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High H5N1 Levels In Patient Throats in Vietnam
Recombinomics Commentary

September 11, 2006

The highest viral RNA levels occurred in H5N1-infected individuals who died (Table 2), suggesting that the level of viral replication influences outcome. Viral RNA could also be detected in blood specimens from 9 of 16 H5N1-infected individuals and in the rectal swabs from 5 of 7 of these individuals (Table 2). It remains unclear whether the detection of virus in rectal specimens reflects genuine gastrointestinal infection. However, symptoms of diarrhea frequently occur during influenza H5N1 (refs. 1,2,15) and were present in three of the five individuals with detectable viral RNA in the rectum. No viral RNA was present in the blood of surviving H5N1-infected individuals or in those with human influenza. Individuals with detectable H5N1 RNA in blood also had higher pharyngeal viral loads (median 7.4 log10 copies per ml; range 5.1–7.4) than those without evidence of H5N1 RNA (6.0 log10 copies per ml; range 4.3–7.0; P = 0.021), suggesting that the presence of viral RNA in blood reflects an overall high viral burden.

An E627K substitution in the viral polymerase basic protein 2 (PB2), which is associated with adaptation and virulence of H5N1 viruses in mammals12, was present in five of eight isolates from fatal cases and in three of four isolates from patients who survived

Notably, three of four viruses without this substitution, but none of the viruses containing Lys627, contained an D701N substitution in PB2 that has been associated with adaptation of H7N7 viruses to mammalian cells

The study also showed that some of the flu strains isolated in Vietnam had particular genetic changes that virologists have been watching for, fearing that these changes would make them more lethal.

But those changes appeared in only some patients, and in those who died as well as those who lived, “so I wouldn’t make too much of it,” Dr. Moscona said.

Henry L. Niman, a Pittsburgh biochemist who has been tracking viral changes and raised earlier alarms about E627K, agreed.

“Lethality in the virus may rely on several changes,” he said. “But it’s got several different paths to the same end. That’s what makes it so efficient.”

The above comments from a recent paper in Nature Medicine on H5N1 infected patients in Vietnam as well as comments in a Donald McNeil article in today’s NY Times raise a number questions concerning recent H5N1 infections and human-to-human transmission in familial clusters.

The paper describes high levels of H5N1 in the throat of H5N1 infected patients in Vietnam in 2004 and 2005.  These high levels were also associated with H5N1 in the blood and rectum as well as increases in cytokine levels.

Almost all H5N1 isolates from these changes had one of two changes in PB2, E627K or D701N, that were associated with increased H5N1 levels in mammals.  E627K has been shown to increase levels 20 fold, while D701N has increased levels 3-4 fold in laboratory mouse experiments.

The levels of H5N1 in the throat of patients was higher than H3N2 in the throat of seasonal flu patients suggesting that in some locations in the upper respiratory tract, the replication of H5N1 is quite efficient.  The spillage of virus into other sites, such as the blood or rectum, create an increased number of transmission opportunities to relatives who care for these patients or stay in close contact with the patent, especially if there is frequently coughing.

The presence of two different changes, each associated with increased H5N1 replication in mammals, suggests selection from several independent genetic alterations can generate high levels of H5N1 in patients.

Although patients in Indonesia generally do not have the PB2 changes described above, the patients in the Karo cluster had a set of tandem alterations, K660R and A661T, between the two changes seen in patients in Vietnam.  Similarly, the remaining H5N1 patients in Indonesia, who had a novel HA cleavage site, also had a pair of changes, T524I and K526R, just upstream from changes in Pb2 in the patients in Vietnam.  Although these changes in H5N1 isolates from Indonesian patients have not been reported to be associated with increased H5N1 levels in mammals, media reports have indicated that the patients in Karo did have increased levels of H5N1 in their upper respiratory tract.

The high levels of H5N1 in patients are cause for concern.  H5N1 in Vietnam appears to have used two distinct genetic changes in PB2 to generate very high levels of H5N1 in patients.  These two changes are not found in patients in Indonesia, but it is likely that additional changes contribute to the high lethality rate and large and frequent familial clusters.

The use of Tamiflu to reduce viral load may contribute to the recovery of some patients and may also serve to halt production and limit detection of H5N1 in other patients, such as those linked to H5N1 positive patients in the Garut cluster.

Since H5N1 has already generate changes that allow for very high levels of virus in the throats of patients, a similar change that allows for high levels in the noses of patients may significantly increase transmission beyond familial clusters.

Such changes at S227N in HA may have contributed to the size of clusters in Turkey.  These patients already had PB2 E627K, but the changes in the receptor binding domain may have altered tissue tropism.

In any event, it is clear that H5N1 can make minor changes in its genome to produce high levels of replicating virus in humans, with severe consequences that are frequently lethal.  The high levels in recent patients also serve as a warning to persons caring for these patients without proper safeguards.

Moreover, the associate of the high levels of H5N1 in humans with an extremely high case fatality rate remains a cause for concern.  The recent data does not indicate that a more efficiently transmitted H5N1 would necessarily be a less lethal virus, since the lethality is in part linked to an internal polymerase gene that is independent of the surface proteins affecting viral entry and exit from cells.

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