Screening Of Drug-resistant Sites In Patients With Severe H1N1pdm09 Influenza Pneumonia And Research Of The Association Between H275Y Mutation And Oseltamivir-resistance

ObjectiveTo explore the prevalence of oseltamivir-resistant viruses in patients withsevere H1N1pdm09 influenza pneumonia and effects of H275Y mutation on the efficacy of oseltamivir.Methods1.Clinical information and samples of H1N1pdm09 influenza patients confirmed by the laboratory of the First Affiliated Hospital,Zhejiang University School of Medicine during December 2017 to March 2018 were collected,and patients were divided into severe pneumonia group and nonsevere-pneumonia group.Viral neuraminidase(NA)genes were sequenced for screening of viral oseltamivir-resistance mutations in patients.2.Viruses that had oseltamivir-resistance mutations,some viruses that did not have oseltamivir-resistance mutation,and H1N1pdm09 influenza virus referencestrain A/California/04/2009 were isolated.Then the NA activity assays and oseltamivir NA inhibition assays of viruses isolations were conducted,and the level of oseltamivir-resistance in vitro were defined according to the WHO criterion.3.Six-weeks-old female Balb/c mice were infected with the sameconcentrations of oseltamivir-resistant viruses and the reference strain respectively.In the meantime,mice were administrated with oseltamivir.Mice lungs were collected at day 2,4 and 6 post infection(pi)and the viral loads were determinedrespectively,and lungs collected at day 6 pi were stained by HE for observation.Rusults1.Viral NA genes from 43 patients with severe H1N1pdm09 influenza pneumoniaand 257 H1N1pdm09 influenza patients without severe pneumonia were sequenced.The only oseltamivir-resistant mutation detected in the two groups of patients was H275Y mutation,which was detected in 6 patients(14.0%)with severe pneumoniaand 1(0.4%)in patients without severe pneumonia.The detection rate of H275Y mutation in patients with severe H1N1pdm09 influenza pneumonia was significantly higher than that in patients without severe H1N1pdm09 influenza pneumonia(P<0.001).2.According to the results of the NA activity assays,3 of the 7 isolations with H275Yoseltamivir-resistant mutation mentioned above could not perform oseltamivir NA inhibition assaysbecause of the low NA activity.Furthermore,in oseltamivir NA inhibition assays,oseltamivir showed highly reduced inhibition to viruses with H275Yoseltamivir-resistant mutation and normal inhibition to viruses withoutoseltamivir-resistant mutation.3.The viral loads of H275Y mutated viruses in the lungs of mice at day 2 post infection(pi)was significantly higher than that of the reference strain(P=0.035),and there was no statistical difference in the viral loads of viruses in the micelungs at day 4and 6 pi.HE staining of mice lungs showed that boththe mutated viruses and the reference strain could induce pathological changes,suchas widening of alveolar walls,infiltration of inflammatory cells,and necrosis ofbronchiolar epithelial cells.Conclusions1.The frequency of viral gene oseltamivir-resistance mutations in patients with severe H1N1pdm09 influenza pneumonia was significantly higher than thatin patientswithout severe H1N1pdm09 influenza pneumonia.2.Inhibition of oseltamivir against H1N1pdm09 influenza viruses with H275Y mutationhighly reduced in vitro.3.H275Y oseltamivir-resistant mutated H1N1pdm09 viruses could delay the anti-viral efficacy of oseltamivir in vivo,and show similar pathogenicity to oseltamivir-sensitive viruses in mice lungs.4.Attentions should be paid to oseltamivir-resistant viruses in patients during the prevention and treatment of severe H1N1pdm09 influenza pneumonia.