Quantitative SNP‐Detection Method for Estimating HIV‐1 Replicative Fitness: Application to Protease Inhibitor‐Resistant Viruses

Ibe, Shiro; Fujisaki, Saeko; Fujisaki, Seiichiro; Morishita, Tatsuo; Kaneda, Toshio

doi:10.1111/j.1348-0421.2006.tb03852.x

Cited by 5 publications

(7 citation statements)

References 22 publications

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“…To bypass the time- and labor-intensive process of cloning the desired genes from the mixed populations and choosing an arbitrary number of clones for genotypic analysis, we used a new method, relative quantification of SNP, to determine the ratio of wild-type to mutant populations. This method showed high reproducibility in genotyping HIV protease gene [42]. Our study is the first to use this method to genotypically analyze influenza viruses.…”

Section: Discussionmentioning

confidence: 89%

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Oseltamivir–Resistant Pandemic H1N1/2009 Influenza Virus Possesses Lower Transmissibility and Fitness in Ferrets

et al. 2010

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The neuraminidase (NA) inhibitor oseltamivir offers an important immediate option for the control of influenza, and its clinical use has increased substantially during the recent H1N1 pandemic. In view of the high prevalence of oseltamivir-resistant seasonal H1N1 influenza viruses in 2007–2008, there is an urgent need to characterize the transmissibility and fitness of oseltamivir-resistant H1N1/2009 viruses, although resistant variants have been isolated at a low rate. Here we studied the transmissibility of a closely matched pair of pandemic H1N1/2009 clinical isolates, one oseltamivir-sensitive and one resistant, in the ferret model. The resistant H275Y mutant was derived from a patient on oseltamivir prophylaxis and was the first oseltamivir-resistant isolate of the pandemic virus. Full genome sequencing revealed that the pair of viruses differed only at NA amino acid position 275. We found that the oseltamivir-resistant H1N1/2009 virus was not transmitted efficiently in ferrets via respiratory droplets (0/2), while it retained efficient transmission via direct contact (2/2). The sensitive H1N1/2009 virus was efficiently transmitted via both routes (2/2 and 1/2, respectively). The wild-type H1N1/2009 and the resistant mutant appeared to cause a similar disease course in ferrets without apparent attenuation of clinical signs. We compared viral fitness within the host by co-infecting a ferret with oseltamivir-sensitive and -resistant H1N1/2009 viruses and found that the resistant virus showed less growth capability (fitness). The NA of the resistant virus showed reduced substrate-binding affinity and catalytic activity in vitro and delayed initial growth in MDCK and MDCK-SIAT1 cells. These findings may in part explain its less efficient transmission. The fact that the oseltamivir-resistant H1N1/2009 virus retained efficient transmission through direct contact underlines the necessity of continuous monitoring of drug resistance and characterization of possible evolving viral proteins during the pandemic.

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Section: Discussionmentioning

confidence: 89%

“…Competitive growth assay by co-infection is a method of evaluating the growth fitness of two viruses [41], [42]. In the present study, we inoculated a ferret with equal doses of oseltamivir-sensitive and -resistant H1N1/2009 viruses to compare their relative growth fitness within the host.…”

Section: Discussionmentioning

confidence: 99%

Oseltamivir–Resistant Pandemic H1N1/2009 Influenza Virus Possesses Lower Transmissibility and Fitness in Ferrets

et al. 2010

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“…However, the quantification capability of the SNaPshot assay was not investigated in these studies because human genome in somatic cells can be only either homozygous or heterozygous. The assay had previously been described only once in the virology literature, to detect two drug resistance markers in the HIV protease gene; this study also explored the quantitative analysis of different genotypes (14). In this study, concentration-response curves of different genotypes differed in their kinetics and plateaus, and the ratios of different genotypes were calculated based on the normalization of signal intensity of G, A, and T relative to C at certain concentrations.…”

Section: Discussionmentioning

confidence: 99%

“…The fluorescence signals must always be normalized. To do this, we used the equations described; another study used the relative ratios of the different signals (14). A novel method must always be compared with other established methods.…”

Section: Discussionmentioning

confidence: 99%

Novel Genotyping and Quantitative Analysis of Neuraminidase Inhibitor Resistance-Associated Mutations in Influenza A Viruses by Single-Nucleotide Polymorphism Analysis

Duan

Boltz

et al. 2011

Antimicrob Agents Chemother

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“…To bypass the time-and labor-intensive process of cloning the desired genes from the mixed populations and choosing an arbitrary number of clones for genotypic analysis, we used a new method, relative quantification of SNP, to determine the ratio of wild-type to mutant populations. This method showed high reproducibility in genotyping HIV protease gene (126). The resistance to NA inhibitors can significantly limit the infection control options for influenza, so the susceptibility of circulating human influenza viruses to NA inhibitors has been closely monitored.…”

Section: Discussionmentioning

confidence: 99%

The Molecular Basis of Fitness and Transmissibility of Neuraminidase Inhibitor Resistant Influenza A Viruses

Duan¹

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DEDICATIONThis dissertation is dedicated to my parents and my own family who have been always giving me unconditional love, indissoluble strength, endless inspiration. iv ACKNOWLEDGEMENTSAll of this would not have been possible had Dr. Robert G. Webster not taken me as his graduate student. I will always remember my first conversation with Dr. Webster about my rotating his lab and the first conversation with Dr. Webster at the day I officially joined the lab: "Work hard, young people." I have been doing nothing but following this simple instruction through all my studies during these years. I will be eternally grateful for Dr. Webster for having faith in me to take me as his student and mentoring my Ph.D study. I thank Dr. Webster for being my mentor in every way, for always giving me the biggest vision about the research and career, for teaching me to start strong and end strong a scientific story, for enlightening me with the most important and critical questions for now and for future in the world of influenza virology.This dissertation would not have been completed in its present way without Dr. Elena G. Govorkova's supervision. The greatest appreciation is to Elena as my supervisor in all kinds of trainings: from introducing me the knowledge of the antiviral field, to helping me experiments in person, to supporting me to develop different projects, to teaching me to write manuscripts, to educating me to be a philosopher, and always reminding me to look pretty as a woman. I will always value all these precious training and experience.Special deep thanks to Dr. Richard J. Webby for providing me different training opportunities in other aspects of influenza virology, for giving me different visions to see different questions. Especially many thanks for his biggest push to greatly move forward the second study in this thesis by adding new angles and much more depth.Special thanks to my committee members, Dr. Paul Thomas, Dr. Michael Whitt and Dr. Tony Marion for giving me different perspectives to look the virology field, for always teaching me to think questions harder, deeper and more precisely.Special thanks to Scott Krauss for his general support for all studies.Special thanks to Dr. Subrata Barman and Dr. Charles Russell for kindly providing related experimental materials and working instructions.Many thanks to a former postdoctor fellow in our lab, Dr. David Boltz, for being a good teacher and good friend to me, for introducing me the influenza genome analysis, for helping me to develop the methodology project and for giving me encouragement to through the ups and downs of the studies. Many thanks to another former postdoctor fellow, Dr. Hui-ling Yen, for also being a great teacher and friend to me, for her unique comments and inspiring discussion on many influenza questions and for her dear encouragement for me all the time. Many thanks to other current and former postdoctor fellows in our flu group for helping me with their respective expertise and those include Dr. Hassan, Zarakat, Dr. Henju Marjuki, D...

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Quantitative SNP‐Detection Method for Estimating HIV‐1 Replicative Fitness: Application to Protease Inhibitor‐Resistant Viruses

Cited by 5 publications

References 22 publications

Oseltamivir–Resistant Pandemic H1N1/2009 Influenza Virus Possesses Lower Transmissibility and Fitness in Ferrets

Oseltamivir–Resistant Pandemic H1N1/2009 Influenza Virus Possesses Lower Transmissibility and Fitness in Ferrets

Novel Genotyping and Quantitative Analysis of Neuraminidase Inhibitor Resistance-Associated Mutations in Influenza A Viruses by Single-Nucleotide Polymorphism Analysis

The Molecular Basis of Fitness and Transmissibility of Neuraminidase Inhibitor Resistant Influenza A Viruses

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