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Hidden H5N1 Reservoir Raises Pandemic Concerns in Indonesia
Recombinomics Commentary
September 17, 2006

Sequence analysis of H5N1 sequences from birds and patients in Indonesia provides compelling evidence that the human infections are linked to a hidden reservoir.  The evidence had been building throughout the year because the sequences from the human isolates did not match the sequences from poultry isolates.  The vast majority of human isolates had a novel cleavage site that was not found in the bird isolates.  This change was associated with a number of additional changes on the HA gene segment, as well as the other seven gene segments.  A phylogenetic tree of HA isolates was presented at WHO meeting on H5N1 in Indonesia held in June, 2006 in Jakarta.  That tree showed that the isolates with the novel cleavage site formed a separate branch that contained no avian isolates.  This branch had all human isolates except for the second confirmed case in Indonesia, and the Karo cluster.  The Karo cluster was on another branch and although there were bird isolates on that branch, the Karo cluster was quite distinct from the other bird isolates, so the origins of virtually all human bird flu cases were not clear.

At that time there were about 40 bird HA sequences that were public and they represented isolates collected between 2003-2005 from a variety of locations in Indonesia.  Although all isolates had a number of markers that distinguished the Indonesian isolates from other H5N1 isolates, the Indonesian isolates were quite heterogeneous and fell into distinct sub-clades.  Because of the diversity, the failure to match may have been due to a lack of recent samples collected from appropriate locations.  Therefore, approximately 100 recent samples were sent to a WHO affiliated lab in Australia for sequencing.

Nineteen of these sequences were released last week, raising the number of recent sequences from Australia to 34.  Included in these sequences were matches for both the human sequences with the novel cleavage site as well as the Karo sequences.  However, the time and location of these isolates pointed toward a hidden reservoir for the human infections.

The initial sequences generated in Australia did not match the Karo cluster, even though several of the samples were from 2006 or from regions in Northern Sumatra, the location of the Karo cluster.  However, last week a duck sample from East Java provided a match.  There were only three differences in the 1705 HA nucleotide and all were silent.  The HA protein sequence was an exact match.  However, the sample was not from 2006 or from Northern Sumatra, but was a 2005 isolate from East Java raising serious questions of the origin of the Karo cluster infections.

Last week’s sequences raised similar questions about the origin of the vast majority of H5N1 human isolates from Indonesia.  As noted above, all isolates on Java in 2006 had the novel cleavage site.  However, initial sequences from birds in Indonesia did not have this change.  A  2006 duck isolate from Indramayu had the change, but that sequence was most closely related to a small subset of human cases from late 2005 or January 2006.  These isolates had an additional silent change in the HA cleavage site, which was also present in the duck isolate.  In addition, there were 8 additional changes in HA that were in the vast majority of the human isolates, but only three of the changes were in the duck isolates, which was also true of the small subset with the silent HA cleavage site change.  Thus, the duck isolate was not a good match for most of the human isolates. 

Last week however, two additional sequences with the novel cleavage site were made public and these two chicken isolates had 7 of the 8 changes in the HA that were rarely found in any other bird isolates.  Thus, the two chicken isolates were solid matches of the human cases.  However, both of the matches were from 2005 and were from two distinct locations on Sumatra, indicating these matches were rare. 

Moreover, there were still no matches in poultry from Java which matched the human cases from Java.

The match failures in poultry isolated in the same location as the human cases strongly point toward a hidden reservoir.  On Java all but one human isolate has the novel cleavage site and the first human isolate was from July, 2005 and extend for over a year.  These isolates are from multiple locations, including Jakarta, West Banten, Bekasi and Garut.  The only two close matches for these isolates are from two 2005 chickens on Sumatra.

The human isolates have a number of polymorphisms that are shared with Qinghai isolates, highlighting the role of migratory birds and recombination.  The shares sequences are on an Indonesian genetic background, indicating the sequences were acquired via recombination.  There are no wild bird H5N1 sequences from Indonesia.  Similarly, there is a lack of mammalian sequences.  The only non-human close match on Java is from a cat, but that is the only public cat sequences.  H5N1 has been reported from a dog as well as wild and domestic cats in Thailand as well as a variety of mammals in Europe and the Middle East where the Qinghai strain of H5N1 is widespread.

The surveillance in these other mammals, as well as humans not linked to dead or dying poultry, has been poor.  The number of familial clusters in Indonesia is high, and WHO has recently acknowledge the possibility of human-to-human transmission in Karo and West Sumatra.  Gaps in disease onset dates indicate human-to-human transmissions within families are common.  In several instances, such transmission has produced mild cases.  These transmissions coupled with the poultry match failures suggest that the level of undetected H5N1 in human populations is significant.

Sequences from a more diverse population are required to identify the source of the hidden H5N1 reservoir in Indonesia.

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