Validated RealTime reverse transcriptase PCR methods for the diagnosis and pathotyping of Eurasian H7 avian influenza viruses

Abstract

Background: Avian influenza (AI) caused by H7 AI viruses (AIVs) of both low pathogenicity (LP) and high pathogenicity (HP) are notifiable poultry diseases.

Objectives: Design and validate two RealTime reverse transcriptase polymerase chain reactions (RRT PCRs) for Eurasian H7 AIV detection and pathotyping.

Methods: The H7 RRT PCRs amplified within the (i) HA2 and (ii) cleavage site CS regions of the haemagglutinin gene. Both were validated against 65 H7 AIVs, 57 non-H7 AIVs and 259 poultry swabs in comparison to M gene (AI generic) RRT PCR and virus isolation (VI). An additional 38 swabs and 20 tissue specimens extended validation against M gene RRT PCR.

Results: Both H7 RRT PCRs amplified all 61 Eurasian lineage H7 AIVs and none of 57 non-H7 AIVs. A total of 297 poultry swabs were used to determine diagnostic sensitivity and specificity relative to M gene RRT PCR, sensitivity was 95.4% and 64.6% for the HA2 and CS RRT PCRs respectively, and specificity 97.9% and 99.6% respectively. The H7 HA2 RRT PCR was more sensitive than VI. This was emphasized by analysis of 37 swabs from turkeys infected experimentally with HPAI H7N1 virus sampled at 24 hours post-inoculation and LPAI H7N1 chicken infections sampled at 40-64 hours. Although less sensitive, usefulness of the H7 CS RRT PCR was confirmed by the correct molecular pathotyping for all 61 Eurasian lineage H7 AIVs tested.

Conclusions: The high sensitivity of H7 HA2 RRT PCR confirms its suitability for use in poultry surveillance and disease diagnosis. H7 CS RRT PCR provides an opportunity for rapid pathotyping of H7 AIVs.

Figure 1

Distribution of H7 RRT PCR Ct values for swabs collected 24 hours pi from H7N1 HPAI directly infected turkeys (Group C, Table 3). Ct values are shown for both the H7 HA2 (n = 9) and CS (n = 2) RRT PCRs where open and filled symbol indicate VI positive and VI negative swabs respectively. Swab results from this group which were negative by H7 HA2 (two, both VI negative) and H7 CS (nine, i.e. five VI negative and four VI positive) RRT‐PCRs are not shown.

Figure 2

Distribution of H7 RRT PCR Ct values for swabs collected 40–64 hours pi from H7N1 LPAI directly infected chickens (Group A, Table 3). Ct values are shown for both the H7 HA2 (n = 23) and CS (n = 11) RRT PCRs where open and filled symbol indicate VI positive and VI negative swabs respectively. Swab results from this group which were negative by H7 HA2 (three, all VI negative) and H7 CS (15, i.e. 14 VI negative and one VI positive) RRT PCRs are not shown.