- "Plummer said his lab has been doing genetic sequencing
on genes from the viruses, but that isn't helping to clarify the picture.
- ''The sequencing's been problematic because the RNA
is mixed up,'' he said.
- ''And it makes it difficult because you get two sets
_ or three sets _ of sequences from this one sample. And they're all jumbled
up. It's very hard to sort out.''
- ''Say if you get an H5 and an N1, and an H7 and an
N3 from a sample, you don't know which H goes with which N.''
- Although the above comments on H5 mixtures from wild
ducks in several Canadian provinces are true, determining if any of the
isolates are the Asian HPAI H5N1 should be fairly straightforward. The
Asian H5N1 has characteristic insertions or deletions that would lead to
size differences of insets generated by RT-PRC, which should be evident
on gels prior to sequencing. These difference can be identified in the
absence of cloning and can be verified by sequencing of the inserts.
- The most well known change in H5N1 is the additional
basic amino acids at the HA cleavage site. All H5N1 HPAI wild bird sequences
from Asia have the characteristic RRRKKR sequence The multi-basic amino
acids at this site are diagnostic for HPAI and are routinely used in diagnostic
labs. If none of the H5 mixtures have this sequence, then that result
should be noted. If any do have the sequence, then the isolate will almost
certainly be H5N1 since that sequences has never been reported in any flu
sero-type other than HPAI H5N1.
- Similarly, the N1 in HPAI H5N1 has a 20 amino acid deletion.
This deletion is found in all recent Asian H5N1's including all reported
sequences from wild birds. Thus, an insert representing this region would
have a unique size. Identity with N1 from HPAI H5N1 could be verified
by sequencing of the insert. Again, because of the unique size, cloning
is not required.
- The same can be said for the NS gene, which has a 5 amino
acid deletion and would also create a novel sized insert that could also
be verified by sequencing.
- Sequencing of a portion of yet another gene, PB2, could
also answer questions about the H5 in Canada. So far all HPAI H5N1 from
wild birds have the E627K polymorphism. This polymorphism has never been
isolated in an H5 from a bird prior to the sequencing of the Qinghai Lake
isolates. Since then, the only bird isolates with that polymorphism have
been closely related isolates from Novosibirsk.
- Winnipeg has had the H5 isolates for at least two weeks
now and it is time for straight answers to simple questions regarding H5N1
wild bird flu in Canada.
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