Preparation of a new monoclonal antibody against nucleocapsid protein of swine acute diarrhea syndrome coronavirus and identification of its linear antigenic epitope.

Swine acute diarrhea syndrome coronavirus (SADS-CoV), which causes severe diarrhea in newborn piglets, was first identified in Southern China in 2017. Since the Nucleocapsid (N) protein in SADS-CoV is highly conserved and plays a key role in virus replication, it is often used as a target protein in scientific research. In this study, the N protein of SADS-CoV was successfully expressed, and a new monoclonal antibody (mAb), 5G12, against the protein was generated successfully. The mAb 5G12 can be used to detect SADS-CoV strains by indirect immunofluorescence assay (IFA) and western blotting. The mAb 5G12 epitope was located to amino acids 11 EQAESRGRK 19 by evaluating the antibody for reactivity with a series of truncated N protein segments. The biological information analysis showed that the antigenic epitope had a high antigenic index and conservation. This study will help further understand the protein structure and function of SADS-CoV and in the establishment of specific SADS-CoV detection methods.

Single-dose rAAV5-based vaccine provides long-term protective immunity against SARS-CoV-2 and its variants.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus and its variants, has posed unprecedented challenges worldwide. Existing vaccines have limited effectiveness against SARS-CoV-2 variants. Therefore, novel vaccines to match mutated viral lineages by providing long-term protective immunity are urgently needed. We designed a recombinant adeno-associated virus 5 (rAAV5)-based vaccine (rAAV-COVID-19) by using the SARS-CoV-2 spike protein receptor binding domain (RBD-plus) sequence with both single-stranded (ssAAV5) and self-complementary (scAAV5) delivery vectors and found that it provides excellent protection from SARS-CoV-2 infection. A single-dose vaccination in mice induced a robust immune response; induced neutralizing antibody (NA) titers were maintained at a peak level of over 1:1024 more than a year post-injection and were accompanied by functional T-cell responses. Importantly, both ssAAV- and scAAV-based RBD-plus vaccines produced high levels of serum NAs against the circulating SARS-CoV-2 variants, including Alpha, Beta, Gamma and Delta. A SARS-CoV-2 virus challenge showed that the ssAAV5-RBD-plus vaccine protected both young and old mice from SARS-CoV-2 infection in the upper and lower respiratory tracts. Whole genome sequencing demonstrated that AAV vector DNA sequences were not found in the genomes of vaccinated mice one year after vaccination, demonstrating vaccine safety. These results suggest that the rAAV5-based vaccine is safe and effective against SARS-CoV-2 and several variants as it provides long-term protective immunity. This novel vaccine has a significant potential for development into a human prophylactic vaccination to help end the global pandemic.

Establishment of a recombinase polymerase amplification (RPA) fluorescence assay for the detection of swine acute diarrhea syndrome coronavirus (SADS-CoV).

BACKGROUND: Swine acute diarrhea syndrome coronavirus (SADS-CoV) causes acute vomiting and diarrhea in piglets, leading to significant financial losses for the pig industry. Recombinase polymerase amplification (RPA) is a rapid nucleic acid amplification technology used under constant temperature conditions. The study established a real-time reverse transcription (RT)-RPA assay for early diagnosis of SADS-CoV.  RESULTS: The detection limit of the real-time RT-RPA was 74 copies/µL of SADS-CoV genomic standard recombinant plasmid in 95% of cases. The assay was performed in less than 30 min and no cross-reactions were observed with eight other common viruses that affect swine, including classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudo rabies virus (PRV), swine influenza virus (SIV), seneca valley virus (SVA), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV). The coefficient of variation (C.V.) values of the two standards dilutions and three positive clinical sample ranged from 2.95% to 4.71%. A total of 72 clinical fecal samples from swine with diarrheal symptoms were analyzed with the developed RT-RPA and quantitative RT-PCR. There was 98.61% agreement between the RT-RPA and the quantitative real-time PCR results. CONCLUSIONS: These results indicated that the developed RT-RPA assay had good specificity, sensitivity, stability and repeatability. The study successfully established a broadly reactive RT-RPA assay for SADS-CoV detection.

Kinetics of IgM and IgG antibody specific to SARS-CoV-2 nucleocapsid and spike proteins

：Objective To study the kinetics of IgM and IgG antibodies based on nucleocapsid（N） and spike（S） protein of SARS-Co V2-in COVID-19 patients. Methods Immunofluorescent kits were used to detect N and S protein specific IgM and IgG antibodies from Jan.21 to Feb.11, 2020 for the 60 hospitalized COVID-19 patients（48 mild, 12 severe cases） with a total of 290 plasma samples collected 9 weeks after the onset of the disease. Results The level of antibodies specific for S protein varied significantly with the course of disease（Ig M from 27.32 to 110.10 TU/ml, IgG from 56.85 to 135.00 TU/ml）, but not for N protein.Higher level of Ig M/Ig G antibodies specific to S protein was observed during the 2-7 week than that to N protein.The seropositive rate of antibodies gradually increased during the early stage of disease.IgM/IgG antibodies specific to N protein changed from 12.50% at the first week to peak level（51.72% and 86.21% respectively） at the 4 th week and those for S protein from 25.00% and 14.58% to 100.00%, and then declined.The seropositive rate of Ig M antibody specific to S protein was higher than that for N protein during 2-8 th week and that for Ig G antibody at 2, 3, 4, 6 and 7 th week.The seropositive rate of Ig G antibody specific to N protein in severe patients at the third week was higher than that in mild patients（100.00% vs 59.52%,χ2=9.67, P=0.001 9）, and the same as to Ig G antibody for S protein at the second week after disease onset（80.00% vs 46.58%, χ2=5.57, P=0.018 2）. Conclusions SARS-Co V2-S protein can induc stronger antibody response than N protein, and the antibody level was related to the severity of the disease.