Airborne transmission of influenza A/H5N1 virus between ferrets

Abstract

Highly pathogenic avian influenza A/H5N1 virus can cause morbidity and mortality in humans but thus far has not acquired the ability to be transmitted by aerosol or respiratory droplet ("airborne transmission") between humans. To address the concern that the virus could acquire this ability under natural conditions, we genetically modified A/H5N1 virus by site-directed mutagenesis and subsequent serial passage in ferrets. The genetically modified A/H5N1 virus acquired mutations during passage in ferrets, ultimately becoming airborne transmissible in ferrets. None of the recipient ferrets died after airborne infection with the mutant A/H5N1 viruses. Four amino acid substitutions in the host receptor-binding protein hemagglutinin, and one in the polymerase complex protein basic polymerase 2, were consistently present in airborne-transmitted viruses. The transmissible viruses were sensitive to the antiviral drug oseltamivir and reacted well with antisera raised against H5 influenza vaccine strains. Thus, avian A/H5N1 influenza viruses can acquire the capacity for airborne transmission between mammals without recombination in an intermediate host and therefore constitute a risk for human pandemic influenza.

Fig. 1

In experiment 1, we inoculated groups of six ferrets intranasally with 1 × 10⁶ TCID₅₀ of (A) influenza A/H5N1_wildtype virus and the three mutants (B) A/H5N1_{HA N182K}, (C) A/H5N1_{HA Q222L,G224S}, and (D) A/H5N1_{HA N182K,Q222L,G224S}. Three animals were euthanized at day 3 for tissue sampling and at day 7, when this experiment was stopped. Virus titers were measured daily in nose swabs (top) and throat swabs (middle) and also on 3 and 7 dpi in respiratory tract tissues (bottom) of individual ferrets. Virus titers in swabs and nasal turbinates (NT), trachea (T), and lungs (L) were determined by end-point titration in MDCK cells. [One animal inoculated with A/H5N1_{HA N182K,Q222L,G224S} died at 1 dpi due to circumstances not related to the experiment (D).] (Top two rows) Virus shedding from the URT as determined by virus titers in nasal and throat swabs was highest in A/H5N1_wildtype-inoculated animals. The mutant that yielded the highest virus titers during the 7-day period was A/H5N1_{HA Q222L,G224S}, but titers were ~1 log lower than for the A/H5N1_wildtype-inoculated animals. In the first 3 days, when six animals per group were present, no significant differences were observed between A/H5N1_{HA N182K}- and A/H5N1_{HA Q222L,G224S}-inoculated animals, as calculated by comparing the viral titer (Mann-Whitney test, P = 0.589 and 0.818 for nose and throat titers, respectively). (Bottom row) No marked differences in virus titers in respiratory tissues were observed between the four groups. Each bar color denotes a single animal.

Fig. 2

Experiment 3, virus passaging in ferrets (P1 to P10, passages 1 to 10). Because no airborne transmission was observed in experiment 2, A/H5N1_wildtype and A/H5N1_{HA Q222L,G224S PB2 E627K} were serially passaged in ferrets to allow adaptation for efficient replication in mammals. Each virus was inoculated intranasally with 1 × 10⁶ TCID₅₀ in one ferret (2 × 250 μl, divided over both nostrils). Nose and throat swabs were collected daily. Animals were euthanized at 4 dpi, and nasal turbinates and lungs were collected. Nasal turbinates were homogenized in virus-transport medium, and this homogenate was used to inoculate the next ferret, resulting in passage 2 (fig. S6). Subsequent passages 3 to 6 were performed in the same way. From passage six onward, nasal washes (NW) were collected at 3 dpi in addition to the nasal swabs. To this end, 1 ml of PBS was delivered drop wise into the nostrils of the ferrets, thereby inducing sneezing. Approximately 200 μl of the sneeze was collected in a Petri dish, and PBS was added to a final volume of 2 ml. For passages 7 through 10, the nasal-wash sample was used for the passages in ferrets. The passage-10 nasal washes were subsequently used for sequence analyses and transmission experiments to be described in experiment 4. For details, see the supplementary materials.

Fig. 3

Virus titers in (A) the nasal turbinates collected at day 4 and (B) nose swabs collected daily until day 4, from ferrets inoculated with A/H5N1_wildtype (blue) and A/H5N1_{HAQ222L,G224S PB2 E627K} (red) throughout the 10 serial passages described in Fig. 2. Virus titers were determined by end-point titration in MDCK cells. After inoculation with A/H5N1_wildtype, virus titers in the nasal turbinates were variable but high, ranging from 1.6 × 10⁵ to 7.9 × 10⁶ TCID₅₀/gram tissue (A), with no further increase observed with repeated passage. After inoculation with A/H5N1_{HA Q222L,G224S PB2 E627K}, virus titers in nasal turbinates averaged 1.6 × 10⁴ in the first three passages, 2.5 × 10⁵ in passages four to seven, and 6.3 × 10⁵ TCID₅₀/gram tissue in the last three passages, suggestive of improved replication and virus adaptation. A similar pattern of adaptation was observed in the virus titers in the nose swabs of animals inoculated with A/H5N1_{HA Q222L,G224S PB2 E627K} (B). These titers also increased during the successive passages, with peak virus shedding of 1 × 10⁵ TCID₅₀ at 2 dpi after 10 passages. Altogether, these data indicate that A/H5N1_{HA Q222L,G224S PB2 E627K} adapted to more efficient replication in the ferret URT upon repeated passage, with evidence for such adaptation by passage number 4. In contrast, analyses of the virus titers in the nose swabs of the ferrets collected at 1 to 4 dpi throughout the 10 serial passages with A/H5N1_wildtype revealed no changes in patterns of virus shedding. Asterisks indicate that a nose wash was collected before the nose swab was taken, which may influence the virus titer that was detected.

Fig. 4

Summary of the substitutions detected upon serial passage and airborne transmission of A/H5N1_{HA Q222L,G224S PB2 E627K} virus in ferrets. The eight influenza virus gene segments and substitutions are drawn approximately to scale (top to bottom: PB2, PB1, PA, HA, NP, NA, M, NS). Viruses shown in blue, orange, and red represent the initial recombinant A/H5N1_{HA Q222L,G224S PB2 E627K} virus (P0), ferret passage-10 virus (P10), and P10 virus after airborne transmission to recipient ferrets, respectively. Viruses shown in gray indicate that virus was not transmitted to the recipient ferret. First, we tested whether airborne-transmissible viruses were present in the heterogeneous virus population of ferret P10. We inoculated four donor ferrets intranasally, which were then housed in transmission cages and paired with four recipient ferrets. Transmissible viruses were isolated from three out of four recipient ferrets (F1 to F3). Next, we took a throat-swab sample from F2 (this sample contained the highest virus titer among the positive recipient ferrets), and this sample was used to inoculate two more donor ferrets intranasally. In a transmission experiment, these donors infected two recipient ferrets via airborne transmission (F5 and F6). Virus isolated from F5 was passaged once in MDCK cells and was subsequently used in a third transmission experiment in which two intranasally inoculated donor ferrets transmitted the virus to one of two recipient ferrets (F7). The genetic composition of the viral quasi-species present in the nasal wash of ferret P10 was determined by sequence analysis using the 454/Roche GS-FLX sequencing platform. Conventional Sanger sequencing was used to determine the consensus sequence in one high-titer nasal- or throat-swab sample for each ferret. Thick and thin black vertical bars indicate amino acid and nucleotide substitutions, respectively; substitutions introduced by reverse genetics are shown in yellow; substitutions detected in passage 10 and all subsequent transmissions are shown in green.

Fig. 5

Airborne transmission of A/H5N1 viruses in ferrets. Transmission experiments are shown for A/H5N1_wildtype (A and B) and A/H5N1_{HA Q222L,G224S PB2 E627K} (C and D) after 10 passages (P10) in ferrets. Two or four ferrets were inoculated intranasally with nasal-wash samples collected from P10 virus of A/H5N1_wildtype and A/H5N1_{HA Q222L,G224S PB2 E627K}, respectively, and housed individually in transmission cages (A and C). A naïve recipient ferret was added to each transmission cage adjacent to a donor ferret at 1dpi (B and D). Virus titers in throat (black bars) and nose swabs (white bars) were determined by end-point titration in MDCK cells. Geometric mean titers and SDs (error bars) of positive samples are shown. The number of animals infected via airborne transmission is indicated in (D) for each time point after exposure; the drop from three animals infected at day 7 to one animal at day 9 and no animals at day 11 is explained by the fact that the animals that became infected via airborne transmission had cleared the virus by the end of the experiment and, therefore, detectable amounts of virus were no longer present. The dotted lines indicate the lower limit of virus detection.

Fig. 6

Comparison of airborne transmission of experimental passaged A/H5N1 and 2009 pandemic A/H1N1 viruses in individual ferrets. A throat-swab sample from ferret F2 at 7 days postexposure (dpe) (Fig. 5D) was used for the transmission experiments shown in (A) and (B), and a virus isolate obtained from a nose swab collected from ferret F5 at 7 dpi (Fig. 6A) was used for the experiments in (C) and (D). For comparison, published data on transmission of 2009 pandemic A/H1N1 virus between ferrets is shown in (E) to (H) (27). Data for individual transmission experiments is shown in each panel, with virus shedding in inoculated and airborne virus–exposed animals shown as lines and bars, respectively. For the transmission experiments with airborne-transmissible A/H5N1 (A to D), nose or throat swabs were not collected at 2 dpi and 2 dpe. White circles and bars represent shedding from the nose; black circles and bars represent shedding from the throat. The asterisk indicates the inoculated animal that died 6 days after intranasal inoculation.