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More Recombination in PB2 Gene of H1 Canadian Swine
March 18, 2006
This week new H1 swine sequences from Canada were placed on deposit at GenBank in association with the publication, " Identification of Human H1N2 and Human-Swine Reassortant H1N2 and H1N1 Influenza A Viruses among Pigs in Ontario, Canada (2003 to 2005)" Seven of the isolates, listed below had an avian PB2 gene and as indicated earlier, several were recombinants and had sequences matching portions of three swine isolates from 1977, 1998, and 2002, also listed below:
As noted earlier, Ontario/11112 was an exact match with North Carolina/35922 for positions 756 through at least 1601 (only a partial sequences from North Carolina was available - sequence numbers adjusted to reflect longer sequences).. Ontario/53518 similarly was an exact match with Korea/CY02 from position 568 to the end of the sequence. Ontario/23866 matched Ontario/53518 through position 568 and then matched Tennessee/24 for positions 1008-1326. These data showed that each of the above recent isolates exactly matched an earlier isolate indicating each was a recombinant. The data also provided evidence for absolute fidelity in copying a large gene segment for 6 years and a shorter segment for 26 years. These data raised serious questions that attributed rapid influenza evolution to random mutation.
Analysis of the remaining sequences eliminated "random mutations" as a viable mechanism for the rapid influenza development observed on an annual basis. The remain isolates all contained an exact match with the 1977 isolate from Tennessee. Ontario/561 had an exact match over the same 318 BP region as Ontario/57561. Ontario/23866 matched Tennessee/24 for a larger region, 768-1354 which overlapped the 318 BP region. This 586 BP region has been copied with absolute fidelity for over 26 years, and this larger region includes sequences not essential for the other two Ontario isolates.
However, the remaining two isolates have even longer regions which exactly matched Tennessee/24. Ontario/48235 was an exact match over position 274-1880 while Ontario/55383 was an exact match over positions 274-1931. Thus each of these two isolates matched the 1977 sequences exactly for over 1600 BP. Maintaining an exact match for over 26 years in these five isolates for over 26 years is not a property of an error prone polymerase that produces evolving genes via random mutation. The regions maintained in these two isolates are not immutable, because most of the 1600 BP has been replaced in the other isolates, eliminating arguments invoking differential evolution.
These data show that all seven isolates evolved via recombination and random mutations were rare or non-existent over this time period. The recombination mechanism was supported by analysis of the PA, which was also a recombinant in all seven sequences, and six of the seven had exact matches with the same 1977 sequence from Tennessee. The regions of identity ranged from 598 to 1866BP.
The above data destroys the rapid evolution by random mutation hypothesis repeatedly stated by WHO and consultants. WHO does not analyze H5N1 bird flu sequences for homolgous recombination, which is the primary driver of H5N1 evolution. Most recombinants in H5N1 involve shorter sequences in part because of the high frequency of recombination, which reduces the length of the exact matches. Most of the differences in the recombined regions are reduce to single nucleotide changes. However, these small changes can be used to track where the isolates have been and where they are going. Moreover, since the parental strains are frequently known, future recombined sequences can be determined.
Determining future recombinants requires a robust and current database. WHO has sequestered many of the most recent H5N1 sequences, including human sequences from Turkey and most of the H5N1 sequences from Europe. These sequences are controlled by the WHO affiliated lab at Weybridge. Similarly, the WHO affiliated lab in Hong Kong has sequestered all human H5N1 sequences from Indonesia.
It is time for the WHO to obtain permission to release these data. If these labs are unwilling to release the existing data, the WHO should seek alternative labs that are willing to release data from samples received in the mail. The existing situation significantly delays the proper analysis of these valuable sequences.