A Strand-Specific Quantitative RT-PCR Method for Detecting vRNA, cRNA, and mRNA of H7N9 Avian Influenza Virus in a Mouse Model

Abstract

Avian influenza virus (AIV) remains a persistent threat to both the poultry industry and human health. Among the AIV subtypes posing public health threats, H7N9 AIV is responsible for five epidemic waves of human infection in China. Here, a detection system based on a mouse model was established, which can simultaneously and quantitatively analyze the dynamic changes in the viral genomic RNA (vRNA), complementary RNA (cRNA), and messenger RNA (mRNA) of H7N9 AIV by using reverse transcription primers with tag sequences to reverse transcribe the three species of RNAs into corresponding cDNA templates, which are then absolutely quantified using the TaqMan quantitative PCR method. This system specifically targets the PB2 and NA genes and, for the first time, enables a spatiotemporal analysis of all three viral RNA species within an animal model. Our results revealed that H7N9 AIV exhibits characteristic replication kinetics, with all three species of viral RNAs showing a rapid increase followed by a certain degree of decline. This system offers a powerful tool for us to further advance our understanding of the replication dynamics of AIV in mice.

Keywords: H7N9; avian influenza virus; qRT-PCR; replication kinetics.

Figure 1

Cross-amplification of cDNAs derived from vRNA, cRNA, and mRNA of H7N9 AIV using three primer pairs by TaqMan qRT-PCR. (a) Schematic diagram of the TaqMan qRT-PCR method for detecting vRNA, cRNA, and mRNA of H7N9 AIV. Strand-specific RT was performed using primers containing the vRNA-tag (purple), cRNA-tag (light blue), or mRNA-tag (rose red) to generate three distinct types of cDNA. Each tag sequence, together with a gene-specific primer, formed a primer pair for amplification. Fluorescent probes were added, and quantification was conducted using TaqMan qRT-PCR. (b) Strand-specific cDNAs of H7N9 AIV were generated from the total RNA extracted from the mouse lungs using primers containing tag sequences, each of which contained a 20-nucleotide sequence unrelated to the influenza virus at the 5′ end. The tagged cDNA was amplified by TaqMan qRT-PCR using the tag portion of the cDNA as the forward primer, a segment-specific oligonucleotide as the reverse primer, and a corresponding probe. Each primer pair was considered as an analytical group, and the Ct values were normalized to the average Ct value of the corresponding targeted cDNA to calculate the relative RNA levels within the group. Values below the dashed line indicate undetectable fluorescence or the absence of amplification curves.

Figure 2

TaqMan qRT-PCR assay for the quantification of the viral RNAs of PB2 and NA genes of H7N9 AIV in mice. (a) Standard curves for the three types of RNAs of PB2 and NA genes, generated by plotting the Ct values against the copy numbers of the input transcribed RNA. Tenfold serial dilutions of the cDNA derived from the transcribed viral RNAs were used to generate standard curves. The standard curve parameters and amplification efficiency are shown in the lower left corner. (b) Synthesis kinetics of vRNA, cRNA, and mRNA of the PB2 and NA genes in the lungs of H7N9 AIV-infected mice. RNA copy numbers were quantified by TaqMan qRT-PCR using the standard curves of the cDNA derived from the transcribed viral RNA. Values represent viral RNA copies in 2 μg of the total RNA of the mouse lungs. Error bars indicate the standard deviation of three biological replicates.