Review of diagnostic tests for SIV s and pandemic H1N1 in pigs. Ian Brown. Veterinary Laboratories Agency- Weybridge

Transcription

1 Review of diagnostic tests for SIV s and pandemic H1N1 in pigs Ian Brown Veterinary Laboratories Agency- Weybridge

2 Protocols submitted to OFFLU scientist (BC) VLA Weybridge CDC, Atlanta SEPRL, Athens FLI Reims CVI, Lelystad NCFAD, Winnipeg Health Protection Agency, UK

3 Aim: To rapidly provide via OFFLU website recommended protocols for tests for application to detection of pandemic H1N virus (primarily in pigs)

4 Approach All assays relate to real time RT-PCR Assay target M, NP, HA Evaluation & Validation Broadly assays were assessed for their suitability for application within the continent/region where they were designed Application to pigs (+poultry) would need to take account of global variations in virus epidemiology

5 Origin of pandemic H1N1 2009

6 VLA M gene: Perfect match Combo (50:50) Spackman/perfect match primers Standard EU M gene test for AI HA: H1.118 differentiates H1 pandemic in European pigs

7 VLA (contd) Evaluation using defined populations of samples Swine positive population Represent of contemporaneous viruses in European pigs All subtypes N = 50 Swine clinical specimens (ex pandemic H1N infection study) N= 38 (c1000 perfect match assay): +Singapore field Negative population of swine clinical samples N=121 Avian positive population Including all subtypes; n= 44 Human pandemic reference strains

8 Good specificity >98% in all assays M gene: Perfect match : Highest sensitivity for pandemic virus but loss off up to 2 CT s for Euro SIV Combo (50:50) Good sensitivity for diversity of swine viruses; loss of c2 CT s for AIV s Standard EU M gene test for AI: significantly reduced sensitivity for pandemic viruses HA: H1.118 equal sensitivity to CDC, SEPRL, HPA, VLA M gene methods. Low sensitivity for ch1n1

9 Modified M gene This is an update of Spackman et al., 2002 that modifies the reverse primer to account for mismatches with the 2009 emergent H1N1 virus. The modified version includes two reverse primers ( 2002 and 2009 ) to increase reactivity with as many isolates as possible. This test will detect most type A influenza viruses. Emergent H1N1 lineage - N1 This assay is specific for the pandemic (H1N1) 2009 lineage and will not detect North American classic swine lineage viruses, European swine influenza viruses, human seasonal influenza viruses, or avian viruses including the Asian H5N1 HPAIV. NVSL reported this assay to be ~1 log10 less sensitive than SEPRL 2009 M-gene. Classical SIV (North American) N1 This assay is specific for the North American classic swine lineage and will not detect 2009 emergent H1N1 lineage viruses, European swine influenza viruses, human seasonal influenza viruses, or avian viruses including the Asian H5N1 HPAIV.

10 NADC, Canada M gene Comparison carried out at VLA indicate this test to be slightly less sensitive (~1CT) to modified SEPRL 2009 M-gene in detecting A(H1N1)v and avian-like SIV. NVSL observed the assay to be less specific for Influenza A. NVSL also reported that although sensitivity was comparable to SEPRL 2009 M-gene obtaining quality primers for the CFIA test may be problematic.

11 CDC, Atlanta M gene (ph1n1) NVSL reported SEPRL M-gene to be equally sensitive. VLA reports tests ability to detect avian-like and conventional swine H1N1. NP gene (ph1n1) NVSL reported SEPRL M-gene to be more sensitive. HA gene (ph1n1) VLA reported equal sensitivity of CDC, HPA and VLA test in detecting A(H1N1)v. Specificity to A(H1N1)v cf N.American SIV s with classical HA?

12 FLI, Riems NP gene Generic influenza A virus rrt PCR. This rrt PCR has been in use at the FLI for more than two years with excellent performance values. Analytical tests have revealed a sensitivity which is similar to that of the Spackman M PCR (non modified). In contrast to the M gene PCR, the novel A/H1N1 virus is detected by the NP PCR without any primer/probe modifications HA gene (IVA H1N1-1; IVA H1N1-2) FLI report these PCRs have been evaluated with a large panel of standard influenza A virus isolates, including several Eurasian porcine strains and two novel A/H1N1 isolates from human patients. The performance characteristics appear to be similar to the assay published by the CDC, Influenza viruses of avian and swine origin (H1 and non-h1 strains) were not detected by this assay. Specificity to cf N.American/Asian SIV s with classical HA?

13 CVI, Lelystad HA gene (ph1n1) CVI report discriminates between European SIV strains and pandemic (H1N1) CVI reports CDC recommended PCR, acdc nh1n1-2009, and CVI nh1n are all app. 10x more sensitive compared to non modified Spackman PCR. So far tested, all three PCRs are able to discriminate between European SIV strains and nh1n HPA, UK H1 HA gene This assay appears to detect most H1, however further investigation is required for non-human isolates. VLA reported equal sensitivity of CDC, HPA and VLA test in detecting A(H1N1)v and ability to detect avian-like SIV. Ellis et al., (2009) reported that in comparison with CDC H1 qpcr, HPA protocol showed greater sensitivity

14 EUROPEAN TESTING ALGORITHM FOR PIGS (OFFLU website?) Screen all samples with modified pan Flu A real time RT-PCR (modified 50:50 M-gene as per SEPRL/VLA; perfect match VLA; FLI NP). All positives can be rapidly screened by a pandemic H specific real time RT-PCR (applicable to European setting only) targeting HA (VLA H1; FLI HA) AND simultaneously inoculate sample(s) into embryonated eggs/mdck s etc to provide virus for further characterization Conventional virus typing by HI/NI if PCR at step 2 is negative Further identification at whole genome level if PCR at step 2 is positive to confirm identity and detect any changes that may have occurred via pigs.

15 Thank you for your attention