Controversial Influenza Research

REFERENCE: Imai et al. "Experimental Adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets." Nature (2012), epub ahead of print.  LINK

Amedeo Post offering more background and editorials related to this work: LINK

                This week in Nature sees the publication of the long discussed influenza research paper from the University of Wisconsin-Madison lab of Yoshihiro Kawaoka.  In a recent editorial, Kawaoka urges for publication and further research in this area by saying “[s]ome people have argued that the risks of such studies…outweigh the benefits.  I counter that H5N1 viruses circulating in nature already pose a threat because influenza viruses mutate constantly and can cause pandemics with great losses of life.  …I believe it would irresponsible not to study the underlying mechanisms.”  This published paper focuses on the hemaglutinin (HA) protein from the H5N1 virus circulating primarily in Southeast Asia that retains specificity and virulence in birds.  To date, 578 humans have become infected with this virus after direct contact with infected animals.  340 have died, but human-to-human transmission has, so far, not been an issue, but the potential for a pandemic caused by an evolving H5N1 virus is still present.  Kawaoka and colleagues sought to determine what mutations within this H5 would allow the protein to bind human receptors, if an influenza virus bearing these mutations could both efficiently infect and transmit the virus among mammals, and finally if current vaccines/antiviral therapies would be useful against such a virus.

                Human cells of the respiratory tract display α2,6-linked sialic acid with galactose while avian tracts have α2,3 linkages.  As the main receptors for HA binding, the ability of an influenza virus that was specific for birds to infect humans means, in part, that the specificity for binding switched from α2,3 to α2,6.  The authors began by introducing random mutations into the H5 globular head region where receptor binding occurs.  Turkey red blood cells were treated with sialidase to remove α2,3 linked sialic acid and preferentially leave α2,6.  Viruses were generated bearing the mutated H5s and tested for their ability to bind treated turkey red blood cells.  The identified viruses were screened again for α2,6 binding to root out false-positives and identified HAs were then further tested in solid-phase binding experiments for α2,6-specificity.  In the end, an H5 bearing mutations at E119, V152, N224 and Q226 was identified as binding only α2,6 linkages.  The authors further confirmed that N224 and Q226 mutations were critical for the shift in specificity.

                The hemaglutinin from H1N1, which was isolated from a human patient, was replaced with an H5 bearing the appropriate N224 and Q226 mutations.  Ferrets were infected with this reassortant virus and found that, after a period of 6 days, a new mutation at position N158 was observed. Viruses bearing an H5 with this new additional mutation could replicate well in ferrets and were mildly transmissible between ferrets.  Intriguingly, the viruses were found to contain yet another mutation as position T318 after ferret infection.  A new H1N1 virus bearing a quadruple H5 mutant protein was found to be highly transmissible between ferrets.  No ferrets died as a result of infection with either virus.  Encouragingly, the authors also showed that a prototype H5N1 vaccine was reactive with the mutant viruses created here and the viruses were susceptible to a licensed NA inhibitor.

                Specific mutations at N224 and Q226 are mostly likely changing the binding pocket to accommodate α2,6 linkages; a specific N158 mutation removes a glycosylation site on H5 that could be improving transmissibility.  These three mutations destabilize the protein in an acidic environment but the T318 mutation returns that stability.  Membrane fusion of the influenza virus with the host cell occurs at low pH so stability at these [H⁺] is necessary.

                It should be noted that hemaglutinin, while highly involved, is only one protein involved in the virulence of an influenza virus.  Studies show that neuraminidase also plays a role.  It was also stressed that the remaining genes in the mutant virus came from H1N1, not the avian-virulent H5N1.  It’s possible that the remaining genes in the influenza also contribute to the virulence of a virus in new hosts.  The hope is that the amino acid mutations identified here will help those keeping an eye on current H5N1 viruses.  Should any of these mutations arise, they will have the tools to predict pandemic potential and, knowing that current therapies are effective against these viruses, that proper safeguards can be put in place quickly.  As a final thought, the authors realize that a pandemic virus may not even show these mutations and something else entirely, but the work has identified important areas of the HA protein that could and most likely will change as an influenza virus evolves from avian to mammal specificity.