key: cord-0764447-47o02vxg
authors: Winter, A.L.; Eshaghi, A.; Farrell, D.J.; King, A.; Li, A.; Li, Y.; Gubbay, J.B.
title: Variant influenza A (H1N1) virus infection in Canada
date: 2013-04-15
journal: J Clin Virol
DOI: 10.1016/j.jcv.2013.03.011
sha: dcc1ce80a3f9e9d2fbb9d8d98ef3e949c8ac7dc1
doc_id: 764447
cord_uid: 47o02vxg

There has been an increase in influenza A variant detections in the US in recent years. In September 2012, an Ontario resident was diagnosed with influenza A (H1N1) variant infection. The demonstrated cross reactivity with the A(H1N1)pdm09 H1 gene CDC realtime PCR suggests that laboratories that only use the pdm09 H1 gene PCR to confirm this subtype would incorrectly report this variant as a A(H1N1)pdm09 subtype unless they were doing further molecular investigations.

There has been an increase in influenza A variant detections in the US in recent years. In September 2012, an Ontario resident was diagnosed with influenza A (H1N1) variant infection. The demonstrated cross reactivity with the A(H1N1)pdm09 H1 gene CDC realtime PCR suggests that laboratories that only use the pdm09 H1 gene PCR to confirm this subtype would incorrectly report this variant as a A(H1N1)pdm09 subtype unless they were doing further molecular investigations.

© 2013 Elsevier B.V. All rights reserved.

There has been an increase in influenza A variant detections in the US in recent years. [1] [2] [3] Influenza viruses that normally circulate in swine are called "variant" viruses when they are found in people. 4 From December 2005 to September 21, 2012, 326 influenza A(H3N2) variant (v), 14 A(H1N1)v and 5 A(H1N2)v cases have been identified. 5 In September 2012, a 37 year old male Ontario resident was diagnosed with influenza A H1N1v infection. This was the first detection of this variant influenza strain in an individual in Canada. The case, who has an unknown influenza immunization history, had recently traveled within Canada during which time he had close contact with swine, and to the United States where he had close contact with cattle; attendance at swine fairs could not be ascertained. At the beginning of September, he became ill with gastrointestinal symptoms of unknown etiology, followed by acute respiratory symptoms and on September 9 was admitted to a local area hospital with pneumonia. Following a 2-day admission he was discharged from hospital, but was re-admitted on September 13 with worsening respiratory symptoms, and subsequently was transferred to a tertiary care center where he required admission to an intensive care unit. The case had two separate treatment courses of oseltamivir administered.

The first course was for five days (75 mg twice daily) starting on September 16, 2012. He had a second seven-day course of 150 mg twice daily starting on September 21, 2012. At time of writing the case had been discharged home following an extended stay in intensive care, however additional travel and exposure information was unattainable. Only one close contact of the case reported symptoms of influenza-like illness (ILI); that person tested negative for influenza.

PHOL performs a large proportion of primary respiratory viral testing for the province of Ontario from a variety of clinical settings including ambulatory, hospital and outbreaks. Specimens from all hospitalized and outbreak patients are tested for influenza A matrix gene and influenza B NS1 gene using CDC protocols. If influenza A-positive, subtyping for seasonal influenza A (H3N2) HA gene (CDC assay) and influenza A (H1N1)pdm09 NA gene (in-house assay) are performed. 6 Following the emergence of H3N2v infections in the US in 2011, PHOL implemented screening of a selection of influenza A-positive, H3-positive specimens with the swine NP gene PCR to screen for H3N2v. Samples that are influenza-A positive, but negative in the initial subtyping assays are designated unsubtypeable, and are investigated further including other seasonal (N2, H1, N1, pdm09H1), swine (NP) and avian (H5, H7, +/− H9) rRT-PCR subtyping targets using CDC protocols, in addition to an end-point swine influenza matrix (M) gene PCR developed by Canada's National Microbiology Laboratory (NML). Influenza-negative outbreak and ICU samples are also tested using A nasopharyngeal swab from the case collected on September 10 tested positive for influenza A in viral culture, and specimens collected on September 12 and 15 during a bronchoalveolar lavage was also positive for influenza A in the MRVP assay. A subsequent nasopharyngeal swab collected on September 17 tested negative for influenza. The September 10 sample was initially unsubtypeable by the influenza A subtyping assays routinely used at PHOL -influenza A(H1N1)pdm09 NA gene and seasonal HA3 gene. Realtime subtyping PCRs for seasonal HA1, seasonal NA1, seasonal NA2, HA5, and HA7 were all negative. Real-time PCRs for swine nucleoprotein (NP) gene and A(H1N1)pdm09 HA1 gene were both positive, as was a PCR for swine M gene. These results suggested that the influenza A virus was of swine origin, and of H1 subtype.

Whole genome sequencing was commenced at PHOL, and sequence data shared with the National Microbiology Laboratory (NML), Canada's reference laboratory. The NML received the primary sample and culture material for further investigation. At NML, testing was performed on the culture material, which was negative for seasonal H3N2, pandemic NA, H3N2 variant (v), and H1N2v. Antiviral susceptibility testing showed that the isolate was resistant to amantadine but sensitive to oseltamivir and zanamivir. Complete sequencing analysis of PB1, PB2, PA, HA, NA, and M genes on viral RNA extracted from the first passage in rhesus monkey kidney cells revealed that all sequences are very similar (between 98.9 and 99.5%) to viruses that have been found in US swine in 2011 and 2012 ( Table 2 ). The even lower nucleotide homology to A/California/7/2009 (H1N1)-like virus NA gene (79.6%) is consistent with this virus not being detected by the PHOL in-house A(H1N1)pdm09 NA gene real-time PCR due to several scattered mismatches in primer and probe binding sites. 6 Comparison . Antigenicity of the H1N1v isolate was examined using ferret antisera to A/California/7/09 by HI assay; it cross-reacted with an HI titer of 160 (reference HI titer is 640). This serological profile suggests close antigenic homology between A(H1N1)pdm09 and the H1N1v strain reported here. Based on the testing results from PHOL and NML, we concluded that this patient was infected with an influenza A (H1N1)v virus (Table 3) . and molecular characterization and the need for a representative selection of influenza viruses to have further molecular investigation by sequencing, which would ultimately detect any significant circulation of such variants. Such infrastructure will also enable rapid detection of more virulent influenza viruses, such as highly pathogenic avian influenza A (H5N1). We also suggest that a representative sample of influenza viruses from a variety of settings (e.g. critical care, outbreak-related, community-based) be screened by molecular methods in order to detect variants. Following the emergence of H3N2v infections in the US in 2011, PHOL does screen a selection of influenza A, subtype H3 specimens, with the swine NP gene PCR to screen for H3N2v. However, the NP gene PCR is positive in influenza A (H1N1)pdm09 as well as H1N1v, as both are of swine origin. In general, exposure and travel-related information is poorly completed on laboratory requisitions, and not consistently asked of patients presenting with influenza-like illness, particularly during influenza season, hence many variant cases would be missed. It is anticipated that finding a variant virus would be very rare, thus it is not essential to test all patients for both the HA and NA genes of influenza A (H1N1)pdm09. However systematic sequencing, which is primarily conducted in order to detect changes in the influenza genome which could impact vaccine effectiveness, could optimize detection of variants. This work should be conducted by reference laboratories as part of influenza surveillance.

There were no external sources of funding for this study.

Jonathan B. Gubbay has received a research grant from Glaxo-SmithKline Inc. to work on resistance to neuraminidase inhibitors. In June 2010, Public Health Ontario received a research grant from GlaxoSmithKline to study phenotypic resistance in the influenza virus.

Research ethics approval was not required.

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