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High Levels of Human H5N1 in Ferret Upper Respiratory Tract
August 1, 2006
Indon05 virus exhibited severe illness with substantial weight loss (18.8% mean maximum 7 days p.i.) and dyspnea but no sneezing; none of the ferrets survived past day 7 p.i. (Table 1). Although high titers of infectious virus were detected in the upper respiratory tract of Indon05-inoculated ferrets, virus was not detected in any of the nasal washes from the contact ferrets through day 9 p.c.
The above comments from the ahead of the press PNAS publication,
"Lack of transmission of H5N1 avian-human reassortant influenza viruses in a ferret model", indicate the human H5N1 isolate from Indonesia, A/Indonesia/5/05(H5N1), grows to high titer in the upper respiratory tract in inoculated ferrets. This property is cause for concern because the data indicate such a change can be achieved in the absence or reassortment or changes in the receptor binding domain.
WHO and consultants have been offering reassurances that new H5N1 human isolates do not have human genes or changes in the receptor binding domain. However, the publication above uses avian / human reassortants in an ferret model to monitor transmissibility of such reassortants. The data, which includes earlier data on reassortants using a 1997 isoalte, indicate that the reassorted genes do not offer selective advantage. The human genes decrease the ability of the H5N1 to grow in tissue culture or transmit under the experimental conditions that allow for transmission of human flu.
Although most of the human sequences from Indonesia have been withheld, the HA and NA genes of the above isolate have been released. The HA sequence does not have changes in the receptor binding domain. WHO and consultants have been monitoring changes at positions 226 and 228. These changes are thought to affect receptor binding specificities and increase binding for receptors found in the upper respiratory tract of mammals, However, although the human H5N1 isolate from Indonesia does not have these changes, it is able to grow to high titers in the upper respiratory tracts of the ferrets.
Thus, although the parameters being monitored by WHO and consultants have not changed in the Indonesia isolate, the high titers in the ferret upper respiratory tracts indicates other changes have led to this increased ability.
The HA sequence that has been released has a novel glycosylation and cleavage site in HA. The novel cleavage site has caused concern because it has not been reported in Indonesian poultry isolates. The frequency of H5N1 in humans or other mammals by be underestimated because surveillance in animals in Indonesia as poor and human cases are generally not tested for H5N1 bird flu unless there is a history of contact with dead or dying poultry.
Changes in the HA cleavage site can impact tissue tropism because HA cleavage is required for infection, and the cleavage is controlled by tissue specific proteases which cleavage at basic amino acids, but the surrounding sequence regulates the protease target. Most of the human isolates on the large island of Java have the novel cleavage site.
H5N1 evolves via acquisition of new sequences via recombination. The high concentration of H5N1 in Indonesia creates conditions for dual infections and H5n1 evolution. These changes do not require reassortment or changes in the receptor binding domain, the parameters being monitored by WHO.
Instead, H5N1 has been gradually changing and the number of distinct versions of H5N1 that have bee shown to cause fatal infections in humans continue to grow. None of these newly reported isolates reported reassortment with human genes. Instead the lack of such changes have been cited by governments claiming that the human isolates have not "mutated".
These reassurances are far from reassuring because H5N1 can evolve and increase efficiencies in the absence of these changes, as indicated by the high levels of H5N1 in the upper respiratory tract of experimental ferrets.