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.

The pathogenicities of influenza viruses are determined by multiple viral genes. The results of our previous study indicated that hemagglutinin (HA) gene substitutions of the two genetically similar H1N1 swine influenza viruses altered their pathogenicities in mice. This study aimed to further identify the key amino acids affecting viral pathogenicity.

After analyzing the amino acid differences of HA protein between the two H1N1 viruses, the reassortant viruses bearing the single amino acid mutations were constructed using the site-directed mutagenesis primers, and their EID₅₀ values were determined. To determine the growth of the parental, reassortant and mutant viruses in vitro, MDCK cells and A549 cells were infected with the indicated viruses at a multiplicity of infection (MOI) of 0.001 and 0.1, respectively. The BALB/c mice was further intranasally (i.n.) inoculated with 10⁶ EID₅₀ of each virus, and three mice were euthanized at 3 days post-infection (dpi). The organs, including brain, nasal turbinate, lung, kidney and spleen, were collected from the mice and titrated in eggs to evaluate the viral replication abilities in vivo. The MLD₅₀ values of the indicated viruses were determined by inoculating i.n. groups of five mice with 10¹-10⁶ EID₅₀ of viruses. The body weight was measured daily for 14 dpi, and the mice that lost more than 25% of their original weight were euthanized for humane reasons.

The HA proteins of the ZD71 and SY130 viruses differed at four amino acids at positions 4, 138, 144, and 225 (H3 numbering). Four reassortants were rescued, followed by whole-genome sequencing to ensure the absence of unwanted mutations. The viral replication abilities of the reassortant viruses (rZD71-HA/G225E and rSY130-HA/E225G) were significantly affected in MDCK, as well as in A549 cells, when G225E and E225G substitutions were introduced into the rZD71 and rSY130 virus, respectively. In contrast, the mutations of the other three amino acids had little effect on viral replication in vitro. Further mouse infection experiments also demonstrated that amino acid substitutions at site 225 of HA protein significantly affected the viral pathogenicities in mice. In particular, the substitution G225E increased the pathogenicity of rZD71-HA/G225E virus, with the MLD₅₀ value of rZD71-HA/G225E virus decreasing from 4.32 log₁₀EID₅₀ to 3.0 log₁₀EID₅₀, compared with that of rZD71 virus. And the virus replicated well not only in the nasal turbinate and lung, but also in the spleen and kidney.

A single amino acid at position 225 in the HA protein significantly affects the viral replication capacity and virulence of these two H1N1 swine influenza viruses. It is suggested that close monitoring for this residue should be paid in the future virological surveillance, so as to provide a scientific basis for better prevention and control of animal influenza, and even human influenza pandemic.

The antigenicity of influenza virus is mainly determined by the hemagglutinin (HA), a surface glycoprotein of the virus. Our previous study indicated that amino acid changes at positions 190, 230 and 269 (H3 numbering) of HA protein resulted in antigenic escape by using the monoclonal antibody (mAb) against the HA protein of Eurasian avian-like H1N1 swine influenza virus (EA H1N1 SIV). These three amino acid substitutions widely existed in the HA protein of the recently isolated EA H1N1 SIVs.

This study aimed to explore which amino acids played a key role in the antigenicity of the virus, and further provide scientific basis for the control of influenza.

In this study, A/swine /Liaoning /SY72/2018 (H1N1) (SY72) was selected as the model virus, and its reverse genetic system (RGS) was established. Then, using SY72 virus as backbone, three reassortant viruses were rescued by introducing the respective single amino acid mutation at position 190, 230, and 269 into HA protein. Antisera were raised by inoculating the rSY72-inactivated vaccine into specific-pathogen-free (SPF) chickens and non-immunized pigs. The effects of each of these substitutions on viral antigenicity were determined by measuring the neutralization and hemagglutination inhibition (HI) titers with mAbs and polyclonal sera raised against the rSY72 virus. Then their effects on viral replication capacities and receptor binding properties were further evaluated.

Sequence analysis showed that the HA of SY72 virus carried 190D, 230M, and 269R, respectively. The viruses, including rSY72, rSY72HA/D190N, rSY72HA/M230I, and rSY72HA/R269M, were rescued by RGS. The results of neutralization test showed that all the three mutant viruses could react with two mAbs, to some extent, compared with the rSY72 virus. The HI results indicated that the HI antibody titers of the rSY72HA/D190N reacted with the rSY72–immunized chicken and pig sera were 4- and 8-fold lower than those of the rSY72 virus with the respective sera. However, the rSY72HA/M230I and rSY72HA/R269M virus reacted well with these two sera. The results indicated that residue 190 in the HA had the important effects on the viral antigenicity. Results of the viral plaque assay and growth curve experiments demonstrated that plaque sizes generated by the rSY72HA/D190N virus were smaller than those by the rSY72 virus in MDCK cells. The replication ability of the rSY72HA/R269M virus was significantly decreased in MDCK cells, compared with that of the rSY72 virus. Furthermore, the three amino acid mutations had no impact on the viral receptor-binding preference.

The amino acid at position 190 of HA protein played an important role in determining the antigenicity of EA H1N1 SIV. The mutation of amino acid at position 269 reduced the viral replication ability in MDCK cells. These results suggested that more attentions should be paid for monitoring these residue changes in the influenza surveillance, so as to improve early warning of influenza in animals.