Identification of conserved linear epitopes in the SARS-CoV-2 receptor-binding region using monoclonal antibodies.

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused millions of cases of infections, leading to a global health emergency. The SARS-CoV-2 spike (S) protein plays the most important role in viral infection, and S1 subunit and its receptor-binding domain (RBD) are widely considered the most attractive vaccine targets. The RBD is highly immunogenic and its linear epitopes are important for vaccine development and therapy, but linear epitopes on the RBD have rarely been reported. In this study, 151 mouse monoclonal antibodies (mAbs) against the SARS-CoV-2 S1 protein were characterized and used to identify epitopes. Fifty-one mAbs reacted with eukaryotic SARS-CoV-2 RBD. Sixty-nine mAbs reacted with the S proteins of Omicron variants B.1.1.529 and BA.5, indicating their potential as rapid diagnostic materials. Three novel linear epitopes of RBD, R6 (391CFTNVYADSFVIRGD405), R12 (463PFERDISTEIYQAGS477), and R16 (510VVVLSFELLHAPAT523), were identified; these were highly conserved in SARS-CoV-2 variants of concern and could be detected in the convalescent serum of COVID-19 patients. From pseudovirus neutralization assays, some mAbs including one detecting R12 were found to possess neutralizing activity. Together, from the reaction of mAbs with eukaryotic RBD (N501Y), RBD (E484K), and S1 (D614G), we found that a single amino acid mutation in the SARS-CoV-2 S protein may cause a structural alteration, exerting substantial impact on mAb recognition. Our results could, therefore, help us better understand the function of the SARS-CoV-2 S protein and develop diagnostic tools for COVID-19.

Characterization and application of a series of monoclonal antibodies against SARS-CoV-2 nucleocapsid protein.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has a significant global social and economic impact, and the emergence of new and more destructive mutant strains highlights the need for accurate virus detection. Here, 90 monoclonal antibodies (MAbs) that exclusively reacted with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP) were generated. These MAbs did not cross-react with NPs of common human coronaviruses (HCoVs, i.e., 229E, OC43, HKU1, and NL63) and Middle East Respiratory Syndrome Coronavirus. Subsequently, overlapped peptides in individual fragments (N1-N4) of NP were synthesized. N1-3 (25-GSNQNGERSGARSKQ-39), N3-1 (217-AALALLLLDRLNQL-230), and N4-8 (393-TLLPAADLDDFSKQL-407) were identified as major epitopes using enzyme-linked immunoassay (ELISA) and recognized by 47, 1, and 18 MAbs, respectively. The 24 remaining MAbs exhibited no reactivity with all synthetic peptides. Among MAb-epitope pairs, only MAbs targeting epitope N1-3 displayed no cross-reaction with NPs of SARS-CoV-1 and other SARS-related CoVs. All Omicron variants contained a three-amino acid deletion (31ERS33) in the N1-3 region. Thus, MAbs targeting N1-3 failed to recognize these variants. Furthermore, a double-antibody sandwich ELISA for antigen detection was established using the optimal MAbs. Overall, a series of MAbs targeting SARS-CoV-2 NP was prepared, characterized with epitope mapping, and applied for the detection of SARS-CoV-2 antigens, and some novel B-cell epitopes of the viral NP were identified.

Improving the efficiency and biosafety of respiratory syncytial virus identification using a nucleic acid extraction-free reagent.

Respiratory syncytial virus (RSV) is the most important virus that causes lower respiratory tract disease in children; efficient viral identification is an important component of disease prevention and treatment. Here, we developed and evaluated a ready-to-use (RTU) nucleic acid extraction-free direct reagent for identification of RSV (RTU-Direct test) in clinical samples. The limit of detection (LOD) of the RSV RTU-Direct test was consistent with the LOD of the standard test using extracted nucleic acids. The virus inactivation ability of RTU-Direct reagent was confirmed by viral infectivity assays involving RTU-Direct-treated samples containing RSV and human coronavirus OC43. RSV RNA stability was significantly better in RTU-Direct reagent than in conventional virus transport medium (VTM) at room temperature and 4°C (p < 0.05). The clinical performance of the RTU-Direct test was evaluated using 155 respiratory specimens from patients with suspected RSV infection. Positive agreement between the RTU-Direct test and the VTM standard test was 100% (42/42); negative agreement was 99.1% (112/113), and the kappa statistic was 0.968 (p < 0.001). The distributions of Ct values did not significantly differ between the RTU-Direct test and the standard test (p > 0.05). Overall, the RTU-Direct reagent can improve the efficiency and biosafety of RSV detection, while reducing the cost of detection.

Human adenovirus (HAdV) infection in children with acute respiratory tract infections in Guangzhou, China, 2010-2021: a molecular epidemiology study.

BACKGROUND: Human adenovirus (HAdV) infection can cause a variety of diseases. It is a major pathogen of pediatric acute respiratory tract infections (ARIs) and can be life-threatening in younger children. We described the epidemiology and subtypes shifting of HAdV among children with ARI in Guangzhou, China. METHODS: We conducted a retrospective study of 161,079 children diagnosed with acute respiratory illness at the Guangzhou Women and Children's Medical Center between 2010 and 2021. HAdV specimens were detected by real-time PCR and the hexon gene was used for phylogenetic analysis. RESULTS: Before the COVID-19 outbreak in Guangzhou, the annual frequency of adenovirus infection detected during this period ranged from 3.92% to 13.58%, with an epidemic peak every four to five years. HAdV demonstrated a clear seasonal distribution, with the lowest positivity in March and peaking during summer (July or August) every year. A significant increase in HAdV cases was recorded for 2018 and 2019, which coincided with a shift in the dominant HAdV subtype from HAdV-3 to HAdV-7. The latter was associated with a more severe disease compared to HAdV-3. The average mortality proportion for children infected with HAdV from 2016 to 2019 was 0.38% but increased to 20% in severe cases. After COVID-19 emerged, HAdV cases dropped to 2.68%, suggesting that non-pharmaceutical interventions probably reduced the transmission of HAdV in the community. CONCLUSION: Our study provides the foundation for the understanding of the epidemiology of HAdV and its associated risks in children in Southern China.

Establishment and evaluation of a 30-minute detection method for SARS-CoV-2 nucleic acid using a novel ultra-fast real-time PCR instrument.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic is still raging worldwide. Efficient, fast and low-cost severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid detection methods are urgently needed. METHODS: A rapid PCR temperature change mode was explored by moving the reaction tube between the independent temperature modules with large temperature differences and a portable ultra-fast real-time PCR instrument were developed. We established a rapid SARS-CoV-2 test method using the ultra-fast real-time PCR instrument, a China Food and Drug Administration-certified SARS-CoV-2 reagent and optimized reaction condition. The analytical and clinical performances of the rapid tests were evaluated by comparing with the standard SARS-CoV-2 tests. RESULTS: The new temperature change mode can effectively shorten the amplification reaction time and be successfully used in the development of the ultra-fast real-time PCR instrument. The rapid SARS-CoV-2 test method was established and the time to yield results were greatly shortened from 81 min of the standard test to 31 min. Specificity of the rapid test was assessed and no non-specific amplification (0/63) was observed. The limits of detection of the rapid and standard tests were similar. Clinical performance was evaluated using 184 respiratory specimens from patients with suspected SARS-CoV-2 infection. The positive agreement between the rapid and standard tests was 100% (67/67), the negative agreement was 97.4% (114/117), and the kappa statistic was 0.965 (P<0.001). No significant differences in the Ct values for each target gene were observed between the rapid test and the standard test (P>0.05). CONCLUSIONS: We had developed a 30-minute detection method for SARS-CoV-2 nucleic acid using a novel ultra-fast real-time PCR instrument. The rapid test method may impact on patient management.

Epitope mapping of severe acute respiratory syndrome-related coronavirus nucleocapsid protein with a rabbit monoclonal antibody.

The emergency SARS-CoV-2, a member of severe acute respiratory syndrome-related coronaviruses (SARSr-CoV), is still greatly harming the health of mankind. SARS-CoV-2-specific monoclonal antibodies (MAbs), which can identify SARS-CoV-2 from common human coronaviruses, are considered to extensively apply to developing rapid and reliable antigen assays. In this study we generated a rabbit MAb (RAb) detecting SARS-CoV-2 nucleocapsid protein (NP), which has cross-reaction with SARS-CoV-1 NP, but not with NPs of MERS and common human CoVs (OC43, NL63, 229E, and HKU1). With truncated NP fragments and synthesized peptides, the linear epitope detected by RAb was mapped in peptide N4-8, 393-407 amino acid residue (TLLPAADLDDFSKQL) of SARS-CoV-2 NP. This epitope N4-8 was highly conserved in SARSr-CoVs, including SARS-CoV-2, SARS-CoV-1, and bat CoV RaTG13 strain. However, the corresponding peptide of bat SARSr-CoV BtKY72 strain could not be recognized by RAb, which indicates amino acid D399 may be critical for N4-8 epitope detected by RAb. The present study will be conducive to developing reliable diagnosis for SARS-CoV-2 and gaining insights into the function of the SARS-CoV-2 N protein.