Extracts from letter:
From: Clark Tibbetts, Ph.D.
Subject: Tessarae, LLC efforts to provide emergency assistance to Public and Animal Health agencies in response to the multinational outbreak of “Swine Flu”.
Date: April 29, 2009
Concerns about the current H1N1 swine flu epidemic have prompted numerous inquiries about the ability of the TessArray® RPM-Flu assay to accurately detect swine flu, based on its comprehensive coverage of human and avian influenza types. Although we have not had the opportunity to evaluate actual swine flu specimens, we are confident that the RPM-Flu assay will accurately detect and identify swine flu sequences with the current product configuration. While the results may not provide the same strain level identification, or perhaps even serotype information, that you are accustomed to obtaining with human and avian flu specimens, the detected sequences will be definitively identified as swine flu. Please see the section below for more detailed technical information regarding the detection and identification of swine flu using RPM-Flu.
Given the absence of definitive swine flu diagnostic assays, we have requested Emergency Use Authorization from the US FDA to allow RPM-Flu results to be used from patient care/treatment decisions by health care providers, and we will let you know if our request is approved.
The relaxed amplification conditions and degenerate primer sequences favor detection of the swine flu sequences. There is high similarity between multiple human and avian target gene detector sequences on the array to facilitate identification and differentiation of the swine flu sequences.
we are confident that the RPM-Flu assay will provide a positive result for swine flu, minimizing false positive detection events.
What is going on with the 2009 swine flu outbreak strains?
We have systematically reviewed the most recent gene sequencing data from 20 isolates of the current swine flu outbreak (A/CA, TX, KS, OH or NY/#/2009(H1N1)) that have been submitted to GenBank and GISAID databases. These sequences are very similar to one another across all eight viral genome segments, with only 94 mutations (SNPs) across more than 150,000 bp of aggregate sequence from the 20 isolates.
We have evaluated consensus outbreak strain viral genome sequences by BLAST analysis to reveal the corresponding 20 most similar sequence records from GenBank matching each of the 8 viral genome segments. The host species of origin for these 160 most similar strains are swine (135), avian (22) and human (3). For each of the eight genome segments, the most likely frequently identified host of origin is swine.
The top 20 matching sequence records for the outbreak strain NA1 gene were all from swine hosts in European countries. The most similar M gene sequence records were from swine hosts in Europe (11) or Asia (9). The most similar sequence records for HA, NP, NS, PA, PB1 and PB2 were predominately from swine hosts in North America and Asia.
It is important to note that the plurality of the most similar sequence records for all of the eight genome segments of the outbreak cluster were from isolates collected and identified more than ten years ago - none are particularly novel or unknown swine flu sequences. It is of further interest that the majority of the most similar sequence records for the HA1 gene were from A/H1N2 swine flu strains. Those for the NA1 gene were exclusively from A/H1N1 strains, and those for the remaining six gene segments were isolates from similar numbers of either A/H1N2 or A/H3N2 strains.
All of this suggests that the non-HA and non-NA gene segments of the outbreak cluster are drawn from a widely circulating and gene segment-reassorting pool of Type A influenza viruses that may appear as components of influenza viruses that infect swine, avian, human and/or other vertebrate hosts. There is no reliable correlation of the sequences of these six gene segments with the particular A/HN subtype of any Type A strain, including the 2009 swine flu outbreak cluster.
The NA1 gene of the outbreak cluster does seem to be an outlier with respect to the origins and sequence similarity profiles of the other seven genome segments. This is also noteworthy in that the dominant phenotype of circulating human A/H1N1 strains is now tamiflu resistant (2008-2009) while the 2009 swine flu outbreak cluster appears to be tamiflu sensitive. Tamiflu resistance now apparent in human A/H1N1 strains is associated with NA1 codon TAT/Tyrosine at position 275 of NA1. The sensitive human strains (pre-2008) have the codon CAT/Histidine at this locus. The NA1 gene of the outbreak cluster strains has codon CAC/Histidine at this position, consistent with assertions of tamiflu sensitivity. We note that the same genotype (CAC/Histidine-275) is present in the NA1 gene of circulating avian influenza A/H5N1 strains.
How can TessArae help?
If you want to analyze clinical specimens with suspected 2009 outbreak swine flu, we are available to support your use of the TessArray RPM-Flu 3.1 assay. If you are willing and able to share your assay results with the community, then we are willing to replace a few of your TessArray RPM-Flu 3.1 assay kits at no charge.
We are configuring a new Multiplex Cocktail E for the TessArray RPM-Flu 3.1 kits that will enhance the capability of the protocol to amplify, detect and identify the HA1, NA1, M, NS and PB2 genes of swine flu. Please let us know if you are interested in testing this simple modification of the standard protocol.
Finally, if you anticipate upcoming orders of additional TessArray RPM-Flu 3.1 kits, please let us know as soon as possible so that we can ensure sufficient inventory of RPM-Flu kits for your needs.
Clark Tibbetts, PhD
Executive Vice President and CTO