Study On Inhibitory Effect Of Favipiravir On Influenza Virus

Background:Influenza viruses can cause seasonal influenza epidemics and occasional influenza pandemics.Anti-influenza drugs are an important method for preventing and controlling influenza virus infection.There are three types of drugs having achie-ved market approval,including M2 ion-channel blockers,neuraminidase inhibitors(NAIs)and RNA-dependent RNA polymerase(RdRp)inhibitors.Owing to the appearance of widespread drug resistance and the lack of efficacy against influenza B viruses,M2 ion-channel blockers are no longer recommended to use;therefore,NAIs(oseltamivir,zanamivir,peramivir and laninamivir)become the major class of antiviral agents.However,the emergence of NAI-resistant mutants can reduce their efficacy.RdRp inhibitor is a new type of anti-influenza drug.Among them,Favipiravir,also known as T-705,was approved for marketing in Japan in 2014.One of the goals of influenza surveillance is to detect the susceptibility of influenza virus to antiviral drugs,but WHO has not yet recommended an uniform method for the detection of polymerase inhibitors.Plaque reduction assay is a classic method for quantifying viruses,which is greatly affected by the virus,the number of cells,and other experiment conditions.At present,there is still a lack of comparative studies of various test conditions when plaque reduction assay is used to detect the susceptibility of influenza virus to polymerase inhibitors.In addition,the process of influenza virus replication requires host cells,which may affect polymerases and their inhibitors.Although previous studies have shown that T-705 could inhibit the replication of influenza virus in Madin-Darby canine kidney(MDCK)cells,the effect of T-705 on influenza virus has not been comprehensively evaluated on human cell lines.Moreover,T-705 has been tested to be efficacious against influenza B virus in vitro,however,its efficacy against oseltamivir-resistant influenza B virus has never been demonstrated in animal models.Objectives:(1)Optimize the various test conditions when the plaque assay and plaque reduction assay are used to detect the susceptibility of influenza virus to T-705.(2)Evaluate the effect of T-705 on different types/subtypes of seasonal influenza viruses in MDCK and human lung adenocarcinoma(A549)cells.(3)Study the inhibitory effect of T-705 on wild-type and oseltamivir-resistant influenza B viruses in a mouse model.Methods:1.Optimize the test conditions of T-705 susceptibility:first,the incubation time,drug concentration and other test conditions were determined when different types/subtypes of seasonal influenza viruses were inoculated in different culture plates,and immuno-staining and crystal violet staining were compared.On this basis,the susceptibility of seasonal influenza viruses to T-705 was evaluated by plaque reduction assay in different culture plates.2.Evaluate the anti-influenza virus activity of T-705 at the cellular level:the cell viability assay was used to evaluate the cytotoxicity of T-705 on MDCK and A549 cells;the 50%tissue culture infective dose(TCID50)assay was used to determine the replication kinetic of influenza virus in MDCK and A549 cells;the cell viability assay and plaque reduction assay were used to determine the 50%inhibitory concentration(IC50)of T-705 against influenza virus in MDCK and A549 cells;on this basis,the effect of T-705 on influenza virus infection in MDCK and A549 cells was determined by TCID50,digital PCR and Western blotting assays.3.Study on the inhibitory effect of T-705 on wild-type and oseltamivir-resistant influenza B viruses at the animal level:C57BL/6 mice were infected with wild-type(NA R152)or oseltamivir-resistant(NA R152K)influenza B/Memphis/20/96 virus.Starting 2 hours post inoculation,T-705 was orally administered to mice twice a day for 5 days.Each virus corresponds to 5 groups:virus control,50mg/kg/day T-705,150mg/kg/day T-705,300mg/kg/day T-705 and 60mg/kg/day oseltamivir.Survival rate and body weight of mice were monitored daily Viral load in nasal turbinate,trachea and lung of mice was measured by digital PCR assay.Histopathology experiment was performed to evaluate the degree of lung injury in mice.ELISA kits were used to detect the levels of cytokines/chemokines in the lungs of infected mice.The IC50 of T-705 to viruses recovered from mice after T-705 treatment was determined by cell viability assay,and Hemagglutination(HA)and Hemagglutination inhibition(HI)assays were used to determine HI titers in serum of surviving mice.Results:1.The results of experiment conditions optimization:in 12-well tissue culture plates,the optimal incubation time was 2 days for influenza A viruses A/Beijing/2/2018(pdmH1N1)and A/Switzerland/8060/2018(H3N2),and 2.5 days was the best time for influenza B virus B/Beijing/1/2018(Yamagata).In 96-well plates,the optimal incubation time for A/Beijing/2/2018(pdmH1N1)and A/Switzerland/8060/2018(H3N2)was 20h.For B/Beijing/1/2018(Yamagata),the optimal incubation time was 24h.0.5 log10 was appropriate dilution for T-705.The 96-well plate has high throughput and the results can be read automatically.Our research suggests that the 96-well plate can be used to detect the susceptibility of seasonal influenza virus to T-705.2.Studies at the cellular level demonstrated that T-705 at a concentration of 100?M or lower hardly affected cell viability of MDCK and A549 cells;four different types/subtypes of influenza viruses A/Michigan/45/2015(Hlpdm),A/Singapore/INFIMH/16-0019/2016(H3N2),B/Colorado/06/2017(Victoria)and B/Phuket/3073/2013(Yamagata)all replicated well in MDCK and A549 cells.For these four strains,the IC50 values obtained by cell viability assay were higher than those obtained by plaque reduction assay.In addition,IC50 values in A549 cells were higher than those in MDCK cells,but there was no statistical difference.In MDCK and A549 cells.10 and 25?M T-705 significantly inhibited the production of infectious virus particles and reduced the expression of MP/NS1 gene and nucleoprotein(NP)in a dose-dependent manner.3.Studies at the animal level showed that the susceptibility of B/Memphis/20/96(R152K)to T-705 was similar to that of B/Memphis/20/96(R152),while B/Memphis/20/96(R152K)was about 150 times less susceptible than B/Memphis/20/96(R152)to oseltamivir carboxylate.T-705 treatment protected mice from lethal challenge with influenza virus B/Memphis/20/96(R152)or B/Memphis/20/96(R152K)and improved survival of mice in a dose-dependent manner.T-705 treatment reduced viral load,lung weight gain,lung lesion and cytokine/chemokine level in infected mice.Moreover,phenotypic analysis showed that T-705 treatment did not result in the emergence of resistant mutant of influenza virus.T-705 administration did not hinder the humoral immune response in infected mice.Conclusions:In summary,first of all,the present study suggested that it is necessary to strictly control the incubation time of viruses and drug concentration to detect the susceptibility of influenza viruses to polymerase inhibitors.In addition to the commonly used 6-well and 12-well plates,96-well plates can also be used to determine the susceptibility to polymerase inhibitors.Second,in human lung-derived A549 cells and canine kidney-derived MDCK cells,T-705 can inhibit the replication of different types/subtypes of seasonal influenza viruses,which is not affected by the origin of the cell line.Finally,T-705 is effective in protecting mice from lethal infection with both wild-type and oseltamivir-resistant influenza B viruses,and can be a promising alternative antiviral to treat severe oseltamivir-resistant influenza B virus infections in patients.