H5N1 Tamiflu Resistance
Due To Chance?

By Dr. Henry L. Niman, PhD

"The H5N1 virus continues to change. It will continue to change," Paul Gully, a senior WHO adviser, told the conference.
"Influenza A viruses replicate but they don't replicate very well. They are bad at replicating themselves so ... mutations continue to occur," he said.
Gully said the mutation of the virus in Egypt "probably occurred by chance".
The above comments show that WHO advisors continue to cling to random mutations to explain H5N1 evolution and Tamiflu (oseltamivir) resistance. However as additional sequences become public, it becomes increasingly clear that the changes in H5N1 are not due to recent mutations, but are linked to transmission and transport by wild birds leading to dual infections and recombination, resulting in novel gene sequences in H5N1, which are evolving at an increasing rate.
The N294S polymorphism in the Gharbiya cluster is an example of such evolution, that is not directly linked to Tamiflu treatment of the fatally infected patient, but may very well be linked to the increased use of a Tamiflu blanket in H5N1 infected areas.
N294S was identified in collections from two related patients in Gharbiya. Samples were collected prior to Tamiflu treatment as well as two days after the start of treatment. All four samples had H5N1 with the Tamiflu resistance marker, N294S. However, it is unlikely that the change was due to chance, as indicated above.
Potential problems with Tamiflu resistance were evident in initial in vitro data linked to analysis of IC50 values for the nine N serotypes. N2 was among the serotypes most sensitive to oseltamivir, while N1 was among the serotypes least sensitive to oseltamivir. The IC50 for N1 was 20-40 fold higher than N2.
The potential problems identified in the in vitro tests were again signaled by in vitro studies. Mice pre-treated with Tamiflu that was 10-20 fold higher than the FDA approved dose of Tamiflu were only partially protected from challenge with H5n1. Although the elevated concentration was linked to bioavailability differences between mice and humans, the failure of the Tamiflu to protect the mice raised additional concerns that the dose approved for seasonal flu might lead to Tamiflu resistance in humans, in part because H5N1 was lethal and virulent in humans.
Tamiflu treatment in humans began in Southeast Asia in 2004, in part because the Clade 1 H5N1 was already resistant to amantadines, which were ion channel blockers that targeted the M2 protein.
Tamiflu resistance was reported in early 2005 in a patient who had been initially treated with the lower prophylactic dose for Tamiflu.
Two Tamiflu resistance markers, H274Y and N294S were identified in separate clones from the patient. Although these changes were assumed to have arisen via de novo mutations in the patient, because wild type H5N1 was also found, an alternative explanation involving isolates with changes was supported by the prior identification of both markers in H5N1 infected birds in Hong Kong.
H274Y had been previously identified in A/chicken/Hong Kong/ 3123.1/2002. Similarly, N294S had been previously identified in A/ Duck/Hong Kong/380.5/2001. Thus, both Tamiflu resistance markers were present in H5N1 infected birds in Hong Kong. N294S was also detected in China, A/duck/Zhejiang/bj/2002.
The presence of Tamiflu resistance markers in H5N1 birds in the region offer opportunities for acquisitions of these markers via recombination instead of de novo mutations.
These linkages have now extended outside of Asia. In May, 2005 H5N1 was identified in long range migratory birds at Qinghai Lake.
Although the H5N1 was readily distinguished from prior H5N1 isolates, it did share polymorphisms with H5N1 infected wild birds in Hong Kong and Japan.
The long range migratory birds carried the Qinghai strain of H5N1 to nature reserves in Mongolia and southern Siberia, which link back to migration paths in eastern Asia. The presence of the Tamiflu resistance markers soon followed. H274Y was identified in Qinghai H5N1 swan isolates from Astrakhan, A/swan/Astrakhan/1/2005 and A/swan/Astrakhan/Russia/Nov-2/2005 while N294S has been identified in A/Egypt/14724-NAMRU3/2006 and A/Egypt/14725- NAMRU-3/2006 as well as corresponding isolates from samples collected prior to Tamiflu treatment.
Thus, the Tamiflu resistant marker, N294S, described as a chance occurrence is unlikely to be due to chance or recent copy errors as suggested by the WHO consultant above. Tamiflu blankets hae been applied to the 2006 H5N1 in Turkey. This widespread useage can lead to an increased incidence of Tamiflu resistance markers, which may have lead to teh presence of N294S in the Gharbiya cluster.
WHO consultants have uniformly indicated that H5N1 evolves via random mutations due to copy errors. However, as the sequence database grows the tracking of these changes follow well defined patterns in H5N1 isolates that are easily explained by homologous recombination.
The failure of WHO consultants to understand the underlying mechanism of H5N1 evolution and drug resistance remains a cause for concern.



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