Impact of Amino Acid Mutations in PB2, PB1-F2, and NS1 on the Replication and Pathogenicity of Pandemic (H1N1) 2009 Influenza Viruses

BACKGROUND Influenza viruses can generate novel reassortants in coinfected cells. The global circulation and occasional introductions of pandemic H1N1/2009 virus in humans and in pigs, respectively, may allow this virus to reassort with other influenza viruses. These possible reassortment events might alter virulence and/or transmissibility of the new reassortants. Investigations to detect such possible reassortants should be included as a part of pandemic influenza surveillance plans. METHODS We established a real-time reverse-transcription (RT)-PCR–based strategy for the detection of reassortment of pandemic H1N1/2009 virus. Singleplex SYBR green–based RT-PCR assays specific for each gene segment of pandemic H1N1/2009 were developed. These assays were evaluated with influenza viruses of various genetic backgrounds. RESULTS All human pandemic H1N1 (n = 27) and all seasonal human (n = 58) isolates were positive and negative, respectively, for all 8 segments. Of 48 swine influenza viruses isolated from our ongoing surveillance program of influenza viruses in swine, 10 were positive in all reactions. All 8 viral segments of these 10 samples were confirmed to be of pandemic H1N1 origin, indicating that these were caused by zoonotic transmissions from human to pigs. The 38 swine viruses that were nonpandemic H1N1/2009 had 1–6 gene segments positive in the tests. Further characterization of these nonpandemic H1N1/2009 swine viruses indicated that these PCR-positive genes were the precursor genes of the pandemic H1N1/2009 virus. CONCLUSIONS Our results demonstrated that these assays can detect reintroductions of pandemic H1N1/2009 virus in pigs. These assays might be useful screening tools for identifying viral reassortants derived from pandemic H1N1/2009 or its precursors.

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Scoring Amino Acid Mutations to Predict Avian-to-Human Transmission of Avian Influenza Viruses

Molecules ◽

10.3390/molecules23071584 ◽

2018 ◽

Vol 23 (7) ◽

pp. 1584 ◽

Cited By ~ 6

Author(s):

Xiaoli Qiang ◽

Zheng Kou ◽

Gang Fang ◽

Yanfeng Wang

Keyword(s):

Amino Acid ◽

Avian Influenza ◽

Influenza Viruses ◽

Avian Influenza Viruses ◽

Amino Acid Mutations

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Predicting potentially permissive substitutions that improve the fitness of A(H1N1)pdm09 viruses bearing the H275Y NA substitution

10.1101/2021.03.21.436293 ◽

2021 ◽

Author(s):

Rubaiyea Farrukee ◽

Vithiagaran Gunalan ◽

Sebastian Maurer-Stroh ◽

Patrick C Reading ◽

Aeron C Hurt

Keyword(s):

Amino Acid ◽

A549 Cells ◽

Computational Approach ◽

Influenza Viruses ◽

Single Amino Acid ◽

Oseltamivir Resistance ◽

Amino Acid Mutations ◽

H275y Mutation ◽

Key Residues

Oseltamivir-resistant influenza viruses arise due to amino-acid mutations in key residues, but these changes often reduce their replicative and transmission fitness. Widespread oseltamivir-resistance has not yet been observed in A(H1N1)pdm09 viruses. However, it is known that permissive mutations in the neuraminidase (NA) of former seasonal A(H1N1) viruses from 2007-2009 buffered the detrimental effect of the NA H275Y mutation, resulting in fit oseltamivir-resistant viruses that circulated widely. This study explored two approaches to predict permissive mutations that may enable a fit H275Y A(H1N1)pdm09 variant to arise. A computational approach used phylogenetic and in silico protein stability analyses to predict potentially permissive mutations, which were then evaluated by in vitro NA enzyme activity and expression analysis, followed by in vitro replication. The second approach involved the generation of a virus library which encompassed all possible individual 2.9 x 104 codon mutations in the NA whilst keeping H275Y fixed. To select for variant viruses with the greatest fitness, the virus library was serially passaged in ferrets (via contact and aerosol transmission) and resultant viruses were deep sequenced. The computational approach predicted three NA permissive mutations, and even though they only offset the in vitro impact of H275Y on NA enzyme expression by 10%, they could restore replication fitness of the H275Y variant in A549 cells. In our experimental approach, a diverse virus library (97% of 8911 possible single amino-acid substitutions were sampled) was successfully transmitted through ferrets, and sequence analysis of resulting virus pools in nasal washes identified three mutations that improved virus transmissibility. Of these, one NA mutation, I188T, has been increasing in frequency since 2017 and is now present in 90% of all circulating A(H1N1)pdm09 viruses. Overall, this study provides valuable insights into the evolution of the influenza NA protein and identified several mutations that may potentially facilitate the emergence of a fit H275Y A(H1N1)pdm09 variant.

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