Molecular Virology of SARS-CoV-2 and Related Coronaviruses.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The global COVID-19 pandemic continues to threaten the lives of hundreds of millions of people, with a severe negative impact on the global economy. Although several COVID-19 vaccines are currently being administered, none of them is 100% effective. Moreover, SARS-CoV-2 variants remain an important worldwide public health issue. Hence, the accelerated development of efficacious antiviral agents is urgently needed. Coronavirus depends on various host cell factors for replication. An ongoing research objective is the identification of host factors that could be exploited as targets for drugs and compounds effective against SARS-CoV-2. In the present review, we discuss the molecular mechanisms of SARS-CoV-2 and related coronaviruses, focusing on the host factors or pathways involved in SARS-CoV-2 replication that have been identified by genome-wide CRISPR screening.

Quantifying Neutralizing Antibodies in Patients with COVID-19 by a Two-Variable Generalized Additive Model.

Considering the urgent demand for faster methods to quantify neutralizing antibody titers in patients with coronavirus (CoV) disease 2019 (COVID-19), developing an analytical model or method to replace the conventional virus neutralization test (NT) is essential. Moreover, a "COVID-19 immunity passport" is currently being proposed as a certification for people who travel internationally. Therefore, an enzyme-linked immunosorbent assay (ELISA) was designed to detect severe acute respiratory syndrome CoV 2 (SARS-CoV-2)-neutralizing antibodies in serum, which is based on the binding affinity of SARS-CoV-2 viral spike protein 1 (S1) and the viral spike protein receptor-binding domain (RBD) to antibodies. The RBD is considered the major binding region of neutralizing antibodies. Furthermore, S1 covers the RBD and several other regions, which are also important for neutralizing antibody binding. In this study, we assessed 144 clinical specimens, including those from patients with PCR-confirmed SARS-CoV-2 infections and healthy donors, using both the NT and ELISA. The ELISA results analyzed by spline regression and the two-variable generalized additive model precisely reflected the NT value, and the correlation between predicted and actual NT values was as high as 0.917. Therefore, our method serves as a surrogate to quantify neutralizing antibody titer. The analytic method and platform used in this study present a new perspective for serological testing of SARS-CoV-2 infection and have clinical potential to assess vaccine efficacy. IMPORTANCE Herein, we present a new approach for serological testing for SARS-CoV-2 antibodies using innovative laboratory methods that demonstrate a combination of biology and mathematics. The traditional virus neutralization test is the gold standard method; however, it is time-consuming and poses a risk to medical personnel. Thus, there is a demand for methods that rapidly quantify neutralizing antibody titers in patients with COVID-19 or examine vaccine efficacy at a biosafety level 2 containment facility. Therefore, we used a two-variable generalized additive model to analyze the results of the enzyme-linked immunosorbent assay and found the method to serve as a surrogate to quantify neutralizing antibody titers. This methodology has potential for clinical use in assessing vaccine efficacy.

Acyl-Coenzyme A Synthetase Long-Chain Family Member 4 Is Involved in Viral Replication Organelle Formation and Facilitates Virus Replication via Ferroptosis.

Enterovirus infections can cause severe complications, such as poliomyelitis, encephalitis, myocarditis, meningitis, neurological pulmonary edema, and even death. Here, we used genome-wide CRISPR screens to gain new insight into the mechanism by which enteroviruses co-opt host pathways to potentiate replication and propagation. We found that acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) is involved in viral replication organelle formation. ACSL4 is a key component of ferroptosis, an iron-dependent, nonapoptotic programmed cell death. Our results indicated that enteroviruses and coronaviruses can induce ferroptosis via ACSL4. Most importantly, ferroptosis inhibitors, including two FDA-approved drugs, rosiglitazone (ROSI; ACSL4 inhibitor) and pioglitazone (PIO; ACSL4 inhibitor), decreased the viral load of human enteroviruses and coronaviruses, suggesting that ACSL4 is a target for counteracting viral infection. IMPORTANCE We provide the first evidence for the role of ACSL4 in enterovirus replication organelle formation. Moreover, both enteroviruses and coronaviruses induce ferroptosis via ACSL4. These findings establish a novel regulatory mechanism for viral replication. The inhibition of ACSL4 by ferroptosis inhibitors can reduce viral yields of enteroviruses and coronaviruses, including SARS-CoV-2, implying that ACSL4-mediated ferroptosis is a promising therapeutic target for viral diseases.

Stability of SARS-CoV-2 Spike G614 Variant Surpasses That of the D614 Variant after Cold Storage.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) carrying the D614G mutation on the spike protein is the predominant circulating variant and is associated with enhanced infectivity. However, whether this dominant variant can potentially spread through the cold chain and whether the spike protein affects virus stability after cold storage remain unclear. To compare the infectivity of two SARS-CoV-2 variants, namely, SARS-CoV-2 variants with spike protein with the D614 mutation (S-D614) and G614 mutation (S-G614), after different periods of refrigeration (4°C) and freezing (-20°C). We also determined the integrity of the viral RNA and the ability of the spike protein to bind angiotensin-converting enzyme 2 (ACE2) after storage at these conditions. The results showed that SARS-CoV-2 was more stable and infectious after storage at -20°C than at 4°C. Particularly, the S-G614 variant was found to be more stable than the S-D614 variant. The spike protein of the S-G614 variant had better binding ability with the ACE2 receptor than that of the S-D614 variant after storage at -20°C for up to 30 days. Our findings revealed that SARS-CoV-2 remains stable and infectious after refrigeration or freezing, and their stability and infectivity up to 30 days depends on the spike variant. Stability and infectivity are related to each other, and the higher stability of S-G614 compared to that of S-D614 may contribute to rapid viral spread of the S-G614 variant.IMPORTANCE It has been observed that variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are more stable and infectious after storage at -20°C than at 4°C. A SARS-CoV-2 S-D614G variant is currently the most dominant variant in circulation and is associated with enhanced infectivity. We compared the stability of two SARS-CoV-2 variants: the early S-D614 variant carrying the D614 spike protein and the new S-G614 variant carrying the G614 spike protein, stored at both 4°C and -20°C for different periods. We observed that SARS-CoV-2 remains stable and infectious after refrigeration or freezing, which further depends on the spike variant, that is, the ability of the spike protein to bind with the ACE2 receptor with higher efficiency. The high stability of the S-G614 variant also explains its rapid spread and infectivity. Therefore, precautions should be taken during and after handling food preserved under cold conditions.