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Influenza A viruses are well known for their genetic diversity. They are constantly evolving through point mutations in their hemagglutinin (H) and neuraminidase (N) genes and through genetic reassortment of their segmented RNA genome (2). The current pandemic/2009/novel H1N1 influenza A virus (called novel H1N1 herein) is a product of antigenic shift resulting from triple reassortment of human, avian, and swine viruses (7). Between 19 April 2009 and November 2009, 199 countries have reported nearly 6,000 novel influenza A H1N1-related deaths to the World Health Organization (WHO) (3).
Several molecular tests have been developed for detection and discrimination of novel H1N1 and seasonal influenza A subtypes (1, 5, 6, 8). Notably, the Centers for Disease Control and Prevention (CDC) have developed two separate real-time reverse transcriptase PCR (rRT-PCR) TaqMan assays for detection and subtyping of the novel and seasonal influenza viruses. These assay target regions of the matrix (M) and H genes and require multiple PCRs for detection and subtyping (9).
The Mayo Clinic uses a previously described influenza A rRT-PCR (Mayo FLU A) (10), which targets the M protein gene via specific primers and fluorescence resonance energy transfer (FRET) probes. Samples are extracted on the Roche MagNA Pure system and then amplified and detected using the Roche LightCycler 2.0. Reverse transcription and amplification take place in a one-step reaction, and subsequent melting temperature (Tm) analysis allows for confirmation of amplicon identity. Polymorphisms may also be detected through use of Tm analysis, since a single base mutation may cause a shift in the Tm.
Early on in the novel H1N1 pandemic, we performed a pilot study to determine if discrimination between the three influenza A virus subtypes in current circulation (novel H1N1, seasonal H1N1, and seasonal H3N2) could be performed using Tm analysis. This would allow for simultaneous identification of influenza A RNA and subtype discrimination in a single rRT-PCR, without the need for a separate reflex assay. Included in the study were 104 novel H1N1, 37 seasonal H1N1, and 20 seasonal H3N2 isolates. The subtype of each isolate was confirmed by the CDC influenza A and swine flu panels and/or the xTAG respiratory viral panel (unsubtypeable samples positive for M protein but negative for seasonal H1 and H3 were assumed to be novel H1N1). This study showed that each major virus subtype had discrete, reproducible Tm ranges (50.5°C to 53.2°C, 47.1°C to 49.2°C, and 62.6°C to 66.8°C for novel H1N1, seasonal H1N1, and seasonal H3N2 isolates, respectively) (Fig. (Fig.11).
Between 1 May 2009 and 31 October 2009, we detected 1,414 influenza A clinical isolates out of 6,739 tested (20.98% positive), using the Mayo FLU A assay; the majority had a Tm between 50.5°C and 53.2°C and were identified as the novel H1N1 subtype. However, between 14 August and 31 October 2009, 19 clinical samples (19 patients) were identified with Tm values outside the validated range for novel H1N1 (19/1,261 [1.51%] isolates during this period); 18 were within or below the Tm range for seasonal H1N1 (mean ± 1 standard deviation [SD] = 47.0 ± 1.3°C), and one had a Tm between the validated range for novel H1N1 and that for seasonal H3N2 (55.5°C). All atypical Tm samples were identified as novel H1N1 by using the CDC swine flu assay (performed using the Roche LightCycler 480). To identify the mutations responsible for the atypical Tm values, sequencing of the 242-base oligonucleotide amplicon and alignment of the portions of the amplicon under the FRET probes were performed with 12 isolates. Analysis revealed multiple point mutations, compared to the original novel H1N1 gene sequence published by the WHO on 28 April 2009 (4) (Table (Table1).1). Of interest, some degree of geographic clustering was observed among the mutations in this small analysis.
Our results confirm that influenza A virus is constantly evolving and these mutations may have important implications in clinical diagnosis and possibly treatment, management, and vaccine development. In our case, we observed a loss of viral subtype discriminatory ability, using Tm analysis, within just 3 months of the pandemic. This was unexpected, given that the influenza A M gene is relatively conserved and is not thought to be under the same genetic selective pressures as the H and N genes. Fortunately, the longer length of the FRET probes, compared to that of alternative probe formats (e.g., TaqMan), and the ability to perform Tm analysis allows for continued detection of influenza A isolates with the Mayo FLU A assay, although subtyping cannot be performed. PCR-based systems that do not allow for detection of polymorphisms may lose sensitivity over time due to high mutability of the virus, and this may hinder laboratory diagnosis.
Published ahead of print on 16 December 2009.