Enhanced mammalian transmissibility of seasonal influenza A/H1N1 viruses encoding an oseltamivir-resistant neuraminidase

Abstract

Between 2007 and 2009, oseltamivir resistance developed among seasonal influenza A/H1N1 (sH1N1) virus isolates at an exponential rate, without a corresponding increase in oseltamivir usage. We hypothesized that the oseltamivir-resistant neuraminidase (NA), in addition to being relatively insusceptible to the antiviral effect of oseltamivir, might confer an additional fitness advantage on these viruses by enhancing their transmission efficiency among humans. Here we demonstrate that an oseltamivir-resistant clinical isolate, an A/Brisbane/59/2007(H1N1)-like virus isolated in New York State in 2008, transmits more efficiently among guinea pigs than does a highly similar, contemporaneous oseltamivir-sensitive isolate. With reverse genetics reassortants and point mutants of the two clinical isolates, we further show that expression of the oseltamivir-resistant NA in the context of viral proteins from the oseltamivir-sensitive virus (a 7:1 reassortant) is sufficient to enhance transmissibility. In the guinea pig model, the NA is the critical determinant of transmission efficiency between oseltamivir-sensitive and -resistant Brisbane/59-like sH1N1 viruses, independent of concurrent drift mutations that occurred in other gene products. Our data suggest that the oseltamivir-resistant NA (specifically, one or both of the companion mutations, H275Y and D354G) may have allowed resistant Brisbane/59-like viruses to outtransmit sensitive isolates. These data provide in vivo evidence of an evolutionary mechanism that would explain the rapidity with which oseltamivir resistance achieved fixation among sH1N1 isolates in the human reservoir.

Fig 1

The oseltamivir-resistant influenza A isolate NY/08-1326(R) was transmitted subtly but consistently more efficiently among guinea pigs than the oseltamivir-sensitive isolate NY/08-1253(S). (A and B) In a contact transmission model, the oseltamivir-sensitive clinical isolate NY/08-1253(S) was transmitted among 75% of guinea pigs (A), while the oseltamivir-resistant clinical isolate NY/08-1326(R) was transmitted among 100% of guinea pigs (B). (C and D) In an aerosol/droplet model at 5°C and 20% relative humidity, NY/08-1253(S) was transmitted among 25% of guinea pigs (C), while NY/08-1326(R) was transmitted among 38% of guinea pigs. Nasal wash virus titers are plotted as a function of day postinoculation. Each dotted line with open symbols represents the nasal wash titers of a virus-inoculated (donor) guinea pig, and each solid line with closed symbols represents the nasal wash titers of a virus-exposed (recipient) guinea pig. Seroconversion status (positive or negative) is shown next to the 10-dpi nasal wash titer of each exposed guinea pig.

Fig 2

In both contact and aerosol/droplet models, the oseltamivir-resistant isolate NY/08-1326(R) was transmitted more rapidly among guinea pigs than the oseltamivir-sensitive isolate NY/08-1253(S). The mean AUC_{2–6 dpi} of guinea pigs inoculated with the oseltamivir-sensitive isolate NY/08-1253(S) (circular symbols) and those inoculated with the oseltamivir-resistant isolate NY/08-1326(R) (square symbols) were similar. (B) The mean nasal wash AUC_{2–6 dpi} values for guinea pigs exposed to the sensitive isolate NY/08-1253(S) were significantly lower than the mean nasal wash AUC_{2–6 dpi} values for guinea pigs exposed to the resistant isolate NY/08-1326(R). Horizontal lines denote the mean AUC_{2–6 dpi}. *, P < 0.05; **, P < 0.01. S, oseltamivir sensitive; R, oseltamivir resistant.

Fig 3

Clinical isolates and reverse genetics viruses demonstrated similar in vitro growth kinetics. (A) In MDCK cells, the clinical isolates NY/08-1253(S) and NY/08-1326(R) displayed similar growth kinetics, but the oseltamivir-resistant isolate achieved a titer approximately 0.5 log higher by 72 hpi. (B) This growth pattern was reproduced by the reverse genetics clones rg1253(S) and rg1326(R). The 7:1 reassortant rg1253(S):1326(R)-NA attained similar titers as rg1253(S), and the 6:2 reassortant rg1253(S):1326(R)-HA/NA attained similar titers as rg1326(R). (C) All four reverse genetics clones achieved similar 72-hpi titers in human lung epithelial (A549) cells. Cells were inoculated at an MOI of 0.01 and incubated at 33°C and 5% CO₂. Error bars represent 1 SD.

Fig 4

Reverse genetics reassortants expressing the oseltamivir-resistant NA were transmitted more efficiently among guinea pigs than the oseltamivir-sensitive reverse genetics clone rg1253(S). (A) The oseltamivir-sensitive reverse genetics clone rg1253(S) was transmitted among 75% (6 of 8 pairs) of guinea pigs. (B) The 7:1 reassortant rg1253(S):1326(R)-NA was transmitted among 100% (8 of 8 pairs) of guinea pigs. (C) The 6:2 reassortant rg1253(S):1326(R)-HA/NA was transmitted among 88% (7 of 8 pairs) of guinea pigs. Nasal wash virus titers are plotted as a function of day postinoculation. Each dotted line with open symbols represents the nasal wash titers of a virus-inoculated (donor) guinea pig, and each solid line with closed symbols represents the nasal wash titers of a virus-exposed (recipient) guinea pig. Seroconversion status (positive or negative) is shown next to the 10-dpi nasal wash titer of each exposed guinea pig.

Fig 5

The oseltamivir-sensitive reverse genetics viruses rg1253(S) and rg1253(S):Bris/59-NA grew similarly in vitro and were transmitted similarly in vivo. (A) The reverse genetics viruses rg1253(S) and rg1253(S):Bris/59-NA, which differ by two residues in the viral NA (S336N and M430L), displayed comparable growth kinetics in MDCK cells. Cells were inoculated at an MOI of 0.01 and incubated at 33°C and 5% CO₂. Error bars represent 1 SD. (B) The 7:1 reassortant rg1253(S):Bris/59-NA was transmitted among 88% (7 of 8 pairs) of guinea pigs. Nasal wash virus titers are plotted as a function of day postinoculation. Each dotted line with open symbols represents the nasal wash titers of a virus-inoculated (donor) guinea pig, and each solid line with closed symbols represents the nasal wash titers of a virus-exposed (recipient) guinea pig. Seroconversion status (positive or negative) is shown next to the 10-dpi nasal wash titer of each exposed guinea pig.

Fig 6

Viruses encoding the oseltamivir-resistant NA were transmitted more rapidly than viruses encoding the oseltamivir-sensitive NA. (A) The mean AUC_{2–6 dpi} of guinea pigs inoculated with clinical isolates (dark gray symbols) and reverse genetics viruses (white or light gray symbols) were generally similar, with the exception of rg1253(S):1326(R)-NA, which was cleared significantly faster than either rg1253(S) or rg1253(S):1326(R)-HA/NA. (B) The mean nasal wash AUC_{2–6 dpi} of guinea pigs inoculated with viruses expressing the sensitive NA (circular symbols) was similar to that of animals inoculated with viruses expressing the resistant NA (square, diamond, and hexagon symbols). (C) The mean nasal wash AUC_{2–6 dpi} values for guinea pigs exposed to NY/08-1253(S) was significantly lower than that of guinea pigs exposed to NY/08-1326(R), and the mean nasal wash AUC_{2–6 dpi} values for guinea pigs exposed to rg1253(S) or rg1253(S):Bris/59-NA were significantly lower than that of guinea pigs exposed to rg1253(S):1326(R)-NA. (D) Transmission events in guinea pigs exposed to viruses expressing the sensitive NA occurred significantly more slowly, as measured by the AUC_{2–6 dpi}, than did transmission events in those exposed to viruses expressing the resistant NA. Horizontal lines denote the mean AUC_{2–6 dpi}. *, P < 0.05; **, P < 0.01; ***, P < 0.001. S, oseltamivir sensitive; R, oseltamivir resistant.