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Volume 19, Number 9—September 2013

R292K Substitution and Drug Susceptibility of Influenza A(H7N9) Viruses

Katrina Sleeman1, Zhu Guo1, John Barnes, Michael Shaw, James Stevens, and Larisa V. GubarevaComments to Author 
Author affiliations: Centers for Disease Control and Prevention, Atlanta, Georgia, USA

Main Article

Table 2

Susceptibility to neuraminidase inhibitors according to modified NI assay with pH 5.1*

Sample type Subtype Virus name (passage) AA at 292† % K292 IC50 nmol/L (fold)
Oseltamivir Zanamivir Peramivir Laninamivir
Virus Isolate H7N9 A/Anhui/1/2013 (E2/S1) R 0.90 (1) 1.00 (1) 0.12 (1) 1.29 (1)
A/Shanghai/1/2013 (E1) R and K 77 642.78
(714) NT NT NT
Recombinant NA H7N9 A/Anhui1/2013 R 0.53 (1) 0.85 (1) 0.09 (1) 0.92 (1)
K 100 9,078 (>1,000) 54.18 (56) 89.70 (814) 30.50 (25)
A/Shanghai/1/2013 R 1.08 (1) 1.45 (1) 0.19 (1) 1.90 (1)
K 100 8,351 (>1,000) 53.36 (53) 77.01 (642) 29.72 (23)
Reference virus H3N2 Oseltamivir-sensitive A/Washington/01/2007 0.68 (1) 0.45 (1) 0.10 (1) 0.86 (1)
Oseltamivir-resistant A/Bethesda/956/2006 K 100 6,083 (>1,000) 14.57 (32) 8.58 (86) 5.65 (7)
H1N1‡ Oseltamivir-sensitive A/California/12/2012 0.74 (1) 0.23 (1) 0.07 (1) 0.21 (1)
Oseltamivir-resistant A/Texas/23/2012 364.74 (493) 0.31 (1) 30.17 (431) 0.42 (2)

*NI, neuraminidase inhibition; AA, amino acid; IC50, concentration of neuraminidase inhibitor required to reduce enzyme activity by 50%; E, passage in eggs; S, passage in MDCK-SIAT1 cells (/ separates passage before and after arrival to CDC); NA, neuraminidase; NT, not tested; RT-PCR, reverse transcription PCR.
†AA position: R292K (N2 numbering); R294K (straight full-length N9 numbering). Single-nucleotide polymorphism analysis was performed by using the pyrosequencing assay (RT-PCR primers: N9-F731-Bio, 5’-CT GGA CCT GCA GAC ACA AGA ATA-3’, N9-R926, 5’-TGT GTC ATT GCT ACT GGG TCT ATC-3’; sequencing primer: N9–292/294-R889-seq, 5’-TAT TTG AGC CCT GCC-3’) and confirmed by deep sequencing. Pac bio RS sequencing library was constructed by using a 701bp RT-PCR amplicon generated by RT-PCR (N9NA-F204, 5’-CAACATCCAAATGGAAGAGAGAA-3’; N9NA-R903 5’-TGTGTCATTGCTACTGGGTCTATC-3’). A single v3 SMRT cell was used for each library and data was collected on 2 × 55 min movies. Only circular consensus sequencing reads were used in the analysis. Subpopulation detection was analyzed by using CLC Genomics Workbench version 6.01 (CLC Bio, Aarhus, Denmark). Isolates were tested in the NI assay by using the NA-Fluor kit (6). Fold change in IC50 compared with drug-sensitive subtype-specific control. IC50 values represent the average taken from at least 4 replicates, with the exception of A/Shanghai/1/2013 (E1), due to insufficient sample volume. Oseltamivir-susceptible and oseltamivir-resistant reference viruses were used as controls in NI assays. Oseltamivir refers to oseltamivir carboxylate. Reference virus A/Texas/23/2012 contains H275Y oseltamivir,resistance conferring neuraminidase substitution.
‡Pandemic influenza A(H1N1) 2009 virus.

Main Article

1These authors contributed equally to this article.

The footnote "1 " is not cited in the text. Please add an in-text citation or delete the footnote.

Medline reports the last page should be "4" not "5" in reference 4 "Centers for Disease Control and Prevention, 2013".

Medline reports the last page should be "374" not "80" in reference 10 "World Health Organization, 2012".

CrossRef reports the first author should be "McKimmbreschkin" not "McKimm-Breschkin" in reference 14 "McKimm-Breschkin, 2000".

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