Development of a quadruple qRT-PCR assay for simultaneous identification of highly and low pathogenic H7N9 avian influenza viruses and characterization against oseltamivir resistance

Abstract

Background: During the fifth wave of human H7N9 infections, a novel highly pathogenic (HP) H7N9 variant emerged with an insertion of multiple basic amino acids in the HA cleavage site. Moreover, a neuraminidase inhibitor (NAI) resistance (R292K in NA) mutation was found in H7N9 isolates from humans, poultry and the environment. In this study, we set out to develop and validate a multiplex quantitative reverse transcript polymerase chain reaction (qRT-PCR) to simultaneously detect the presence of H7N9 and further identify the HP and NAI-resistance mutations.

Methods: A quadruple qRT-PCR to simultaneously detect the presence of H7N9 and further identify the HP and NAI-resistance mutations was designed based on the analyses of the HA and NA genes of H7N9. This assay was further tested for specificity and sensitivity, and validated using clinical samples.

Results: The assay was highly specific and able to detect low pathogenic (LP)- or HP-H7N9 with/without the NAI-resistance mutation. The detection limit of the assay was determined to be 50 genome-equivalent copies and 2.8 × 10^{- 3} 50% tissue culture infectious doses (TCID₅₀) of live H7N9 per reaction. Clinical validation was confirmed by commercial kits and Sanger sequencing with ten clinical samples.

Conclusions: We developed and validated a rapid, single-reaction, one-step, quadruple real-time qRT-PCR to simultaneously detect the presence of H7N9 and further identify the HP- and NAI-resistance strains with excellent performance in specificity and sensitivity. This assay could be used to monitor the evolution of H7N9 viruses in the laboratory, field and the clinic for early-warning and the prevention of H7N9 infections.

Keywords: H7N9; Highly/low pathogenic avian influenza virus; Molecular diagnostics; NAI-resistance; Oseltamivir; Quadruple qRT-PCR.

Fig. 1

Validation and optimization of the multiplex qRT-PCR assay using different H7N9 viruses. (a) Representative amplification plot for viral RNAs from LP-H7N9 without NAI-resistance mutation, (b) LP-H7N9 with NAI-resistance mutation, (c) HP-H7N9 without NAI-resistance mutation and (d) HP-H7N9 with NA inhibitor-resistance mutation. H7-W: detection target for universal H7; H7-M: detection target for highly pathogenic H7; N9-W: detection target for universal N9; N9-M: detection target for R292K mutation of N9

Fig. 2

Sensitivity of the multiplex qRT-PCR assay using DNA standards. (a) Representative amplification plot of the different concentrations of plasmids expressing the targeted H7 or N9 genes (10-fold dilutions). (b) Standard curve for ten-fold serial dilutions of the DNA standards. The log numbers of plasmids (Copies/μL) are expressed linearly on the x-axis, whereas Ct values obtained from qRT-PCR are expressed linearly on the y-axis. H7-W, H7-M, N9-W and N9-M are defined the same with Fig. 1

Fig. 3

Sensitivity of the multiplex qRT-PCR assay using viral RNAs from LP-H7N9 viruses. (a) Representative amplification plot and standard curves for ten-fold serial dilutions of LP-H7N9 without NAI-resistance mutation strain. (b) Representative amplification plot and standard curves for ten-fold serial dilutions of LP-H7N9 with NAI-resistance mutation strain. The log numbers of live H7N9 viruses (TCID₅₀/mL) are expressed linearly on the x-axis, whereas Ct values are expressed linearly on the y-axis. H7-W, H7-M, N9-W and N9-M are defined the same with Fig. 1

Fig. 4

Sensitivity of the multiplex qRT-PCR assay using viral RNAs from HP-H7N9 viruses. (a) Representative amplification plot and standard curves for ten-fold serial dilutions of HP-H7N9 without NAI-resistance mutation strain. (b) Representative amplification plot and standard curves for ten-fold serial dilutions of HP-H7N9 with NAI-resistance mutation strain. The log numbers of live H7N9 viruses (TCID₅₀/mL) are expressed linearly on the x-axis, whereas Ct values are expressed linearly on the y-axis. H7-W, H7-M, N9-W and N9-M are defined the same with Fig. 1