Multidrug resistant 2009 A/H1N1 influenza clinical isolate with a neuraminidase I223R mutation retains its virulence and transmissibility in ferrets

Abstract

Only two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that influenza virus becomes resistant to these antiviral drugs and spreads in the human population. The 2009 pandemic A/H1N1 influenza virus is naturally resistant to adamantanes. Recently a novel neuraminidase I223R mutation was identified in an A/H1N1 virus showing cross-resistance to the neuraminidase inhibitors oseltamivir, zanamivir and peramivir. However, the ability of this virus to cause disease and spread in the human population is unknown. Therefore, this clinical isolate (NL/2631-R223) was compared with a well-characterized reference virus (NL/602). In vitro experiments showed that NL/2631-I223R replicated as well as NL/602 in MDCK cells. In a ferret pathogenesis model, body weight loss was similar in animals inoculated with NL/2631-R223 or NL/602. In addition, pulmonary lesions were similar at day 4 post inoculation. However, at day 7 post inoculation, NL/2631-R223 caused milder pulmonary lesions and degree of alveolitis than NL/602. This indicated that the mutant virus was less pathogenic. Both NL/2631-R223 and a recombinant virus with a single I223R change (recNL/602-I223R), transmitted among ferrets by aerosols, despite observed attenuation of recNL/602-I223R in vitro. In conclusion, the I223R mutated virus isolate has comparable replicative ability and transmissibility, but lower pathogenicity than the reference virus based on these in vivo studies. This implies that the 2009 pandemic influenza A/H1N1 virus subtype with an isoleucine to arginine change at position 223 in the neuraminidase has the potential to spread in the human population. It is important to be vigilant for this mutation in influenza surveillance and to continue efforts to increase the arsenal of antiviral drugs to combat influenza.

Figure 1. Replication kinetics in MDCK or MDCK-SIAT1 cells.

MDCK (panel A) or MDCK-SIAT1 (panel B) cells were inoculated with 0.001 TCID₅₀ virus per cell of recNL/602 (black circles), isolate NL/2631-R223 (black triangles) and recNL/602-I223R (open circles). Supernatants were harvested after 6, 12, 24, and 48 hours post infections and were titrated in MDCK cells. Geometric mean titers and standard deviations were calculated from two independent experiments. The lower limit of detection is indicated by the dotted line.

Figure 2. Ferret relative weight loss and virus shedding from the ferret upper respiratory tract.

Ferrets were inoculated intratracheally with 1×10⁶ TCID₅₀ of NL/602 or NL/2631-R223. Body weights for NL/602 (Panel A) and NL/2631-R223 (Panel B) inoculated animals are depicted as percentage of body weight relative to the time of inoculation. Data are shown for individual animals until the animals were euthanized at day 4 or 7 p.i.. Virus detection in throat (panel C) and nose swabs (panel D) is indicated for NL/602 (white bars), and NL/2631-R223 (black bars). Geometric mean titers from 6 (day 1 to 4) or 3 animals (day 5 to 7) are displayed and the error bars indicate the standard deviations. The number of influenza virus positive animals per day is depicted in each bar. The lower limit of detection is indicated by the dotted line.

Figure 3. Semi-quantitative lung scores and histological examination of the infected ferret respiratory tract.

Lung virus titers (panel A), percentage of affected lung tissue (panel B) and relative lung weights (panel C) were determined for lungs of ferrets inoculated with NL/2631-R223 (triangles) or NL/602 (circles) that were euthanized at day 4 or 7 p.i.. Semi-quantitative assessment of the extent and severity of the tracheitis (panel D), bronchiolitis (panel E) and alveolitis (panel F) are shown. Individual values are displayed. In panel A, the lower limit of detection is indicated by a dotted line.

Figure 4. Transmission of NL/602 and NL/2631-R223 by aerosol or respiratory droplets in ferrets.

Virus titers in throat (black bars) and nose swabs (grey bars) are displayed for inoculated (Panel A and C) and exposed ferrets (Panel B and D). The geometric mean titers of positive samples are displayed and the error bars indicate the standard deviations. The number of positive exposed animals per day is depicted. The lower limit of detection is indicated by the dotted line.

Figure 5. Transmission of recNL/602 and recNL/602-I223R by aerosol or respiratory droplets in ferrets.

Virus titers in throat (black bars) and nose swabs (grey bars) are displayed for inoculated (Panel A and C) and exposed ferrets (Panel B and D). The geometric mean titers of positive samples are displayed and the error bars indicate the standard deviations. The number of positive exposed animals per day is depicted. The lower limit of detection is indicated by the dotted line.