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78% of US Adolescent 2011/2012 Flu A Sequences Are Novel
Recombinomics Commentary 07:45
December 12, 2011

The latest two cases of novel influenza (trH3N2 and trH1N2) have increased the frequency of novel virus in children in the United States to 78% of the published sequences for the 2011/2012 season (see chronological list below), which is higher than the 67% frequency for the period between the end of July to the official start of the flu season in October, 2011.

This season there has only been one seasonal H3N2 sequence (A/Indiana/09/2011, as well as one H1N1pdm11 sequence (A/Florida/27/2011).  In contrast, there have been seven novel sequences (five H3N2pdm11 – A/Maine/06/2011, A/Maine/07/2011, A/Iowa/07/2011, A/Iowa/08/2011, A/Iowa/09/2011; one trH3N2 – A/West Virginia/06/2011; one trH1N2 – A/Minnesota/19/2011).  Moreover, only the first two cases from Maine were associated with “swine exposure”, although, like the four H3N2pdm11 cases over the summer (A/Indiana/08/2011, A/Pennsylvania/09/2011, A/Pennsylvania/10/2011, A/Pennsylvania/11/2011), none were linked to swine with confirmed SOIV, and only one case was linked to symptomatic swine, which tested negative for SOIVs.

The reduced frequency in novel influenza cases linked to swine exposure is related to the CDC’s program for the identification of novel viruses which is largely focused on testing of cases during the off season, when novel viruses are more easily detected because the background of seasonal flu is greatly reduced.  Thus, cases with flu-like symptoms who are loosely tied to swine are tested further by the CDC, in part because these viruses are identified via cross reactivity, which can lead to false negatives, or a misdiagnosis of seasonal flu.

The first case confirmed in 2011, A/Pennsylvania/40/2010, was a September 2010 case which initially tested as seasonal H3N2, because samples with low RNA can be positive for H3N2, but negative for the swine component NP in the PCR testing panels because the NP target is based on sequences from H1N1pdm09, and detection relies on cross reactivity with related sequences, such as those in the above trH3N2 case.  This sample came from a patient with swine exposure, which was the likely cause for the additional testing by the CDC, and the low RNA led to attempts to grow the virus, which delayed reporting until February 4, 2011, and delayed the sequence until Sunday, April 17, 2011.  The sequence was closely related to all 8 gene segments of A/Wisconsin/12/2010, which was also a September, 2010 case, and was one of the two case cited in a WHO page alert in November 2010.  The CDC report to the FDA advisory committee on vaccine selection listed the Pennsylvania case prior to the Minnesota case from 2010 (A/Minnesota/11/2010) suggesting the mis-diagnosed trH3N2 case was also known to be another novel influenza based on partial sequencing, which can be generated by direct sequencing of the sample and the CDC was aware of this second case from Pennsylvania when the WHO pager alert was issued..

This case was similar to the second case from Maine, A/Maine/07/2011, which also involved low levels or RNA.  The newly approved CDC PCR test was used, but only the seasonal H3N2 target was positive.  The sample tested negative for the NP swine target, but since the the case had multiple swine “exposures” the sample, which tested as seasonal H3N2, was sent to the CDC, where partial sequencing showed it was a H3N2pdm11 case and was virtually identical to the Maine/06/2011.  However, only short segments of 4 of the 8 gene segments were released due to the low level of RNA, and full sequences have not been released, which is likely due to problems with isolation of a virus from that sample.  Similar delays are linked to the three samples from Pennsylvania, highlighting the low levels of RNA in these “off season” samples, and a need for sequencing to confirm that these cases were infected with a novel influenza.

Thus, the association with swine is not due to infections by swine, but due to detection via sequencing of samples which would normally simply be misdiagnosed as seasonal H3N2 or negatives (two of the Pennsylvania case were initially classified as unsubtyable, as were many 2010 samples, however, these earlier cases had no swine exposure and therefore were not forwarded to the CDC and did not appear in the CDC FluViews in the 2010/2011 season).

Thus, the swine exposure positives are linked to more intense testing (sequencing) because of the CDC interest in samples from cases with a swine exposure.

The sequence data  represent a more accurate determination of what is and what isn’t circulating, and those sequences indicate the levels of novel influenza in children is alarmingly high, and the absence of a program targeting such cases without swine exposure, leads to a gross underestimate the frequency of such cases.

As RNA levels increase with the start of the flu season, more cases will test positive for the NP component because the sensitivity of the PCR test will increase, and all seven novel cases are reassortants of the 2010 sequences from A/Wisconsin/12/2010 and A/Pennsylvania/40/2010.
The H3N2pdm11 cases have reassorted with an H1N2 related to A/swine/Ohio/FAH10-1/2010, which contributed PB1, NA, and the H1N1pdm09 M gene.  The trH1N2 reassorted with the same trH1N2 parent, but swapped out the H3 and N2 genes from the trH3N2 human cases for the H1 and N2 in the swine trH1N2.  Similarly, the 2011 trH3N2 case exchanged the N2 in H3N2pdm11 for the N2 found in trH3N2 swine, which is a lineage that is distinct from the human trH3N2 cases, but still traces back to human H3N2 from 2003.

Thus, all of the 2011 novel cases have seasonal H3, H1, or N2.  The H3 traces back to seasonal H3N2 from the mid 1990’s, the H1 traces back to seasonal H1N2 from 2003, while the N2 traces back to seasonal H3N2 from 2003, although the N2 is found in three different lineages circulating in swine (the N2 from 2010 human trH3N2 is from one version in trH3N2 swine, while the N2 from the 2011 human trH3N2 is from another trH3N2 lineage), while the 2011 H3N2pdm11 cases as well as  the trH1N2 case have an N2 from swine H1N2.  The 2010 trH3N2 is from a different lineage, but not found in any of the 2011 novel cases, which all have an N2 from the same trH1N2 swine lineage.

All seven novel cases this season, as well as the four adolescent H3N2 pdm11 cases from the summer, and the adult case from this season (A/Indiana/10/2011) have 2010 trH3N2 parent genes (5 or 6), and the five most recent case have no swine exposure, emphasizing the need for aggressive testing, including sequencing, of cases under 10 without swine exposure.

Novel Influenza in Bold

A/West Virginia/06/2011   1F  trH3N2             11/21
A/Iowa/07/2011                   3F  H3N2pdm11   11/14
A/Iowa/08/2011                  1M  H3N2pdm11   11/14
A/Iowa/09/2011                  2M  H3N2pdm11   11/14
A/Minnesota/19/2011      U5F  trH1N2            11/04
A/Florida/27/2011             10F   H1N1pdm09  10/30
A/Maine/07/2011               8M   H3N2pdm11   10/24
A/Maine/06/2011               8M   H3N2pdm11   10/10
A/Indiana/09/2011             1M    H3N2               10/02

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