Although H5N1 viruses continue to cause outbreaks in poultry with human cases in Indonesia, Viet Nam, Egypt and elsewhere (http://www.who.int/influenza/human_animal_interface/H5N1_cumulative_table_archives/en/index.html
), they have not acquired the ability to cause human-to-human transmission. Investment in H5N1 vaccines has therefore been questioned. However, since humans lack immunity to influenza viruses possessing an H5 HA, the emergence of a transmissible H5 HA-possessing virus would likely cause a pandemic. To better prepare for such a scenario, it is critical to understand the molecular changes that may render H5 HA-possessing viruses transmissible in mammals. Such knowledge would allow us to monitor circulating or newly emerging variants for their pandemic potential, focus eradication efforts on viruses that already have acquired subsets of molecular changes critical for transmission in mammals, stockpile antiviral compounds in regions where such viruses circulate, and initiate vaccine generation and large-scale production prior to a pandemic. Here, we therefore studied the molecular features that would render H5 HA-possessing viruses transmissible in mammals.
Previous studies suggested that HA plays a major role in host range restriction of influenza A viruses1–3
. The HA of human isolates preferentially recognizes sialic acid linked to galactose by α2,6-linkages (Siaα2,6Gal), whereas the HA of avian isolates preferentially recognizes sialic acid linked to galactose by α2,3-linkages (Siaα2,3Gal)3
. A small number of avian H5N1 viruses isolated from humans show limited binding to human-type receptors, a property conferred by several amino acid changes in HA4–9
. None of the H5N1 viruses tested transmitted efficiently in a ferret model10–13
, although, while our paper was under review, Chen et al.14
reported that a virus with a mutant H5 HA and a neuraminidase (NA) of a human virus in the H5N1 virus background caused respiratory droplet transmission in one of two contact ferrets. A previous study reported that H5N1 and 2009 pandemic H1N1 viruses show high genetic compatibility15,16
, providing an opportunity for the generation of avian-human reassortant H5N1 viruses. To assess the pandemic potential of such reassortants, we generated a virus possessing the HA from an H5N1 virus and the seven remaining gene segments from a 2009 pandemic H1N1 virus (H5HA/pdm09). In receptor-binding studies and animal experiments, we identified a derivative of this reassortant virus that possessed four mutations in its HA protein.
The mutant H5 HA reassortant (H5HA-mutant/pdm09) was capable of respiratory droplet transmission in ferrets, which are widely accepted as an animal model for influenza virus transmissibility and pathogenesis studies. In this transmission experiment, we placed naïve ferrets in wireframe cages next to ferrets inoculated with 106
plaque-forming units (PFU) of virus. This experimental setting allowed the exchange of respiratory droplets between the inoculated and non-inoculated ferrets, but prevented transmission by direct or indirect contact. Similar to previous transmission experiments17
, a pdm09 virus was efficiently transmitted via respiratory droplets to all three contact ferrets, as evidenced by the detection of virus in nasal washes and haemagglutinin-inhibition (HI) antibody in these animals ( and ). As expected, the H5HA/pdm09 virus was not transmitted; neither virus shedding nor seroconversion was detected in any contact animals. In the H5HA-mutant/pdm09-inoculated group, virus was recovered from 4 of the 6 contact ferrets between days 3 and 7 post-contact. Moreover, seroconversion was detected in all six animals. No animals died in the course of these transmission experiments. Significantly, the transmission pattern with the H5HA-mutant/pdm09 virus was 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 washes of 2 of 3 contact animals (unpublished data). These findings demonstrate that an H5 HA can support respiratory droplet transmission of an avian-human reassortant virus among ferrets.
Transmission in ferrets inoculated with H5 avian-human reassortant viruses
Figure 1 Respiratory droplet transmission of H5 avian-human reassortant viruses in ferrets. Groups of three or six ferrets were inoculated intranasally with 106 PFU of pdm09 (a), H5HA/pdm09 (b), or H5HA-mutant/pdm09 (c). One day post-infection, three or six naive (more ...)
To assess the replication and pathogenicity of the transmissible H5N1 avian-human reassortant virus, we infected ferrets with 106 PFU of virus and determined virus titres at 3 and 6 days post-infection. The pdm09 virus replicated efficiently in the respiratory organs of infected animals, and was isolated from the colon, but not from any other organ tested ( and ). The H5HA/pdm09 virus replicated to titres comparable to those of the pdm09 in nasal turbinates, but substantially less in the lungs. The H5HA-mutant/pdm09 virus replicated efficiently in nasal turbinates and was isolated from brain tissue in one of the three ferrets infected.
Figure 2 Virus replication in respiratory organs. Ferrets were infected intranasally with 106 PFU of virus. Three ferrets per group were euthanized on days 3 and 6 after infection for virus titration. Virus titres in nasal turbinates, trachea, and lung were determined (more ...)
Virus titres in tissues of ferrets infected with H5 avian-human reassortant viruses
Pathological examination revealed similar histological changes and levels of viral antigens in the nasal mucosa of pdm09- and H5HA-mutant/pdm09-infected ferrets (). In the H5HA/pdm09 groups, however, less tissue damage was found in the nasal mucosa compared to the pdm09 group on day 3 post infection (Dunnett’s test; P=0.0057 and 0.0175, respectively; ). In addition, all three viruses caused lung lesions ().
Figure 3 Pathological analyses of H5 avian-human reassortant viruses. a, Representative histological changes in nasal turbinates from influenza virus-infected ferrets. Three ferrets per group were infected intranasally with 106 PFU of virus, and tissues were collected (more ...)
To understand the molecular basis for the transmissibility of the H5HA-mutant/pdm09 virus in ferrets, we evaluated the receptor-binding property of a virus with the mutant H5 HA in solid phase assays. In these experiments, the virus with the mutant H5 HA preferentially bound to human-type receptors, Siaα2,6Gal, as did a virus with the HA of a seasonal human virus (). A virus containing the wild-type H5 HA bound only to avian-type receptors, Siaα2,3Gal.
Figure 4 Characterization of the receptor-binding properties of an H5 HA mutant virus possessing four mutations in its HA protein. a, Binding of an H5 HA-mutant virus to sialylglycopolymers in solid-phase binding assays. A human seasonal H1N1 virus, a wild-type (more ...)
To determine whether the H5HA-mutant/pdm09 virus with human-type receptor specificity could bind to cells in the human respiratory tract, sections of human tracheal and lung tissues were exposed to viruses with a human virus HA, a wild-type H5 HA and the mutant H5 HA that supports virus transmission. All viruses bound extensively to the alveolar epithelial surface of human lung tissue, where both human- and avian-type receptors are present18
(). Unlike the virus with the wild-type H5 HA, the virus with the mutant H5 HA attached to human tracheal epithelia, where predominantly human-type receptors are present, as did the virus with the human virus HA. These results suggest that the recognition of human-type receptors present in human respiratory organs is required for virus transmission in mammals. However, findings by others12,14
as well as information in the full version of this paper19
have shown that human-type receptor-binding specificity is not sufficient to confer respiratory droplet transmission in ferrets. This is in line with our hypothesis that a subset of the HA mutations in the H5HA-mutant/pdm09 virus affected HA stability, thus offsetting the decrease in stability conferred by the mutations that directly affect receptor-binding specificity19
. Together, these findings suggest that a fine balance of mutations affecting different functions in HA may be critical to confer transmissibility in mammals.
Our transmissible H5HA-mutant/pdm09 virus possesses seven segments (all but the HA segment) from a human pandemic 2009 H1N1 virus. Human virus-characteristic amino acids in these seven segments may have critically contributed to the respiratory droplet transmission of the H5HA-mutant/pdm09 virus in ferrets. Examples include amino acids in the PB2 polymerase protein that confer efficient replication in mammalian, but not avian cells20–24
. Since the PB2 gene of the H5HA-mutant/pdm09 virus is of human virus origin, the virus possesses high replicative ability in mammalian cells. In contrast, most avian virus PB2 proteins lack these human-type amino acids, although one of these changes (a glutamic acid-to-lysine mutation at position 627) is found in highly pathogenic avian H5N1 viruses circulating in the Middle East25
As a second example, the viral NA gene may contribute to viral transmissibility. The NA protein cleaves α-ketosidic linkages between a terminal sialic acid and an adjacent sugar residue, an activity that balances the sialic acid-binding activity of HA. A recent study found that a human virus NA gene was critical to confer limited transmissibility to a mutant H5 avian-human reassortant virus14
. In general, a human-type receptor-recognizing H5 HA alone may not be sufficient to confer transmissibility in mammals, but may have to act in concert with other human virus-characteristic traits (in PB2, NA, and/or other viral proteins). Therefore, at this point, we cannot predict whether the four mutations in the H5 HA identified in our study would render a wholly avian H5N1 virus transmissible.
To assess if current control measures may be effective against the mutant transmissible virus, we examined the reactivity of sera from individuals vaccinated with an H5N1 prototype vaccine26
against a virus possessing the mutant H5 HA gene, the NA gene of a H5N1 virus, and the remaining six genes from A/Puerto Rico/8/34 (H1N1) (PR8) (designated H5HA-mutant/PR8). We found that pooled human sera from individuals immunized with this vaccine reacted with the H5HA-mutant/PR8 at a higher titre than with a virus possessing the HA and NA genes of a H5N1 virus and the remaining genes from PR8 (designated H5HA/PR8) (), suggesting that current H5N1 vaccines would be efficacious against H5HA-mutant/pdm09 virus. In addition, the transmissible reassortant mutant virus was highly susceptible to a licensed NA inhibitor, oseltamivir (). These experiments show that appropriate control measures would be available to combat the transmissible virus described in this study.
Haemagglutination inhibition (HI) reactions of H5 HA reassortant viruses against post-vaccination sera
Virus susceptibility to oseltamivir in cell culture
Since highly pathogenic H5N1 viruses first emerged in the late 1990s, pandemic preparations have been underway to develop appropriate vaccines and expand the array of effective antivirals that might be needed should these viruses acquire the ability to transmit in humans. We do not know whether the mutations that we identified in this study that allowed the H5HA-mutant/pdm09 virus to become transmissible in ferrets would also support sustained human-to-human transmission. However, as H5N1 viruses continue to evolve and infect people, receptor-binding variants of H5N1 viruses including avian-human reassortant viruses as tested here may emerge.
Our study suggests the pandemic potential of viruses possessing an H5 HA. Although current H5N1 vaccines may protect against a virus similar to that tested here, the continued evolution of H5N1 viruses reinforces the need to prepare and update candidate vaccines to H5 viruses. We strongly endorse sharing our data globally. They will help individuals conducting surveillance in regions with circulating H5N1 viruses (e.g., Egypt, Indonesia, Viet Nam) to recognize key residues that predict the pandemic potential of isolates. Rapid responses in a potential pandemic situation are essential to generating appropriate vaccines and initiating other public health measures to control infection. Our findings are of critical importance to those making public health and policy decisions.
Our research answers a fundamental question in influenza research: can H5 HA-possessing viruses support transmission in mammals? Details available in the full version of this paper19
advance our understanding of influenza host specificity and the evolution of the HA molecule. These findings could form the basis for additional experiments to increase our understanding of the basic biology of influenza virus transmission.