Rapid PCR-based molecular pathotyping of H5 and H7 avian influenza viruses

Abstract

While the majority of avian influenza virus (AIV) subtypes are classified as low-pathogenicity avian influenza viruses (LPAIV), the H5 and H7 subtypes have the ability to mutate to highly pathogenic avian influenza viruses (HPAIV) in poultry and therefore are the etiological agents of notifiable AIV (NAIV). It is of great importance to distinguish HPAIV from LPAIV variants during H5/H7 outbreaks and surveillance. To this end, a novel and fast strategy for the molecular pathotyping of H5/H7 AIVs is presented. The differentiation of the characteristic hemagglutinin (HA) protein cleavage sites (CSs) of HPAIVs and LPAIVs is achieved by a novel PCR method where the samples are interrogated for all existing CSs with a 484-plex primer mixture directly targeting the CS region. CSs characteristic for HP or LP H5/H7 viruses are distinguished in a seminested duplex real-time PCR format using plexor fluorogenic primers. Eighty-six laboratory isolates and 60 characterized NAIV-positive clinical specimens from poultry infected with H5/H7 both experimentally and in the field were successfully pathotyped in the validation. The method has the potential to substitute CS sequencing in the HA gene for the determination of the molecular pathotype, thereby providing a rapid means to acquire additional information concerning NAIV outbreaks, which may be critical to their management. The new assay may be extended to the LP/HP differentiation of previously unknown H5/H7 isolates. It may be considered for integration into surveillance and control programs in both domestic and wild bird populations.

Fig. 1.

Outline of the scheme for the seminested three-level real-time PCR method designed for avian influenza pathotyping. A triangle marks the CS. Primer concentrations are shown. The preamplification PCR, denoted by I, and the two-level pathotyping PCR are indicated and boxed with dashed and full lines, respectively. The bipartite selection primers are denoted by II, with the tag sequence signifying HP and LP viruses shown with full and dashed lines, respectively. The numbers of distinct primers targeting CSs of HP and LP viruses are shown as n_1–178 and m_1–306, respectively. The detection primers are denoted by III and have sequences identical to the corresponding tag sequence. These primers initiate the amplification of the PCR products originating from selection primers to detectable levels. The dyes tethered to the detection primers that label products originating from HP (FAM) and LP (CO-560) viruses are indicated. Note that the concentration of the reverse preamplification primers when used in the two-level pathotyping step is lowered to 0.3 μM.

Fig. 2.

Pair-wise comparison of the sequence-recognizing portion of the 484 bipartite selection primers produced by partly degenerated primers listed in Table 6. The intervals of percent similarity corresponding to each color are shown in the figure. The sets of sequences corresponding to CSs of HP and LP isolates are compared in the lower and upper diagonal areas, respectively, and are separated by the black lines. The off-diagonal areas show the similarities between the two sets of primers. The highest similarity (90.91%) between two primers belonging to different pathogenicity groups is indicated by a white triangle.

Fig.3.

Amplification curves displayed as CO-560 fluorescence normalized against FAM fluorescence and baseline corrected. Due to the plexor primer fluorescence, which decreases upon product formation, this procedure leads to signal decrease for LP isolates and apparent signal increase for HP strains (see the text). Preamplifications were carried out for Eastern Hemisphere H5 (A) and H7 (B) isolates using the H5 (A)- and H7 (B)-specific primers designed for the Eastern Hemisphere isolates (Table 5). The tested AIV isolates are indicated in the figure and are listed in Table 2. Curves obtained for HP and LP viruses are shown with full and dashed lines, respectively. For Western Hemisphere H5/H7 isolates, the preamplifications were carried out using the H5 (C)- and H7 (D)-specific primers designed for the Western Hemisphere isolates (Table 5). The tested AIV isolates are indicated in the figure and are listed in Tabe 3. Curves obtained for HP and LP viruses are shown with full and dashed lines, respectively. The selection and detection primers were identical in all experiments.

Fig.3.

Amplification curves displayed as CO-560 fluorescence normalized against FAM fluorescence and baseline corrected. Due to the plexor primer fluorescence, which decreases upon product formation, this procedure leads to signal decrease for LP isolates and apparent signal increase for HP strains (see the text). Preamplifications were carried out for Eastern Hemisphere H5 (A) and H7 (B) isolates using the H5 (A)- and H7 (B)-specific primers designed for the Eastern Hemisphere isolates (Table 5). The tested AIV isolates are indicated in the figure and are listed in Table 2. Curves obtained for HP and LP viruses are shown with full and dashed lines, respectively. For Western Hemisphere H5/H7 isolates, the preamplifications were carried out using the H5 (C)- and H7 (D)-specific primers designed for the Western Hemisphere isolates (Table 5). The tested AIV isolates are indicated in the figure and are listed in Tabe 3. Curves obtained for HP and LP viruses are shown with full and dashed lines, respectively. The selection and detection primers were identical in all experiments.

Fig. 4.

Amplification curves for the 2010 EU AI PCR proficiency ring trial. The investigated samples are indicated and are listed in Table 4. The 10 samples were preamplified with the H5 (A)- or H7 (B)-specific primer designed for Eastern Hemisphere isolates (Table 5). Curves obtained for HP and LP viruses are shown with full and dashed lines, respectively.

Fig. 5.

Amplification curves for the three indicated H7 influenza viruses with (curves with symbols) and without (curves without symbols) a selection primer specifically accounting for the CS of the equine influenza virus A/equine/Prague/1/57 (H7N7) (thick lines). The samples were preamplified with the H7-specific primers for the Eastern Hemisphere (Table 5).