Virulence-affecting amino acid changes in the PA protein of H7N9 influenza A viruses

Abstract

Novel avian-origin influenza A(H7N9) viruses were first reported to infect humans in March 2013. To date, 143 human cases, including 45 deaths, have been recorded. By using sequence comparisons and phylogenetic and ancestral inference analyses, we identified several distinct amino acids in the A(H7N9) polymerase PA protein, some of which may be mammalian adapting. Mutant viruses possessing some of these amino acid changes, singly or in combination, were assessed for their polymerase activities and growth kinetics in mammalian and avian cells and for their virulence in mice. We identified several mutants that were slightly more virulent in mice than the wild-type A(H7N9) virus, A/Anhui/1/2013. These mutants also exhibited increased polymerase activity in human cells but not in avian cells. Our findings indicate that the PA protein of A(H7N9) viruses has several amino acid substitutions that are attenuating in mammals.

Importance: Novel avian-origin influenza A(H7N9) viruses emerged in the spring of 2013. By using computational analyses of A(H7N9) viral sequences, we identified several amino acid changes in the polymerase PA protein, which we then assessed for their effects on viral replication in cultured cells and mice. We found that the PA proteins of A(H7N9) viruses possess several amino acid substitutions that cause attenuation in mammals.

FIG 1

Phylogenetic tree and inferred ancestral changes of influenza A virus PA. A phylogenetic tree of the PA gene, which includes the A(H7N9) viruses and neighboring and ancestral sequences that may have played a role in the genesis of A(H7N9) PA, is shown. A(H7N9) PA genes are shown in red; the group of chicken H9N2 viruses is indicated in green, while the groups of duck and wild-bird viruses are indicated in orange. The Anhui/1 virus used in the present study is indicated by a purple arrow. The MRCA of A(H7N9) PA genes is indicated by a red circle. Ancestral inference was carried out for branches on the path to A(H7N9) PA located between an ancestral node (blue circle) and the MRCA; all significant bootstrap values (i.e., those >70) are shown. Also shown is the potentially mammalian-adapting PA-V100A substitution, which occurred within the group of A(H7N9) PA genes.

FIG 2

Viral polymerase activity in vitro. A549 or DF-1 cells were transfected with plasmids encoding the PB2, PB1, NP, and wild-type or mutant PA proteins, with a plasmid for the expression of a virus-like RNA encoding the firefly luciferase gene, and with a control plasmid encoding Renilla luciferase. At 24 h posttransfection, firefly and Renilla luciferase activities were measured by means of a dual-luciferase assay. Polymerase activity was calculated by normalization of the firefly luciferase activity to the Renilla luciferase activity. The data are shown as the relative polymerase activity with the standard deviation (SD; n = 3). The polymerase activity of the wild-type PA was set to 100%. * and **, P < 0.05 and P < 0.01, respectively, according to a one-way analysis of variance (ANOVA), followed by a Dunnett's test.

FIG 3

Growth kinetics of mutant viruses in vitro. A549 (A) or DF-1 (B) cells were infected with the indicated viruses at an MOI of 0.001. The supernatants of the infected cells were collected at the indicated time points. Virus titers were determined by use of a plaque assay in MDCK cells. The virus titers are means ± the SD (n = 3). * and **, P < 0.05 and P < 0.01, respectively, according to a one-way ANOVA, followed by a Dunnett's test.

FIG 4

Virulence of mutant viruses in mice. Four mice per group were intranasally inoculated with 10¹, 10², 10³, 10⁴, 10⁵, or 10⁶ PFU (each in 50 μl) of the indicated viruses. Body weight and survival were monitored daily. Mice that lost >25% of their baseline weight were euthanized. The values represent the average of body weight compared to the baseline weight ± the SD from four mice.