that of the original rg(N224K/Q226L)/CA04 (104.6p.f.u. g–1; Fig. 3, ferret 1 of rg(N224K/Q226L)/CA04), whereas the virus with the N158K/N224K/Q226L mutations (105.6p.f.u.g–1; Fig. 3, ferret 3 of rg(N224K/Q226L)/CA04) grew to one order of magnitude higher than the original mutant. These data indicate that the additional mutation N158D improved the replication of rg (N224K/Q226L)/CA04 in ferrets. To test the effect of this mutation on the replication of H5 reassortant viruses in ferrets, we examined the replicative ability of a virus with the triple N158D/N224K/Q226L HA substitutions in ferrets. This HA(N158D/N224K/Q226L)/CA04 virus replicated efficiently in infected animals, except in the trachea (Fig. 3 and Supplementary Table 2). On day 3 after infection, this virus was isolated from the brain of two of the three animals tested, although we did not observe neurological signs in these animals. These results indicate that the N158D mutation contributed to the efficient growth in the nasal turbinates of ferrets of an H5 reassortant virus with the N224K/Q226L mutations. Removal of the glycosylation site at position 158 has been reported to result in enhanced binding of H5N1 viruses to human-type receptors in combination with the Q226L/G228S mutations7. A previous study showed that H5N1 viruses lacking this glycosylation site transmit efficiently by direct contact among guinea-pigs31. By contrast, H5N1 viruses that acquire this glycosylation site lose the ability to transmit among guinea-pigs. Therefore, we speculated that the loss of the glycosylation site in HA(N158D/N224K/Q226L)/CA04 virus may affect its transmissibility in ferrets.

To assess the ability of H5 reassortant viruses with human-type receptor specificity to transmit between ferrets, we placed naive ferrets in wireframe cages next to ferrets inoculated with 106 p.f.u. of rgCA04, rgVN1203/CA04, rg(N224K/Q226L)/CA04, or HA(N158D/N224K/Q226L)/CA04 (Supplementary Fig. 7). Similar to previous experiments32, rgCA04 was efficiently transmitted via respiratory droplets to all three contact ferrets, as evidenced by the detection of virus in nasal washes and haemagglutination inhibition (HI) antibody in these animals (Table 1 and Fig. 4). By contrast, rgVN1203/CA04 and rg(N224K/Q226L)/CA04 were not transmitted; neither virus shedding nor seroconversion was detected in any contact animals, despite the binding of the latter to Siaα2,6Gal. This result was consistent with that of previous studies in which human-type receptor recognition was shown to be necessary but not sufficient for respiratory droplet transmission of an H5N1 virus in a ferret model12,14. In the HA(N158D/N224K/Q226L)/CA04-inoculated group, virus was recovered from two of the six contact ferrets (pairs 1 and 2) between days 5 and 7 after contact. Moreover, seroconversion was detected in five animals including those from which virus was recovered. No animals died in the course of these transmission experiments. This finding demonstrates the generation of an H5 HA that supports virus transmission by respiratory droplets among ferrets.

To determine whether additional mutations occurred in the HA of HA(N158D/N224K/Q226L)/CA04 during transmission, viral RNA was analysed from nasal washes of inoculated and contact ferrets (Fig. 4 and Supplementary Table 4). On day 5 after infection, the A242S andT318I mutations in HA were present in five (pairs 1,3,4,5 and 6) and one (pair 2) of the six inoculated animals, respectively. Viruses derived from the contact animals of pair 1 on day 7 after contact had two changes in HA (K193N and A242S) (Fig. 1a), whereas those derived from the contact animals of pair 2 contained a single change in HA (T318I) (Fig. 1b), indicating that additional changes in HA occurred during the infection of ferrets with HA(N158D/N224K/Q226L)/CA04. No mutations in the remaining genes were detected in any of these viruses from nasal washes compared with the CA04 virus sequences.

Because HA(N158D/N224K/Q226L)/CA04 was isolated from only one-third of the contact animals, we isolated a virus from the nasal wash of the contact ferret that shed a high titre (107.5 p.f.u. ml–1 ) of virus on day 7 after contact (pair 2) (Fig. 4d) to evaluate the replication and transmissibility of that virus in ferrets. This mutant virus, designated HA(N158D/N224K/Q226L/T318I)/CA04, replicated efficiently in the nasal turbinates and was isolated from brain tissue (Fig. 3 and Supplementary Table 2). In the transmission study, four of the six contact ferrets were positive for virus between days 3 and 7 after contact, and all contact animals were seropositive; no animals died in the course of the transmission experiments (Table 1; Fig. 4e and Supplementary Fig. 8). Notably, this transmission pattern is comparable to that of the 1918 pandemic H1N1 virus when tested under the same experimental conditions; the 1918 pandemic virus was recovered from the nasal wash of two of three contact animals (our own unpublished data). Sequence comparison of viruses from inoculated and contact animals identified mutations at positions 225 and 242 as well as a reversion at position 224 (Fig. 1a and Supplementary Table 5) (in addition to the original four mutations) although the 224 reversion was found only in viruses from inoculated ferrets. Collectively, these findings demonstrate that four amino acid substitutions (N158D/N224K/Q226L/T318I) in H5 HA confer efficient respiratory droplet transmission in ferrets to a virus possessing an H5 HA in a 2009 pandemic H1N1 backbone. We also confirmed that recombinant viruses possessing the three HA mutations N158D, N224K and Q226L, or the four HA mutations N158D, N224K, Q226L and T318I, and the NA of VN1203 in a PR8 backgrand (designated N158D/N224K/Q226L or N158D/N224K/Q226L/T318I, respectively) preferentially bind to Siaα2,6Gal and attach to human tracheal epithelia (Fig. 2c, d).

HA(N158D/N224K/Q226L/T318I)/CA04 transmitted by respiratory droplet more efficiently than HA(N158D/N224K/Q226L)/CA04, raising the possibility that the T318I mutation is involved in the efficient transmission of avian H5N1/pandemic H1N1 reassortants. To explore the functional role of this mutation in respiratory droplet transmission, we generated an H5 reassortant expressing the H5 HA with the T318I mutation and examined its receptor-binding specificity and transmissibility. This reassortant (designated rgT318I/CA04) bound to only Siaα2,3Gal and showed little binding to human tracheal epithelia (Fig. 2c, d). rgT318I/CA04 did not transmit via respiratory droplet among ferrets (Table 1 and Fig. 4f), although it replicated in nasal turbinates and trachea as efficiently as rgCA04 (Fig. 3 and Supplementary Table 2). These results indicate that the T318I mutation alone is not sufficient for H5 reassortant viruses to transmit efficiently among ferrets.

Table 1 | Transmission in ferrets inoculated with H5 avian-human reassortant viruses

Virus Inoculated ferrets Contact ferrets
Weight loss (%)* Peak virus titre in nasal wash (mean log10(p.f.u. ml21)) (days after inoculation) Seroconversion (positive and total numbers) (HI titre){ Virus detection in nasal wash (positive and total numbers) Seroconversion (positive and total numbers) (HI titre)
rgCA04 3 of 3 (15.1) 7.5 (1) 3 of 3 (≥1,280, ≥1,280, ≥1,280) 3 of 3 3 of 3 (≥1,280, ≥1,280, ≥1,280)
rgVN1203/CA04 3 of 3 (5.9) 5.3 (5) 3 of 3 (80, 40, 80) 0 of 3 0 of 3 (,10, ,10, ,10)
rg(N224K/Q226L)/CA04 2 of 3 (7.8){ 3.9 (5) 3 of 3 (≥1,280, ≥1,280, ≥1,280) 0 of 3 0 of 3 (,10, ,10, ,10)
HA(N158D/N224K/Q226L)/ 6 of 6 (5.7) 6.7 (3) 6 of 6 (640, ≥1,280, ≥1,280, 640, 2 of 6 5 of 6 (160, 320, 20, 160, 40, ,10)
CA04 ≥1,280, ≥1,280)
HA(N158D/N224K/Q226L/ 6 of 6 (9.8) 6.1 (5) 6 of 6 (≥1,280, ≥1,280, 640, ≥1,280, 4 of 6 6 of 6 (640, 640, ≥1280, 80,
T318I)/CA04 ≥1 280 ≥1 280) ≥1 280 320)
rgT318I/CA04 3 of 5 (1.5)1 5.6 (3) 5 of 5 (40, 20, 20, 40, 40) 0 of 5 0 of 5 (,10, ,10, ,10, ,10, ,10)

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