In Vitro System for Modeling Influenza A Virus Resistance under Drug Pressure

Abstract

One of the biggest challenges in the effort to treat and contain influenza A virus infections is the emergence of resistance during treatment. It is well documented that resistance to amantadine arises rapidly during the course of treatment due to mutations in the gene coding for the M2 protein. To address this problem, it is critical to develop experimental systems that can accurately model the selection of resistance under drug pressure as seen in humans. We used the hollow-fiber infection model (HFIM) system to examine the effect of amantadine on the replication of influenza virus, A/Albany/1/98 (H3N2), grown in MDCK cells. At 24 and 48 h postinfection, virus replication was inhibited in a dose-dependent fashion. At 72 and 96 h postinfection, virus replication was no longer inhibited, suggesting the emergence of amantadine-resistant virus. Sequencing of the M2 gene revealed that mutations appeared at between 48 and 72 h of drug treatment and that the mutations were identical to those identified in the clinic for amantadine-resistant viruses (e.g., V27A, A30T, and S31N). Interestingly, we found that the type of mutation was strongly affected by the dose of the drug. The data suggest that the HFIM is a good model for influenza virus infection and resistance generation in humans. The HFIM has the advantage of being a highly controlled system where multiplicity parameters can be directly and accurately controlled and measured.

Effect of Half-Life on the Pharmacodynamic Index of Zanamivir against Influenza Virus Delineated by a Mathematical Model

“…We used the hollow-fiber infection model (HIFM) system to study the response of the oseltamivir-resistant pandemic H1N1 influenza A virus, A/Hong Kong (H275Y), to zanamivir exposure. The use of the HFIM system for influenza A viruses has been described previously (6,22). Briefly, 10 2 MDCK cells infected with H1N1 A/Hong Kong influenza virus were mixed with 10 8 uninfected MDCK cells in 25 ml of VGM and were inoculated into the extracapillary space (ECS) of several hollow-fiber (HF) cartridges (FiberCell Systems, Frederick, MD).…”

“…Extracellular virus (V extra ) is the result of the egress of intracellular virus from the last intracellular virus compartment (V i5 ) and was assumed to be subject to a first-order loss rate constant (k loss,V extra ). The latter represents the degradation of virus in medium due to temperature sensitivity (6). The initial condition for extracellular virus was set to 0 PFU/ml, because the inoculum was free of intracellular virus.…”

“…(6), aliquoted, and stored at Ϫ80°C. MDCK cells (ATCC CCL-34) were obtained from the American Type Culture Collection (Manassas, VA) and were grown in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), nonessential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml of penicillin, 100 g/ml of streptomycin, and 1.5 g/liter sodium bicarbonate at 37°C under 5% CO 2 .…”

T-705 (Favipiravir) Induces Lethal Mutagenesis in Influenza A H1N1 Viruses In Vitro

“…Influenza viruses have been shown to develop resistance to both NA inhibitors and adamantanes when serially passaged in the presence of the drugs in vitro (25,26). The drug-resistant viruses are viable, demonstrate decreased susceptibility to the drug in phenotypic assays, and possess mutations in the targeted protein (in the catalytic and/or framework residues of the NA protein after passage with NA inhibitors and in the transmembrane region of M2 protein after serial passage with adamantanes).…”

Pharmacokinetic Determinants of Virological Response to Raltegravir in the In Vitro Pharmacodynamic Hollow-Fiber Infection Model System

“…The HFIM system has been extensively described elsewhere (14)(15)(16). For these studies, H9 cells were infected with the IIIB strain of HIV-1 by incubating viral stocks with H9 cells for 1 week.…”