tetano
Editor, Senior Moderator
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[SUP] 1 [/SUP], Irina Chon[SUP] 2 [/SUP], Wint Wint Phyu[SUP] 2 [/SUP], Keita Wagatsuma[SUP] 2 [/SUP], Nobuo Nagata[SUP] 3 [/SUP], Takashi Kawashima[SUP] 4 [/SUP], Isamu Sato[SUP] 5 [/SUP], Tadashi Saito[SUP] 6 [/SUP], Naoki Kodo[SUP] 7 [/SUP], Hironori Masaki[SUP] 8 [/SUP], Norichika Asoh[SUP] 9 [/SUP], Yoshiko Tuchihashi[SUP] 9 [/SUP], Yutaka Shirahige[SUP] 10 [/SUP], Yasuhiko Ono[SUP] 11 [/SUP], Yasushi Shimada[SUP] 12 [/SUP], Hirotsune Hamabata[SUP] 13 [/SUP], Kousuke Saito[SUP] 2 [/SUP], Reiko Saito[SUP] 14 [/SUP]
Affiliations
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 10[SUP]1[/SUP] to 10[SUP]6[/SUP] 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.
. 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[SUP] 1 [/SUP], Irina Chon[SUP] 2 [/SUP], Wint Wint Phyu[SUP] 2 [/SUP], Keita Wagatsuma[SUP] 2 [/SUP], Nobuo Nagata[SUP] 3 [/SUP], Takashi Kawashima[SUP] 4 [/SUP], Isamu Sato[SUP] 5 [/SUP], Tadashi Saito[SUP] 6 [/SUP], Naoki Kodo[SUP] 7 [/SUP], Hironori Masaki[SUP] 8 [/SUP], Norichika Asoh[SUP] 9 [/SUP], Yoshiko Tuchihashi[SUP] 9 [/SUP], Yutaka Shirahige[SUP] 10 [/SUP], Yasuhiko Ono[SUP] 11 [/SUP], Yasushi Shimada[SUP] 12 [/SUP], Hirotsune Hamabata[SUP] 13 [/SUP], Kousuke Saito[SUP] 2 [/SUP], Reiko Saito[SUP] 14 [/SUP]
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 10[SUP]1[/SUP] to 10[SUP]6[/SUP] 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.