Development of multiplex RT-ddPCR assays for detection of SARS-CoV-2 and other common respiratory virus infections.

BACKGROUND: Measures for mitigation of Coronavirus Disease 2019 (COVID-19) were set to reduce the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). SARS-CoV-2 and other respiratory viruses share similar transmission routes and some common clinical manifestations. Co-circulation of SARS-CoV-2 and other common respiratory viruses is imminent. Therefore, development of multiplex assays for detecting these respiratory viruses is essential for being prepared for future outbreaks of respiratory viruses. METHODS: A panel of three reverse transcription droplet digital PCR (RT-ddPCR) assays were developed to detect 15 different human respiratory viruses. Evaluations of its performance were demonstrated. A total of 100 local and 98 imported COVID-19 cases in Hong Kong were screened for co-infection with other common respiratory viruses. RESULTS: All detected viral targets showed distinct signal clusters using the multiplex RT-ddPCR assays. These assays have a broad range of linearity and good intra-/inter-assay reproducibility for each target. The lower limits of quantification for all targets were ≤46 copies per reaction. Six imported cases of COVID-19 were found to be co-infected with other respiratory viruses, whereas no local case of co-infection was observed. CONCLUSIONS: The multiplex RT-ddPCR assays were demonstrated to be useful for screening of respiratory virus co-infections. The strict preventive measures applied in Hong Kong may be effective in limiting the circulation of other human respiratory viruses. The multiplex assays developed in this study can achieve a robust detection method for clinical and research purposes.

A six-plex droplet digital RT-PCR assay for seasonal influenza virus typing, subtyping, and lineage determination.

BACKGROUND: There are two influenza A subtypes (H1 and H3) and two influenza B lineages (Victoria and Yamagata) that currently co-circulate in humans. In this study, we report the development of a six-plex droplet digital RT-PCR (ddRT-PCR) assay that can detect HA and M segments of influenza A (H1, H3, and M) and influenza B (Yamagata HA, Victoria HA, and M) viruses in a single reaction mixture. It can simultaneously detect six different nucleic acid targets in a ddRT-PCR platform. METHODS: The six-plex ddRT-PCR used in this study is an amplitude-based multiplex assay. The analytical performance of the assay was evaluated. Correlation with standard qRT-PCR methodology was assessed using 55 clinical samples. RESULTS: The assay has a wide dynamic range, and it has good reproducibility within and between runs. The limit of quantification of each target in this assay ranged from 15 copies/reaction for influenza B Victoria M gene to 45 copies/reaction for influenza B Yamagata M gene. In addition, this assay can accurately quantify each of these targets in samples containing viral RNAs from two different viruses that were mixed in a highly skewed ratio. Typing, subtyping, and lineage differentiation data of 55 tested clinical respiratory specimens were found to be identical to those deduced from standard monoplex qRT-PCR assays. CONCLUSIONS: The six-plex ddRT-PCR test was demonstrated to be highly suitable for detecting dual influenza infection cases. This assay is expected to be a useful diagnostic tool for clinical and research use.

Characterization of influenza A viruses with polymorphism in PB2 residues 701 and 702.

The 701 and 702 positions of influenza PB2 polymerase subunit are previously shown to have roles on host range. Limited polymorphisms at these two residues are identified in natural isolates, thereby limiting the study of their role in the polymerase. In this study, we generated 31 viable viruses by random mutagenesis at this region, indicating that these positions can tolerate a wide range of amino acids. These mutants demonstrated varying polymerase activities and viral replication rates in mammalian and avian cells. Notably, some mutants displayed enhanced polymerase activity, yet their replication kinetics were comparable to the wild-type virus. Surface electrostatic charge predication on the PB2 structural model revealed that the viral polymerase activity in mammalian cells generally increases as this region becomes more positively charged. One of the mutants (701A/702E) showed much reduced pathogenicity in mice while others had a pathogenicity similar to the wild-type level. Distinct tissue tropisms of the PB2-701/702 mutants were observed in infected chicken embryos. Overall, this study demonstrates that the PB2-701/702 region has a high degree of sequence plasticity and sequence changes in this region can alter virus phenotypes in vitro and in vivo.