Identification and Functional Analysis of Adaptive Amino Acid Mutations in the Eurasian Avian-Like H1N1 Swine Influenza Virus

Influenza A virus (IAV) can cross species barriers through adaptive mutations, and infect a variety of mammals, including humans, posing a continual threat to global public health. Based on cell passage culture, the adaptive amino acid mutations of influenza virus were identified and their impacts on viral biological characteristics of were further investigated, which could provide a theoretical guidance for the prevention and control of influenza

A/swine/Zhejiang/199/2013 (H1N1) (ZJ199) virus was passaged in MDCK cells, with the adaptive amino acid mutations occurred in the virus after three passages, which was named ZJ199-P3. The replication capacity and infectivity of the ZJ199 and ZJ199-P3 viruses were compared both in vitro and in vivo. A reverse genetics system for ZJ199 was established, and recombinant viruses containing single-site mutation were rescued using rZJ199 as a backbone. The replication capacities of the parental virus and the single-site mutant recombinant viruses were determined in vitro to identify key amino acid sites affecting viral replication. The impacts of the adaptive mutations on viral biological activity were further investigated by measuring viral polymerase activity, neuraminidase activity, and the activity of ISRE and NF-κB reporter genes.

Comparative analysis of the amino acid sequences between the ZJ199 and ZJ199-P3 viruses revealed three mutations in the ZJ199-P3 virus, including A234T in the NP protein, S21G in the NA protein, and M100I in the NS2 protein, respectively. Replication capacity assays demonstrated that the ZJ199-P3 virus exhibited reduced replication abilities both in vitro and in vivo, compared with the ZJ199 virus. Using reverse genetics system, recombinant virus rZJ199 and its single-point mutant viruses including rZJ199-NA-S21G, rZJ199-NP-A234T, and rZJ199-NS2-M100I were rescued. In vitro replication analysis showed that the NP-A234T mutation significantly impaired viral replication capacity compared with the rZJ199 virus, whereas the NA-S21G and NS2-M100I mutations had no apparent effect on viral replication. Further studies demonstrated that the NP-A234T mutation reduced viral polymerase activity, the NA-S21G mutation decreased neuraminidase enzymatic activity, and the NS2-M100I mutation weakened the inhibitory effects on ISRE and NF-κB reporter gene activities.

Through serial passaging in cells, three adaptive mutations were identified in the EA H1N1 SIV. The A234T mutation in the NP reduced viral polymerase activity, resulting in diminished viral replication in vitro. The S21G mutation in the NA protein and the M100I mutation in the NS2 protein both impacted the virus's biological activity. These findings underscored the importance of enhanced surveillance of influenza viruses to provide critical scientific evidence for the development of effective influenza prevention and control strategies.