Influenza viruses cause considerable annual worldwide morbidity and mortality. In the United States alone, greater than 200,000 persons are hospitalized each year due to influenza and approximately 36,000 die from influenza-related disease (16
). Vaccination is considered the first and best defense against influenza. However, the efficacy of protection conferred by annual vaccination can be limited by the strength of the antigenic match of vaccine strains to the circulating strains. In addition, herd (community-level) immunity is limited by less than 100% vaccine coverage. These circumstances allow influenza to easily spread among susceptible persons and through populations. Antiviral drug treatment and prophylaxis are additional and necessary modes of defense against morbidity, mortality, and further spread of the virus.
Widespread resistance against the adamantane class of drugs among A/H3N2 viruses, beginning in the 2003-2004 season, prompted public health officials in the United States to recommend against the use of these drugs during the 2005-2006 season in favor of neuraminidase inhibitors (NAIs) (3
). First approved for clinical use in the United States in 1999 (17
), NAIs target the viral surface protein neuraminidase and are effective against both influenza A and B. There are currently two FDA-approved drugs in this class: oseltamivir (Tamiflu; Roche) and zanamivir (Relenza; GlaxoSmithKline). When given within the first 48 h of a patient becoming symptomatic, NAIs have been shown to reduce the duration and severity of influenza illness in both adults and children (11
In January 2008, nine European countries reported seasonal influenza A/H1N1 isolates showing resistance to oseltamivir (13
). In the following months, additional countries, including the United States, reported oseltamivir-resistant influenza viruses (24
). These findings were alarming because drug resistance testing during the previous influenza season (2006-2007) had revealed no oseltamivir resistance in Europe (13
). Fewer than 1% of North American seasonal A/H1N1 viruses from the same time period tested by the Centers for Disease Control and Prevention (CDC) showed resistance (4
). In addition, during clinical trials with oseltamivir, shedding of drug-resistant virus was noted at a frequency of only 4% in children and in ≤1% in adults (28
). Analysis revealed that all resistant viruses were seasonal A/H1N1, carrying the same C→T transition mutation in the neuraminidase gene, with a resulting histidine-to-tyrosine change at amino acid position 275 (“H274Y” in universal N2 numbering). By the end of the 2007-2008 season, the CDC reported this mutation in 111 of 1,020 tested seasonal A/H1N1 isolates; 4 were found in New York State (4
). Oseltamivir-resistant seasonal A/H1N1 spread extensively, becoming the dominant variant in Oceania and Southeast Asia in May 2008 (10
) and with virtually all seasonal A/H1N1 strains possessing the H274Y mutation during the 2008-2009 influenza season in the United States (5
The need for continual monitoring for antiviral drug resistance among influenza viruses is highlighted by several factors. Use of antiviral medications as a treatment and prophylactic is an integral component of infection control during influenza outbreaks. With the advent of adamantane resistance, use of the neuraminidase inhibitors, most commonly oseltamivir, has risen dramatically. In addition, oseltamivir is a major component of influenza pandemic preparedness stockpiles in the United States. Continual improvement of methods for the survey and detection of oseltamivir resistance in all influenza isolates is imperative to ensure the lasting viability of this drug as an antiviral treatment.
Laboratory testing for NAI resistance can be performed via a phenotypic neuraminidase inhibition assay, giving a 50% inhibitory concentration (IC50
) for a drug of interest. Laboratories can also use nucleic acid testing such as real-time quantitative reverse transcriptase (RT)-PCR as well as dideoxy sequencing and pyrosequencing methods. Sequencing methods provide detailed information about gene regions of interest and enable the identification of new mutations that may have an effect on drug resistance. Pyrosequencing, in particular, has been reported to be capable of detecting minor populations at concentrations as low as 10% within mixtures of neuraminidase gene targets (7
). However, both dideoxy sequencing and pyrosequencing require additional processing steps after an initial PCR, as well as specialized sequencing instruments, software, and training. Real-time quantitative RT-PCR provides a rapid, highly sensitive, and specific alternative to sequencing. This method is also capable of detecting targets in samples with viral loads too low for detection in sequencing assays. Furthermore, real-time platforms are available from multiple manufacturers and are more commonplace in clinical laboratories than sequencing equipment.
Here, we describe the development of a novel, highly sensitive real-time quantitative RT-PCR assay for the detection of H274Y in seasonal A/H1N1 influenza. The assay utilizes MultiCode technology, a system that employs an additional base pair set of 5′-methyl-isocytosine (isoC) and isoguanine (isoG) (12
). An isoC and isoG can only base pair to one another, and neither an isoC-G nor a C-isoG bond can form. In real-time RT-PCR applications, an isoC base is included at the 5′ end of a primer, directly linked to a fluorophore, such as 6-carboxyfluorescein (FAM) or hexachlorofluorescein (HEX) (Fig. ). During PCR, a quencher (Dabcyl) directly linked to an isoG nucleotide is site specifically incorporated into the PCR product complementary to the isoC-fluorophore, resulting in a detectable decrease in fluorescence (23
). Because multiple fluorophores can be used in the same reaction, multiplexed assays can be designed. In addition, because the fluorophores are not cleaved from the oligonucleotides, postamplification melt curve analysis is possible, facilitating analyses of assay specificity. This PCR technology has been successfully employed as part of a PCR panel targeting several respiratory viruses (15
), in assays for other viruses including hepatitis C (20
) and HIV (19
), and in the detection of anthrax-related toxin genes (18
FIG. 1. Primer alignment to target sequence. Primers were designed against a consensus sequence of N1 sequences from seasonal A/H1N1-positive clinical samples submitted for testing to the Wadsworth Center between 2004 and 2008. The genotype-specific primers are (more ...)
This new assay was developed with analyses for both mutation detection and facilitation of resistance evolution in mind. To that end, we developed a novel multiplexed assay with high sensitivity and specificity and with a higher sensitivity for detecting a minor component mutant population compared to that detectable by pyrosequencing. The assay was applied to a collection of A/H1N1 influenza-positive samples collected between 2005 to 2008, in order to determine whether the H274Y mutation was present in clinical samples prior to its apparent emergence during the 2007-2008 season.