Generation of APN-chimeric gene-edited pigs by CRISPR/Cas9-mediated knock-in strategy

The prevalence of porcine enteric coronaviruses (PECs), including transmissible gastroenteritis virus (TGEV), swine acute diarrhea syndrome coronavirus (SADS-CoV), porcine delta coronavirus (PDCoV), and porcine epidemic diarrhea virus (PEDV), poses a serious threat to animal and public health. Here, we aimed to further optimize the porcine aminopeptidase N (pAPN) gene editing strategy to explore the balance between individual antiviral properties and the biological functions of pAPN in pigs. Finally, APN-chimeric gene-edited pigs were produced through a CRISPR/Cas9-mediated knock-in strategy. Further reproductive tests indicated that these gene-edited pigs exhibited normal pregnancy rates and viability. Notably, in vitro viral challenge assays further demonstrated that porcine kidney epithelial cells isolated from F1-generation gene-edited pigs could effectively inhibit TGEV infection. This study is the first to report the generation of APN-chimeric pigs, which may provide a natural host animal for characterizing PEC infection with APN and help in the development of better antiviral solutions.

COVID-19 diagnosis and study of serum SARS-CoV-2 specific IgA, IgM and IgG by a quantitative and sensitive immunoassay (preprint)

Background. The pandemic of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is causing great loss. Detecting viral RNAs is standard approach for SARS-CoV-2 diagnosis with variable success. Currently, studies describing the serological diagnostic methods are emerging, while most of them just involve the detection of SARS-CoV-2-specific IgM and IgG by ELISA or flow immunoassay with limited accuracy. Methods. Diagnostic approach depends on chemiluminescence immunoanalysis (CLIA) for detecting IgA, IgM and IgG specific to SARS-CoV-2 nucleocapsid protein (NP) and receptor-binding domain (RBD) was developed. The approach was tested with 216 sera from 87 COVID-19 patients and 483 sera from SARS-CoV-2 negative individuals. The diagnostic accuracy was evaluated by receiver operating characteristic (ROC) analysis. Concentration kinetics of RBD-specific serum antibodies were characterized. The relationship of serum RBD-specific antibodies and disease severity was analyzed. Results. The diagnostic accuracy based on RBD outperformed those based on NP. Adding IgA to a conventional serological test containing IgM and IgG improves sensitivity of SARS-CoV-2 diagnosis at early stage. CLIA for detecting RBD-specific IgA, IgM and IgG showed diagnostic sensitivities of 98.6%, 96.8% and 96.8%, and specificities of 98.1%, 92.3% and 99.8%, respectively. Median concentration of IgA and IgM peaked during 16-20 days after illness onset at 8.84 g/mL and 7.25 g/mL, respectively, while IgG peaked during 21-25 days after illness onset at 16.47 g/mL. Furthermore, the serum IgA level positively correlates with COVID-19 severity. Conclusion. CLIA for detecting SARS-CoV-2 RBD-specific IgA, IgM and IgG in blood provides additional values for diagnosing and monitoring of COVID-19.