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False Negatives for H5N1 Receptor Binding Domain Changes?
Recombinomics Commentary
November 18, 2006

A/Vietnam/30262III/04 and A/Vietnam/3028II/04 contained a heterogeneous mixture of HAs on sequence analysis, prompting us to plaque-purify the viruses in Madin–Darby canine kidney (MDCK) cells to obtain viral clones with distinct HA sequences

Moreover, two of these changes, lysine at position 182 and arginine at position 192, were present in the HAs of clade-2 H5N1 viruses isolated from two individuals in Azerbaijan and one individual in Iraq, but not in any of the more than 600 avian isolates examined.

The above comments, describing isolates that had several of the changes that increased receptor binding, suggest that false negatives may mask the prevalence of such changes in avian populations. 

The receptor changes were identified by isolating the H5N1 on mammalian cells, which select for H5N1 with mammalian receptor specificity.  It is known that growth of flu in chicken eggs can lead to the loss of mammalian specific sequences, which may account for the failure to detect these changes in avian isolates.

The history of S227N positive H5N1 isolates is instructive.  S227N was first identified in two human H5N1 isolates, A/Hong Kong/212/03 and A/Hong Kong/213/03, from a father and son who had traveled to Fujian province in 2003.  The H5N1 was isolated on MDCK mammalian cells, as described above for the human Vietnam isolates.

The increased affinity of these two isolates for mammalian receptors led to a warning in October, 2005 that donor sequences for the formation of S227N were present in H9N2 isolates in the Middle East.  In January, 2006, the first confirmed human Qinghai case was announced, and the isolate, A/Turkey/12/2006, was positive for S227N.  However, the isolate from his sister, A/Turkey/15/2006 was negative for S227N, although another human isolate from Turkey, A/Turkey/65596/2006, was also positive for S227N, while a fourth isolate, A/Turkey651242//2006 was also negative.

The data for the three clones from A/Vietnam/3028II/04 provide an explanation for the detection of S227N in two of the four isolates from Turkey.  Even though the isolates from Vietnam were grown on mammalian MDCK cells, only clone 3 had S227N, as well as Q196R.  Neither polymorphism was in clone 1 or clone 2, although both had position 138 polymorphisms (A138A and A138T, respectively).  Clone 2 also had N248S.  Thus, S227N was only found in one of three clones from the same patient, even though all isolates were obtained using the same mammalian cell line in the same lab.

This variation using MDCK cells raises the strong possibility that other changes that increase affinity for mammalian receptors and decrease affinity for avian receptors. might be lost during isolation, especially if the isolation is in chicken eggs.

Difference were also seen in sequences from the patient in Thailand, A/Thailand/1-KAN-1/04.  The original sequence had both G143R and N186K.  However, that sequence was replaced with a sequence that had only G143R.  G143R is present in H5N1 avian sequences.  N186K is only in human isolates, including influenza B.

These data suggest that the failure to find some of the receptor binding domain changes in avian isolates, may be linked to the use of chicken eggs to isolate the virus.  The receptor binding changes described in humans in Vietnam, Thailand, Hong Kong, Turkey, Egypt, Azerbaijan, and Iraq have not been described in human H5N1 from Indonesia, but Hong Kong and the CDC labs use chicken eggs to isolate the human H5N1 from Indonesia, which may select against the changes that increase affinity for mammalian receptors.

Isolation of both human and avian H5N1 on mammalian cells may be useful.

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