Avian influenza viruses preferentially recognize sialosugar chains terminating in sialic acid-␣2,3-galactose (SA␣2,3Gal), whereas human influenza viruses preferentially recognize SA␣2,6Gal. A conversion to SA␣2,6Gal specificity is believed to be one of the changes required for the introduction of new hemagglutinin (HA) subtypes to the human population, which can lead to pandemics. Avian influenza H5N1 virus is a major threat for the emergence of a pandemic virus. As of 12 June 2007, the virus has been reported in 45 countries, and 312 human cases with 190 deaths have been confirmed. We describe here substitutions at position 129 and 134 identified in a virus isolated from a fatal human case that could change the receptor-binding preference of HA of H5N1 virus from SA␣2,3Gal to both SA␣2,3Gal and SA␣2,6Gal. Molecular modeling demonstrated that the mutation may stabilize SA␣2,6Gal in its optimal cis conformation in the binding pocket. The mutation was found in approximately half of the viral sequences directly amplified from a respiratory specimen of the patient. Our data confirm the presence of H5N1 virus with the ability to bind to a human-type receptor in this patient and suggest the selection and expansion of the mutant with human-type receptor specificity in the human host environment.

Highly pathogenic H5N1 avian influenza virus has spread through at least 45 countries in three continents. Despite the ability to infect and cause severe disease in humans, the virus cannot transmit efficiently from human to human. The lack of efficient transmission indicates the incompletion of the adaptation of the avian virus to the new host species. The required mutations for the complete adaptation and the emergence of a potential pandemic virus are likely to originate and be selected within infected human tissues. Differential receptor preference plays an important role in the species-tropism of avian influenza. We have analysed quasispecies of sequences covering the receptor-binding domain of the haemagglutinin gene of H5N1 viruses derived from fatal human cases. We employed a likelihood ratio test to identify positive-selection sites within the quasispecies. Nine of seventeen positive-selection sites identified in our analyses were found to be located within or flanking the receptor-binding domain. Some of these mutations are known to alter receptor-binding specificity. This suggests that our approach could be used to screen for mutations with significant functional impact. Our data provide new candidate mutations for the viral adaptation to a human host, and a new approach to search for new genetic markers of potential pandemic viruses.

The codon usage pattern is a specific characteristic of each species; however, the codon usage of all of the genes in a genome is not uniform. Intriguingly, most viruses have codon usage patterns that are vastly different from the optimal codon usage of their hosts. How viral genes with different codon usage patterns are efficiently expressed during a viral infection is unclear. An analysis of the similarity between viral codon usage and the codon usage of the individual genes of a host genome has never been performed. In this study, we demonstrated that the codon usage of human RNA viruses is similar to that of some human genes, especially those involved in the cell cycle. This finding was substantiated by its concordance with previous reports of an upregulation at the protein level of some of these biological processes. It therefore suggests that some suboptimal viral codon usage patterns may actually be compatible with cellular translational machineries in infected conditions.

The highly pathogenic avian influenza A (H5N1) virus is a virulent virus that causes an acute febrile respiratory disease with high mortality in humans. To gain a better insight of H5N1 viral distributions in infected human tissues, the levels of viral RNA were determined in the autopsy tissues from two patients who were infected with H5N1 virus by using real-time reverse transcription-polymerase chain reaction. In one patient who died on day 6 of the illness, the viral load in the lung was extremely high, whereas the levels of viral RNA in the other organs were more than 6 log lower. In the other patient who died on day 17 of the illness, the viral load was similar in the lung and other organs, and was comparable to the viral load in the extra-pulmonary tissues of the first patient. These results suggested that while the H5N1 virus can cause disseminated infection in humans, the lung is still the major site of viral replication, and viral replication in the lung in the later stages may decrease as a result of the depletion of the available target cells. In addition, the mRNA levels of the tumor necrosis factor-α (TNF-α) were found to be associated with the viral titers.