from those specimens.