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Novel Human H5N1 HA Cleavage Site In Jakarta

Recombinomics Commentary

August 10, 2006

The novel HA cleavage site in human H5N1 bird flu in Indonesia has attracted considerable attention.  Although the first reported human case of H5N1 was in July of 2005, the first sequence became public on March 25, 2006.  The sequence was on interest because Indonesia had an unusually high frequency of familial clusters as well as a very high case fatality rate,  However, the human sequences were sequestered at a WHO private database, which could only be viewed by a small group of WHO consultants.

The sequence for the first case was deposited on July 29, 2005 by Hong Kong University and on August 1, 2005 by the CDC in Atlanta.  Both was WHO-affiliated labs that verify H5N1 cases and both regularly receive samples that are positive in Jakarta.  The United States selected the Indonesian sequence as a pandemic vaccine target, and shortly after that announcement the HA and NA sequences were released at Los Alamos.

The sequences raised additional questions because they had a novel cleavage site and a new glcosylation site.  The cleavage site was not found in any H5N1 sequence at GenBank or Los Alamos.  The cleavage site became more curious because the project elader of the Jakarta USRAMRU group, Andrew Jerimijenko, found an H5N1 positive cat near one of the Jakarta victims in early 2006.  The CDC isolated the H5N1 and the sequence was very similar to the novel human H5N1 cleavage site.  He surveyed other scientist who were sequencing H5N1 and none have found the novel cleavage site outside of human cases in Indonesia.

Recently, a meeting on H5N1 in Indonesia was held in Jakarta and data on additional sequences was presented.  The phylogenetic tree from the meeting showed that almost all human sequences from Indonesia had the novel cleavage site.  Moreover, the tree indicated there were many changes in the human sequences that set them apart from the other Indonesian sequences.  The two exceptions were the second reported case, which was linked to fertilizer, and the Karo cluster in northern Sumatra.  All other sequences fell on the lower branch of the tree and that branch had no human sequences.

Recently, the government of Indonesia announced that the sequences from the human cases would be made public.  The password protection of the sequences at Los Alamos was removed on Friday, when over 500 sequences were made public.  These sequences included the 8 gene segments from the cat as well as two bird isolates from late 2005.  The more recent sequences were important because the bird sequences that had been made public were from collections between 2003 and 2005 and most of the 2005 collections were from the first half of the year.  In contrast, the human collections did not begin until the second half of 2005.

Analysis of the released sequences indicated that the differences in the human sequences extended to the other 7 gene segments.  Thus, all 8 gene segments from the human isolates were readily distinguished from the avian isolates, including the more recent sequences released last Friday.

The failure to match caused concern at the Jakarta meeting and more recent samples were sent to Australia for sequencing.  The initial sequences were deposited at Los Alamos today and none matched the human sequences.  Some were close to the two CDC sequences, but the larger number of more recent bird sequences did not produce a match of the novel cleavage site of the other differences in the human sequences.

Today Hong Kong also released additional recent human sequences.  Other than the one sequence from the Karo cluster, the other more recent sequences had the same novel cleavage site, RESRRKKR.

However, the most recent sequence, from a 3.5 year-old girl in Jakarta who died July 6 had an additional change in the cleavage site.  This change did not affect the amino acid sequence, which matched the other human sequences, but did provide another opportunity to identify other sequences at GenBank which matched.  The isolates with the matching sequences are lusted below.  Three of the matches came from another recent isolate from a baby in the Jakarta suburbs.  She was 20 month-old and H5N1 was isolated from three samples and all matched the recent H5N1 from the other young child in Jakarta.  Matches were also found in earlier low path H5 isolates, but the only recent isolates were from a child in the Jakarta area.

The WHO update for the 3.5 year old indocated poultry in the area was H5N1 positive.  These birds would provide yet another opportunity to isolate matching avian sequences, which in this case should also have the novel cleavage site.

The recent human H5N1 sequences provided evidence for additional evolution of the human H5N1 away from the bird sequences in Indonesia.  Most of the human sequences are from patients on Java and a high concentration is in west Java in the Jakarta area.  However, the number of different sequences continues to rise, and these sequences are easily distinguished from the bird sequences.

More sequences from more recent bird isolates are expected.  However, the data continue to point away from an avian source for the human H5N1 infections in Indonesia.

   ISDN187453  A/Indonesia/604H/2006            2006  H5N1  
   ISDN181365  A/Indonesia/CDC523/2006      2006  H5N1  
   ISDN183297  A/Indonesia/CDC523E/2006   2006  H5N1  
   ISDN183305  A/Indonesia/CDC523T/2006    2006  H5N1  
   AF082042      A/Duck/Potsdam/1402-6/86     1986  H5N2  
   M18450          A/Duck/Ireland/113/83               1983  H5N8  
   M18451          A/Turkey/Ireland/1378/83          1983  H5N8  
   AF082039     A/Duck/Hong Kong/698/79        1979  H5N3  
   AF290443     A/Duck/Ho Chi Minh/14/78         1978  H5N3  
   CY005575     A/duck/Hong Kong/342/1978     1978  H5N2  
   CY006028     A/goose/Hong Kong/23/1978     1978  H5N3  
   AF082038     A/Duck/Hong Kong/205/77         1977  H5N3  
   AB241615     A/duck/Miyagi/54/76                    1976  H5N3  
   AY500365     A/turkey/England/N28/73             1973  H5N2 

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