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Drug susceptibility profile and pathogenicity of H7N9 influenza virus (Anhui1 lineage) with R292K substitution.

ABSTRACT: Neuraminidase inhibitors (NAIs) are the only available licensed therapeutics against human H7N9 influenza virus infections. The emergence of NAI-resistant variants of H7N9viruses with an NA R292K mutation poses a therapeutic challenge. A comprehensive understanding of the susceptibility of these viruses to clinically available NAIs, non-NAIs and their combinations is crucial for effective treatment. In this study, by using limited serial passage and plaque purification, an R292K variant of the Anhui1 lineage was isolated from a patient with clinical evidence of resistance to oseltamivir. In vitro and cell-based assays confirmed a high level of resistance conferred by the R292K mutation to oseltamivir carboxylate and a moderate level of resistance to zanamivir and peramivir. Non-NAI antivirals, such as T-705, ribavirin and NT-300, efficiently inhibited both the variant and the wild-type in cell-based assays. A combination of NAIs and non-NAIs did not exhibit a marked synergistic effect against the R292K variant. However, the combination of two non-NAIs (T-705 and ribavirin) exhibited significant synergism against the mutant virus. In experimentally infected mice, the variant showed delayed onset of symptoms, a reduced viral load and attenuated lethality compared with the wild-type. Our study suggested non-NAIs should be tested clinically for H7N9 patients with a sustained high viral load. Possible drug combination regimens, such as T-705 plus ribavirin, should be further tested in animal models. The pathogenicity and transmissibility of the R292K H7N9 variant should be further assessed with genetically well-characterized pairs of viruses and, most-desirably, with competitive fitness experiments.

SUBMITTER: Zhang X

PROVIDER: S-EPMC4274890 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Drug susceptibility profile and pathogenicity of H7N9 influenza virus (Anhui1 lineage) with R292K substitution.

Zhang Xiaonan X Song Zhigang Z He Jing J Yen Hui-Ling HL Li Jianhua J Zhu Zhaoqin Z Tian Di D Wang Wei W Xu Lei L Guan Wencai W Liu Yi Y Wang Sen S Shi Bisheng B Zhang Wanju W Qin Boyin B Cai Jialin J Wan Yanmin Y Xu Chunhua C Ren Xiaonan X Chen Haili H Liu Lu L Yang Yuqin Y Zhou Xiaohui X Zhou Wenjiang W Xu Jianqing J Zhang Xiaoyan X Peiris Malik M Hu Yunwen Y Yuan Zhenghong Z

Emerging microbes & infections 20141112 11

Neuraminidase inhibitors (NAIs) are the only available licensed therapeutics against human H7N9 influenza virus infections. The emergence of NAI-resistant variants of H7N9viruses with an NA R292K mutation poses a therapeutic challenge. A comprehensive understanding of the susceptibility of these viruses to clinically available NAIs, non-NAIs and their combinations is crucial for effective treatment. In this study, by using limited serial passage and plaque purification, an R292K variant of the A ...[more]

PMID: 26038501

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Project description:UnlabelledA novel avian-origin influenza A/H7N9 virus infecting humans was first identified in March 2013 and, as of 30 May 2013, has caused 132 human infections leading to 33 deaths. Phylogenetic studies suggest that this virus is a reassortant, with the surface hemagglutinin (HA) and neuraminidase (NA) genes being derived from duck and wild-bird viruses, respectively, while the six "internal gene segments" were derived from poultry H9N2 viruses. Here we determine the pathogenicity of a human A/Shanghai/2/2013 (Sh2/H7N9) virus in healthy adult mice in comparison with that of A/chicken/Hong Kong/HH8/2010 (ck/H9N2) virus, highly pathogenic avian influenza (HPAI) A/Hong Kong/483/1997 (483/H5N1) virus, and a duck influenza A H7N9 virus of different genetic derivation, A/duck/Jiangxi/3286/2009 (dk/H7N9). Intranasal infection of mice with Sh2/H7N9 virus doses of 10(3), 10(4), and 10(5) PFU led to significant weight loss without fatality. This virus was more pathogenic than dk/H7N9 and ck/H9N2 virus, which has six internal gene segments that are genetically similar to Sh2/H7N9. Sh2/H7N9 replicated well in the nasal cavity and lung, but there was no evidence of virus dissemination beyond the respiratory tract. Mice infected with Sh2/H7N9 produced higher levels of proinflammatory cytokines in the lung and serum than did ck/H9N2 and dk/H7N9 but lower levels than 483/H5N1. Cytokine induction was positively correlated with virus load in the lung at early stages of infection. Our results suggest that Sh2/H7N9 virus is able to replicate and cause disease in mice without prior adaptation but is less pathogenic than 483/H5N1 virus.ImportanceAn H7N9 virus isolate causing fatal human disease was found to be more pathogenic for mice than other avian H9N2 or H7N9 viruses but less pathogenic than the highly pathogenic avian influenza virus (HPAI) H5N1. Similarly, the ability of Sh2/H7N9 to elicit proinflammatory cytokines in the lung and serum of mice was intermediate to ck/H9N2 and dk/H7N9 on the one hand and HPAI H5N1 on the other. These findings accord with the observed epidemiology in humans, in whom, as with seasonal influenza viruses, H7N9 viruses cause severe disease predominantly in older persons while HPAI H5N1 can cause severe respiratory disease and death in children and young adults.

Project description:Wild waterfowl is the natural reservoir of influenza A virus (IAV); hosted viruses are very variable and provide a source for genetic segments which can reassort with poultry or mammalian adapted IAVs to generate novel species crossing viruses. Additionally, wild waterfowl act as a reservoir for highly pathogenic IAVs. Exposure of wild birds to the antiviral drug oseltamivir may occur in the environment as its active metabolite can be released from sewage treatment plants to river water. Resistance to oseltamivir, or to other neuraminidase inhibitors (NAIs), in IAVs of wild waterfowl has not been extensively studied.In a previous in vivo Mallard experiment, an influenza A(H6N2) virus developed oseltamivir resistance by the R292K substitution in the neuraminidase (NA), when the birds were exposed to oseltamivir. In this study we tested if the resistance could be maintained in Mallards without drug exposure. Three variants of resistant H6N2/R292K virus were each propagated during 17 days in five successive pairs of naïve Mallards, while oseltamivir exposure was decreased and removed. Daily fecal samples were analyzed for viral presence, genotype and phenotype.Within three days without drug exposure no resistant viruses could be detected by NA sequencing, which was confirmed by functional NAI sensitivity testing. We conclude that this resistant N2 virus could not compete in fitness with wild type subpopulations without oseltamivir drug pressure, and thus has no potential to circulate among wild birds. The results of this study contrast to previous observations of drug induced resistance in an avian H1N1 virus, which was maintained also without drug exposure in Mallards. Experimental observations on persistence of NAI resistance in avian IAVs resemble NAI resistance seen in human IAVs, in which resistant N2 subtypes do not circulate, while N1 subtypes with permissive mutations can circulate without drug pressure. We speculate that the phylogenetic group N1 NAs may easier compensate for NAI resistance than group N2 NAs, though further studies are needed to confirm such conclusions.

Dataset Information

Drug susceptibility profile and pathogenicity of H7N9 influenza virus (Anhui1 lineage) with R292K substitution.

Publications

Drug susceptibility profile and pathogenicity of H7N9 influenza virus (Anhui1 lineage) with R292K substitution.

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