The R251K Substitution in Viral Protein PB2 Increases Viral Replication and Pathogenicity of Eurasian Avian-like H1N1 Swine Influenza Viruses

Abstract

The Eurasian avian-like swine (EA) H1N1 virus has affected the Chinese swine industry, and human infection cases have been reported occasionally. However, little is known about the pathogenic mechanism of EA H1N1 virus. In this study, we compared the mouse pathogenicity of A/swine/Guangdong/YJ4/2014 (YJ4) and A/swine/Guangdong/MS285/2017 (MS285) viruses, which had similar genotype to A/Hunan/42443/2015 (HuN-like). None of the mice inoculated with 10⁶ TCID₅₀ of YJ4 survived at 7 days post infection, while the survival rate of the MS285 group was 100%. Therefore, a series of single fragment reassortants in MS285 background and two rescued wild-type viruses were generated by using the reverse genetics method, and the pathogenicity analysis revealed that the PB2 gene contributed to the high virulence of YJ4 virus. Furthermore, there were 11 amino acid differences in PB2 between MS285 and YJ4 identified by sequence alignment, and 11 single amino acid mutant viruses were generated in the MS285 background. We found that the R251K mutation significantly increased the virulence of MS285 in mice, contributed to high polymerase activity and enhanced viral genome transcription and replication. These results indicate that PB2-R251K contributes to the virulence of the EA H1N1 virus and provide new insight into future molecular epidemiological surveillance strategies.

Keywords: Eurasian avian-like H1N1 swine influenza viruses; PB2 gene; PB2-R251K; pathogenicity; polymerase.

Figure 1

Weight changes and survival rates of mice infected with MS285 and YJ4 viruses. Six-week-old female BALB/c mice were inoculated intranasally with A/swine/Guangdong/YJ4/2014 and A/swine/Guangdong/MS285/2017 at 10⁶ TCID₅₀. The body weight change rate (A) and survival rate (B) in the infected mouse were continuously recorded for 14 days. Mice with weight loss of more than 25% of their initial body weight were euthanized humanely. Significant body weight changes of the YJ4 viruses-inoculated mice compared with MS285 viruses-inoculated mice (* p < 0.05; *** p < 0.001).

Figure 2

Weight changes and survival rates of mice infected with MS285 reassortant viruses. Six-week-old female BALB/c mice were inoculated intranasally with a series of reassortant viruses at 10⁶ TCID₅₀. The body weight change rate (A) and survival rate (B) in the infected mice were continuously recorded for 14 days. Mice with weight loss of more than 25% of their initial body weight were euthanized humanely. Significant body weight changes of the single fragment recombinant virus-inoculated mice compared with r/MS285 virus-inoculated mice (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 3

Weight changes, mortality, and replication of MS285 mutants in mice. Six-week-old female BALB/c mice were inoculated intranasally with 10⁶ TCID₅₀ of reassortant viruses containing single amino acid mutations. The body weight change rate (A) and survival rate (B) in the infected mice were continuously recorded for 14 days. Mice with weight loss of more than 25% of their initial body weight were euthanized humanely. The average body weight change rates in each group are displayed with error bars representing the standard deviations (±). Significant body weight changes of the mutant virus-inoculated mice compared with r/MS285 virus-inoculated mice (* p < 0.05; ** p < 0.01). Replication efficiency of viruses in the nasal turbinate (C) and lungs (D) of infected mice. At 1, 3, and 5 days post-infection (dpi) with 10⁶ TCID₅₀ of r/MS285 and r/MS285-251K viruses three mice were euthanized to collect the nasal turbinate and lungs. Virus replication was determined by TCID₅₀ assay in MDCK cells.

Figure 4

Growth kinetics of r/MS285 and r/MS285-251K viruses in MDCK and A549 cells. MDCK cells were infected at an multiplicity of infection (MOI) of 0.01 with r/MS285 and r/MS285-251K viruses and cultured at 37 °C (A) or 33 °C (C). A549 cells were infected at an MOI of 0.01 with r/MS285 and r/MS285-251K viruses and cultured at 37 °C (B) and 33 °C (D). Culture supernatants were harvested at 12, 24, 36, 48, and 60 hpi and subjected to TCID₅₀ assay in MDCK cells. The results are expressed as the means ± SD (n = 3) and the statistical significance was calculated using one-way ANOVA. * p < 0.05; ** p < 0.01.

Figure 5

Effect of PB2-R251K on polymerase activity, RNA synthesis, and protein expression. (A) Polymerase activity was analyzed in 293T cells co-transfected with pPolI-NP-luc, pPL-TK, and expression plasmids encoding the MS285 PB1, PA, and NP genes and a PB2 clones (WT or R251K) at 33 °C or 37 °C. Relative polymerase luciferase activity compared to r/MS285 polymerase complex. (B) A549 cells were infected at an MOI of 1 with r/MS285 and r/MS285-251K viruses and cultured at 37 °C. After 24 hpi, the relative levels of viral NP segment viral RNA (vRNA), complement RNA (cRNA), and messenger RNA (mRNA) were quantified by strand-specific real-time RT-qPCR and normalized against 18S rRNA levels. The vRNA, cRNA, and mRNA values were expressed relative to the results for r/MS285. (C) A549 cells were infected at an MOI of 1 with r/MS285 and r/MS285-251K viruses and cultured at 37 °C. Cell lysates were prepared at 3, 6, 9, and 12 hpi to evaluate the expression levels of NP and PB2 by Western blotting. GAPDH was used as a loading control. In (A,B), the results are expressed as the means ± SD (n = 3), and the statistical significance was calculated using one-way ANOVA. ** p < 0.01, *** p < 0.001.