Quantitative analysis of influenza A (H3N2) E119V and R292K variants in clinical specimens by real-time reverse transcription polymerase chain reaction

Sato, Masatoki; Honzumi, Ken; Sato, Toshiko; Hashimoto, Koichi; Watanabe, Masahiro; Miyazaki, Kyohei; Kawasaki, Yukihiko

doi:10.1016/j.jcv.2015.05.018

Cited by 3 publications

(7 citation statements)

References 32 publications

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“…Furthermore, measuring the variant and WT RNA loads in clinical samples using the LNA/qRT -PCR method facilitates NAI susceptibility estimation in clinical specimens, and clinical samples may have clinically low susceptibility when the H1N1 pdm09 H275Y, H3N2 E119V, and R292K variant RNA populations are >50%, >90%, and >60%, respectively. The absolute variant load after antiviral therapy is usually much lower than that before treatment 9,14,15) , and a lower variant viral load has no impact on the clinical course of illness, even if the variant virus population increases during antiviral therapy 9,16,17) . Therefore, it is important to monitor the absolute viral load over time.…”

Section: Discussionmentioning

confidence: 99%

“…The absolute variant load after antiviral therapy is usually much lower than that before treatment 9 , 14 , 15) , and a lower variant viral load has no impact on the clinical course of illness, even if the variant virus population increases during antiviral therapy 9 , 16 , 17) . Therefore, it is important to monitor the absolute viral load over time.…”

Section: Discussionmentioning

confidence: 99%

“…Viral RNAs were extracted from the supernatant of cell cultures using the QIAamp MiniElute Virus Spin Kit (Qiagen, Inc., Valencia, CA, USA). PCR mixtures contained 4×TaqMan ® Fast Virus 1-Step Master Mix (Thermo Fisher Scientific), 900 nM of each primer 9 , 11 , 12) , 270 nM of each probe 9 , 11 , 12) ( Table 1 ), and 2 μL of target RNA, with the volume increased to 20 μL using nuclease-free water. PCR amplification was performed using the following conditions: reverse transcription of cDNA at 50°C for 5 min, followed by reverse transcriptase inactivation at 95°C for 20 s and PCR amplification by 40 two-step cycles (3 s at 95°C for denaturation and 30 s at 60°C for annealing and extension) using the QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

“…Before performing the NA inhibition assay, 1 × 10 5 pfu/mL each of the infectious WT and variant strains as well as 1×10 7 copies/mL of each RNA of the WT and variant strains were prepared to produce 11 different variantmixed samples in which the population level of resistant variants was 0% -100%. These variantmixed samples were used to conduct the NA inhibition assay, which was per-Prediction of drug susceptibility by RT -PCR formed using a NA -XTD chemiluminescencebased NA inhibition assay kit (Thermo Fisher Scientific), and the IC 50 values were calculated using GraphPad Prism 6 software (GraphPad Software Inc., La Jolla, CA, USA), as previously described 9) . Oseltamivir carboxylate was provided by F. Hoffmann -La Roche, Ltd. (Basel, Switzerland).…”

Section: Na Inhibition Assaymentioning

confidence: 99%

“…Recently, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) using specific probes composed of locked nucleic acids (LNA/qRT-PCR) to quantitatively detect major H1N1 pdm09 and H3N2 oseltamivir-resistant variants was demonstrated 9 , 11 , 12) . This PCR method allows for rapid viral susceptibility prediction and is expected to be useful in clinical practice.…”

Section: Introductionmentioning

confidence: 99%

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Population analysis of oseltamivir-resistant variants for the rapid prediction of drug susceptibility by real-time reverse transcription polymerase chain reaction

Sato

Hashimoto

2022

FJMS

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This study investigated whether quantitative realtime reverse transcription polymerase chain reaction (qRT -PCR), using specific probes composed of locked nucleic acids (LNA/qRT -PCR), designed to evaluate H1N1 pdm09 H275Y, H3N2 E119V and R292K variant populations, could replace a neuraminidase (NA) inhibition assay to determine the 50% inhibitory concentration (IC 50 ) of NA activity. For H1N1 pdm09, when the H275Y variant RNA load was 50% or 70% and the infective H275Y variant load was 40% or 70%, the IC 50 were >10 -and 100 -fold higher, respectively, than that of the wildtype (WT) strain. For H3N2, when the E119V RNA load and infective E119V variant load were >90% and >60%, respectively, the IC 50 of the mixed sample was >10 -fold higher than that of the WT strain. The variantmixed samples with a 70% or 80% R292K variant RNA load and a 60% or 70% infective R292K variant load exhibited >10 -and 100 -fold decreased susceptibility, respectively, compared with that of the WT. A positive correlation between the variant RNA load and infective variant load populations was observed. The LNA/qRT -PCR method can be used to improve the treatment and management of patients during antiviral therapy for influenza virus infection.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Na Inhibition Assaymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Population analysis of oseltamivir-resistant variants for the rapid prediction of drug susceptibility by real-time reverse transcription polymerase chain reaction

Sato

Hashimoto

2022

FJMS

Self Cite

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The impact of PA/I38 substitutions and PA polymorphisms on the susceptibility of zoonotic influenza A viruses to baloxavir

Taniguchi,

Noshi,

Omoto

et al. 2024

Arch Virol

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Genetic reassortment of avian, swine, and human influenza A viruses (IAVs) poses potential pandemic risks. Surveillance is important for influenza pandemic preparedness, but the susceptibility of zoonotic IAVs to the cap-dependent endonuclease inhibitor baloxavir acid (BXA) has not been thoroughly researched. Although an amino acid substitution at position 38 in the polymerase acidic protein (PA/I38) in seasonal IAVs reduces BXA susceptibility, PA polymorphisms at position 38 are rarely seen in zoonotic IAVs. Here, we examined the impact of PA/I38 substitutions on the BXA susceptibility of recombinant A(H5N1) viruses. PA mutants that harbored I38T, F, and M were 48.2-, 24.0-, and 15.5-fold less susceptible, respectively, to BXA than wild-type A(H5N1) but were susceptible to the neuraminidase inhibitor oseltamivir acid and the RNA polymerase inhibitor favipiravir. PA mutants exhibited significantly impaired replicative fitness in Madin-Darby canine kidney cells at 24 h postinfection. In addition, in order to investigate new genetic markers for BXA susceptibility, we screened geographically and temporally distinct IAVs isolated worldwide from birds and pigs. The results showed that BXA exhibited antiviral activity against avian and swine viruses with similar levels to seasonal isolates. All viruses tested in the study lacked the PA/I38 substitution and were susceptible to BXA. Isolates harboring amino acid polymorphisms at positions 20, 24, and 37, which have been implicated in the binding of BXA to the PA endonuclease domain, were also susceptible to BXA. These results suggest that monitoring of the PA/I38 substitution in animal-derived influenza viruses is important for preparedness against zoonotic influenza virus outbreaks.

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Development of Digital Droplet PCR Targeting the Influenza H3N2 Oseltamivir-Resistant E119V Mutation and Its Performance through the Use of Reverse Genetics Mutants

Poelvoorde

Dufrasne

Gucht

et al. 2023

CIMB

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The monitoring of antiviral-resistant influenza virus strains is important for public health given the availability and use of neuraminidase inhibitors and other antivirals to treat infected patients. Naturally occurring oseltamivir-resistant seasonal H3N2 influenza virus strains often carry a glutamate-to-valine substitution at position 119 in the neuraminidase (E119V-NA). Early detection of resistant influenza viruses is important for patient management and for the rapid containment of antiviral resistance. The neuraminidase inhibition assay allows the phenotypical identification of resistant strains; however, this test often has limited sensitivity with high variability depending on the virus strain, drugs and assays. Once a mutation such as E119V-NA is known, highly sensitive PCR-based genotypic assays can be used to identify the prevalence of such mutant influenza viruses in clinical samples. In this study, based on an existing reverse transcriptase real-time PCR (RT-qPCR) assay, we developed a reverse transcriptase droplet digital PCR assay (RT-ddPCR) to detect and quantify the frequency of the E119V-NA mutation. Furthermore, reverse genetics viruses carrying this mutation were created to test the performance of the RT-ddPCR assay and compare it to the standard phenotypic NA assay. We also discuss the advantage of using an RT-ddPCR instead of qPCR method in the context of viral diagnostics and surveillance.

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Quantitative analysis of influenza A (H3N2) E119V and R292K variants in clinical specimens by real-time reverse transcription polymerase chain reaction

Cited by 3 publications

References 32 publications

Population analysis of oseltamivir-resistant variants for the rapid prediction of drug susceptibility by real-time reverse transcription polymerase chain reaction

Population analysis of oseltamivir-resistant variants for the rapid prediction of drug susceptibility by real-time reverse transcription polymerase chain reaction

The impact of PA/I38 substitutions and PA polymorphisms on the susceptibility of zoonotic influenza A viruses to baloxavir

Development of Digital Droplet PCR Targeting the Influenza H3N2 Oseltamivir-Resistant E119V Mutation and Its Performance through the Use of Reverse Genetics Mutants

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