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Commenting on the point genetic mutation seen in throat swabs of three
patients who succumbed to the virus, Chadha said, "We didn't see any
change in the genetic make-up of the virus after that." Of the three
patients, two were from Pune and one from Nashik, said Chadha. The
mutation was detected in the haemagglutinin (HA) region of the virus'
gene, she added.
The above comments describe the detection of D225G in the HA sequences from three patients in India. Thirteen HA sequences were released at Genbank in association with a PLOS paper on H1N1 from India. The paper included two samples from Pune, which had D225G (A/Pune/NIV9355/2009 and A/Pune/NIV10278/2009) and both were fatal, indicating the two sequences represented the two Pune patients described above. However, earlier the CDC had released an HA sequence, A/India/8489/2009, which also had D225G, but was distinct from the two sequences above and was from a fatally infected 42 year old female, which matched A/Pune/NIV8489/2009 in sample number, age, and gender. Moreover, the NA sequence from each isolate was identical, and included the rare polymorphism, C765T.
The two HA sequences also had the rare tandem polymorphisms, T11C and T30C, further supporting sequences from the same patient. However, the sequence released by India did not have A716G (encodes D225G) or A424G, which was present as a mixture in the CDC sequence. Thus, the sequence from India appears to represent a clone that did not have the mixed signal or D225G, raising concerns that D225G is under-reported. The CDC sequence would represent the fourth example of D225G in India, all of which were fatal.
The failure to detect/report D225G in one of the fatal Pune infections extends the number of examples of D225G failures. A recent abstract on the death cluster at Duke Medical Center indicated D225G or D225N was found in two of the three fatal cases. However, the three fatal cases, and the one survivor were all infected with H1N1 with H274Y in NA and Y233H in HA indicating all four patients were infected with the same H1N1. However, D225G was only detected in two sequential collections from one patient (and was not found in a partial sequence from the original sample), while D225N was found in another patient, but also undetected in another partial sequence of the original sample. These data once again indicate D225G and D225N can be missed in sequencing that lacks sensitivity.
Similarly, D225G was found in early egg isolates from mild cases, but was not found in original samples or virus grown in mammalian cells. However, D225G is associated with binding to 2,3 receptors, which are at low levels in mammalian cells, but at high levels in chicken embryos. Therefore, D225G will be more easily detected in egg isolates, but many sequences are limited to isolates from mammalian cell lines, even though human lung has 2,3 receptors. Thus, the true levels of D225G in clinical samples remain unclear.
However, a recent report from Norway identified D225G in 11 patients. Eight were fatal and three were severe. Similarly 27/37 autopsy lung sampls from Ukraine had D225G, D225N, or both. Like Norway, virtually all samples positive for D225G/N were from fatal cases.
These data, as well as similar data from other countries raise concerns that D225G/N may be higher in the next wave, since D225G was declared a “low reactor” by Mill Hill. The many examples of false negatives, like the failure to detect D225G in India, continue to raise concerns.