Antiviral Res . Development of cycling probe based real-time PCR methodology for influenza A viruses possessing the PA/I38T amino acid substitution associated with reduced baloxavir susceptibility

[tetano]

Antiviral Res


. 2021 Feb 9;105036.
doi: 10.1016/j.antiviral.2021.105036. Online ahead of print.
Development of cycling probe based real-time PCR methodology for influenza A viruses possessing the PA/I38T amino acid substitution associated with reduced baloxavir susceptibility


Hidekazu Osada ¹ , Irina Chon ² , Wint Wint Phyu ² , Keita Wagatsuma ² , Nobuo Nagata ³ , Takashi Kawashima ⁴ , Isamu Sato ⁵ , Tadashi Saito ⁶ , Naoki Kodo ⁷ , Hironori Masaki ⁸ , Norichika Asoh ⁹ , Yoshiko Tuchihashi ⁹ , Yutaka Shirahige ¹⁰ , Yasuhiko Ono ¹¹ , Yasushi Shimada ¹² , Hirotsune Hamabata ¹³ , Kousuke Saito ² , Reiko Saito ¹⁴



Affiliations

• PMID: 33577807
• DOI: 10.1016/j.antiviral.2021.105036

Abstract

Baloxavir marboxil has been used for influenza treatment since March 2018 in Japan. After baloxavir treatment, the most frequently detected substitution is Ile38Thr in polymerase acidic protein (PA/I38T), and this substitution reduces baloxavir susceptibility in influenza A viruses. To rapidly investigate the frequency of PA/I38T in influenza A(H1N1)pdm09 and A(H3N2) viruses in clinical samples, we established a rapid real-time system to detect single nucleotide polymorphisms in PA, using cycling probe real-time PCR. We designed two sets of probes that were labeled with either 6-carboxyfluorescein (FAM) or 6-carboxy-X-rhodamine (ROX) to identify PA/I38 (wild type strain) or PA/I38T, respectively. The established cycling probe real-time PCR system showed a dynamic linear range of 10¹ to 10⁶ copies with high sensitivity in plasmid DNA controls. This real-time PCR system discriminated between PA/I38T and wild type viruses well. During the 2018/19 season, 377 influenza A-positive clinical samples were collected in Japan before antiviral treatment. Using our cycling probe real-time PCR system, we detected no (0/129, 0.0%) influenza A(H1N1)pdm09 viruses with PA/I38T substitutions and four A(H3N2) (4/229, 1.7%) with PA/I38T substitution prior to treatment. In addition, we found PA/I38T variant in siblings who did not received baloxavir treatment during an infection caused by A(H3N2) that afflicted the entire family. Although human-to-human transmission of PA/I38T variant may have occurred in a closed environment, the prevalence of this variant in influenza A viruses was still limited. Our cycling probe-PCR system is thus useful for antiviral surveillance of influenza A viruses possessing PA/I38T.

Keywords: PA/I38T substitution; antiviral susceptibility; baloxavir marboxil; cycling probe real-time PCR; influenza virus.