Introduction of virulence markers in PB2 of pandemic swine-origin influenza virus does not result in enhanced virulence or transmission

Abstract

In the first 6 months of the H1N1 swine-origin influenza virus (S-OIV) pandemic, the vast majority of infections were relatively mild. It has been postulated that mutations in the viral genome could result in more virulent viruses, leading to a more severe pandemic. Mutations E627K and D701N in the PB2 protein have previously been identified as determinants of avian and pandemic influenza virus virulence in mammals. These mutations were absent in S-OIVs detected early in the 2009 pandemic. Here, using reverse genetics, mutations E627K, D701N, and E677G were introduced into the prototype S-OIV A/Netherlands/602/2009, and their effects on virus replication, virulence, and transmission were investigated. Mutations E627K and D701N caused increased reporter gene expression driven by the S-OIV polymerase complex. None of the three mutations affected virus replication in vitro. The mutations had no major impact on virus replication in the respiratory tracts of mice and ferrets or on pathogenesis. All three mutant viruses were transmitted via aerosols or respiratory droplets in ferrets. Thus, the impact of key known virulence markers in PB2 in the context of current S-OIVs was surprisingly small. This study does not exclude the possibility of emergence of S-OIVs with other virulence-associated mutations in the future. We conclude that surveillance studies aimed at detecting S-OIVs with increased virulence or transmission should not rely solely on virulence markers identified in the past but should include detailed characterization of virus phenotypes, guided by genetic signatures of viruses detected in severe cases of disease in humans.

FIG. 1.

Activity of the NL602 polymerase complex with wild type (wt) PB2, PB2-E627K, PB2-E677G, and PB2-D701N in minigenome assays. Firefly luciferase was expressed from a synthetic viral RNA when the influenza virus polymerase complex was present. The luminescence of the firefly luciferase reporter was standardized using a plasmid constitutively expressing Renilla luciferase protein to correct for differences in transfection efficiencies and sample processing. Relative luminescence was calculated as the percent relative light units (firefly luciferase/Renilla luciferase), and the fold difference in reporter protein expression was calculated with the wild-type polymerase complex as a reference. Averages and standard deviations from two independent experiments performed in duplicate are shown. Neg, negative control.

FIG. 2.

Replication kinetics of NL602, NL602/PB2-627K, NL602/PB2-677G, and NL602/PB2-701N viruses in MDCK cells. MDCK cells were inoculated with 0.01 TCID₅₀/cell of the NL602 (closed circles), NL602/PB2-627K (open circles), NL602/PB2-677G (closed squares), and NL602/PB2-701N (open squares) viruses, and supernatant samples were harvested 6, 12, 24, and 48 h later. The supernatant samples were titrated in MDCK cells. Geometric mean titers and standard deviations were calculated from two independent experiments.

FIG. 3.

Weight loss of BALB/c mice inoculated with NL602, NL602/PB2-627K, NL602/PB2-677G, and NL602/PB2-701N viruses. Mean bodyweights and standard deviations were calculated as percentages of bodyweight compared to bodyweight at the time of inoculation for each group inoculated with NL602 (closed circles), NL602/PB2-627K (open circles), NL602/PB2-677G (closed squares), and NL602/PB2-701N (open squares) viruses.

FIG. 4.

Weight loss of ferrets inoculated with NL602, NL602/PB2-627K, NL602/PB2-677G, and NL602/PB2-701N viruses. Bodyweight is depicted as percentage of bodyweight at time of inoculation. Data are shown for individual animals until the animals were euthanized at 3 or 7 days p.i.

FIG. 5.

Virus shedding from the upper respiratory tract of ferrets inoculated with NL602, NL602/PB2-627K, NL602/PB2-677G, and NL602/PB2-701N viruses. Virus detection in throat swabs (A) and nose swabs (B) is indicated for NL602 (black bars), NL602/PB2-627K (white bars), NL602/PB2-677G (dark-gray bars), and NL602/PB2-701N (light-gray bars). Geometric mean titers for positive samples are displayed, and the error bars indicate the standard deviations. The lower limits of detection are indicated by the dotted lines.

FIG. 6.

Transmission of NL602, NL602/PB2-627K, NL602/PB2-677G, and NL602/PB2-701N viruses by aerosol or respiratory droplets in ferrets. Virus titers in throat (black bars) and nose (white bars) swabs are displayed for inoculated (A to D) and exposed (E to H) ferrets. The geometric mean titers of positive samples are displayed, and the error bars indicate the standard deviations. The number of positive contact animals per day is depicted. The asterisk indicates that one ferret was negative for virus shedding but seroconverted to influenza A virus nucleoprotein in the course of the experiment. The lower limits of detection are indicated by the dotted lines.