1. Executive summary
An exercise to assess the Community Network of Reference Laboratories for Human Influenza in Europe’s (CNRL) capability to detect novel reassortant and circulating triple reassortant swine viruses (TRA) in humans was completed in July 2011. These are viruses that are appearing in the United States in pigs (swine) and are
occasionally seen there in humans but have yet to be reported in Europe in either swine or humans.
A short questionnaire was distributed to 35 laboratories in 29 countries to assess the predicted detection of TRA
A(H3N2) viruses using a theoretical, computer-based (in silico) approach. The objective was to identify gaps in the capability of laboratories and countries to detect and subtype such swine origin viruses, should they appear in Europe. Responses were received from all participating laboratories.
Type A influenza virus detection by real-time reverse transcription-polymerase chain reaction (RT-PCR) is
performed by the majority of responding laboratories (94%) and countries (93%), using the matrix (M) gene as
the target. Where predictions could be made (31 laboratories in 25 countries), the typing real-time RT-PCR assay employed was predicted to be capable of detecting TRA viruses. This predicts that the network has a good capability for detecting TRA viruses when using generic influenza A real-time RT-PCR assays.
Subtype determination of influenza A(H3) by real-time RT-PCR is undertaken by nearly all responding network
laboratories (34) and countries (28). Prediction of the RT-PCR subtyping assays ability to detect TRA H3N2 viruses highlighted that this subtyping capability is significantly reduced compared to type A specific detection. Whilst this signals gaps in the CNRL network for identification of TRA viruses of swine origin, this overall position is as would be expected, with many different protocols in use for subtype determination, which requires the most highly variable genes to be targeted.
The in silico exercise provided a technical performance prediction of individual assays, rather than a reflection of
how they are employed in laboratory algorithms, which may significantly affect the ability of a laboratory/country to detect unusual variants. If laboratories use a screening approach, testing initially with a generic influenza A realtime RT-PCR assay first, then the capability for detecting TRA swine origin H3N2 viruses is good. However, if laboratories use algorithms with a subtyping real-time RT-PCR assay detecting H3 and, or N2 gene segments first, then these viruses could be missed in approximately 30% of laboratories.
The following conclusions and recommendations were made from the results of the analysis of the in silico exercise;
• The exercise proved to be useful in highlighting development requirements and gaps in detection and
identification capabilities for potential emerging influenza A TRA viruses of swine origin. Where countries
have more than one CNRL laboratory, there is a requirement to relate the capability of laboratories to the
overall in-country capabilities. Within Europe, there is a need to upgrade the capability for detection and
identification of swine origin viruses.
• A review of algorithms for influenza virus detection and identification used in CNRL laboratories is required
to determine the detection capabilities of individual countries and within the network overall.
• The in silico assessment should be followed up with a proficiency panel containing the same North American
TRA viruses considered in the in silico exercise, to provide qualitative and quantitative experimental data to
assess the validity of the in silico approach.
• The proficiency panel should be extended to contain more recent TRA H3N2 North American viruses, and
Eurasian swine viruses.
• The discrepancy between high confidence in the ability of M gene RT-PCR assays to detect TRA H3N2
viruses but the lower ability of RT-PCR based subtyping, requires clear algorithms for rapid investigation of
viruses which are not subtypable.
• Although a number of the H3 and N2 subtyping protocols are predicted to be positive with the TRA H3N2
virus, this would not identify the virus as being a TRA H3N2 virus. Therefore, testing algorithms should
include the capability to distinguish seasonal human viruses from new zoonotic influenza viruses.
• Methodology for rapidly assessing laboratory protocols in silico should be developed through web-based
sequence questionnaire tools, to enhance the rapid assessment of diagnostic capability within the CNRL
network in the event of an emerging virus.http://ecdc.europa.eu/en/publications/P ... ercise.pdf