A Rapid Method for Generating Infectious SARS-CoV-2 and Variants Using Mutagenesis and Circular Polymerase Extension Cloning.

The appearance of SARS-CoV-2 variants in late 2020 raised alarming global public health concerns. Despite continued scientific progress, the genetic profiles of these variants bring changes in viral properties that threaten vaccine efficacy. Thus, it is critically important to investigate the biologic profiles and significance of these evolving variants. In this study, we demonstrate the application of circular polymerase extension cloning (CPEC) to the generation of full-length clones of SARS-CoV-2. We report that, combined with a specific primer design scheme, this yields a simpler, uncomplicated, and versatile approach for engineering SARS-CoV-2 variants with high viral recovery efficiency. This new strategy for genomic engineering of SARS-CoV-2 variants was implemented and evaluated for its efficiency in generating point mutations (K417N, L452R, E484K, N501Y, D614G, P681H, P681R, Δ69-70, Δ157-158, E484K+N501Y, and Ins-38F) and multiple mutations (N501Y/D614G and E484K/N501Y/D614G), as well as a large truncation (ΔORF7A) and insertion (GFP). The application of CPEC to mutagenesis also allows the inclusion of a confirmatory step prior to assembly and transfection. This method could be of value in the molecular characterization of emerging SARS-CoV-2 variants as well as the development and testing of vaccines, therapeutic antibodies, and antivirals. IMPORTANCE Since the first emergence of the SARS-CoV-2 variant in late 2020, novel variants have been continuously introduced to the human population, causing severe public health threats. In general, because these variants acquire new genetic mutation/s, it is critical to analyze the biological function of viruses that such mutations can confer. Therefore, we devised a method that can construct SARS-CoV-2 infectious clones and their variants rapidly and efficiently. The method was developed based on a PCR-based circular polymerase extension cloning (CPEC) combined with a specific primer design scheme. The efficiency of the newly designed method was evaluated by generating SARS-CoV-2 variants with single point mutations, multiple point mutations, and a large truncation and insertion. This method could be of value for the molecular characterization of emerging SARS-CoV-2 variants and the development and testing of vaccines and antiviral agents.

Development of a Universal Cloning System for Reverse Genetics of Human Enteroviruses.

Enteroviruses (EVs) have been associated with several human diseases. Due to their continuous emergence and divergence, EV species have generated more than 100 types and recombinant strains, increasing the public health threat caused by them. Hence, an efficient and universal cloning system for reverse genetics (RG) of highly divergent viruses is needed to understand the molecular mechanisms of viral pathology and evolution. In this study, we generated a versatile human EV whole-genome cDNA template by enhancing the template-switching method and designing universal primers capable of simultaneous cloning and rapid amplification of cDNA ends (RACE)-PCR of EVs. Moreover, by devising strategies to overcome limitations of previous cloning methods, we simplified significant cloning steps to be completed within a day. Of note, we successfully verified our efficient universal cloning system enabling RG of a broad range of human EVs, including EV-A (EV-A71), EV-B (CV-B5, ECHO6, and ECHO30), EV-C (CV-A24), and EV-D (EV-D68), with viral titers and phenotypes comparable to those of their wild types. This rapid and straightforward universal EV cloning strategy will help us elucidate molecular characteristics, pathogenesis, and applications of a broad range of EV serotypes for further development of genetic vaccines and delivery tools using various replication systems. IMPORTANCE Due to the broad spread, incidence, and genetic divergence of enteroviruses (EVs), it has been challenging to deal with this virus that causes severe human diseases, including aseptic meningitis, myocarditis, encephalitis, and poliomyelitis. Therefore, an efficient and universal cloning system for the reverse genetics of highly divergent EVs contributes to an understanding of the viral pathology and molecular mechanisms of evolution. We have simplified the important cloning steps, hereby enhancing the template-switching method and designing universal primers, which enable the important cloning steps to be completed in a day. We have also successfully demonstrated recovery of a broad range of human EVs, including EV-A to -D types, using this advanced universal cloning system. This rapid and robust universal EV cloning strategy will aid in elucidating the molecular characteristics, pathogenesis, and applications of a wide range of EVs for further development of genetic vaccines and antiviral screening using various replication systems.

Combination therapy with nirmatrelvir and molnupiravir improves the survival of SARS-CoV-2 infected mice.

As the SARS-CoV-2 pandemic remains uncontrolled owing to the continuous emergence of variants of concern, there is an immediate need to implement the most effective antiviral treatment strategies, especially for risk groups. Here, we evaluated the therapeutic potency of nirmatrelvir, remdesivir and molnupiravir, and their combinations in SARS-CoV-2 infected K18-hACE2 transgenic mice. Systemic treatment of mice with each drug (20 mg/kg) resulted in slightly enhanced antiviral efficacy and yielded an increased life expectancy of only about 20-40% survival. However, combination therapy with nirmatrelvir (20 mg/kg) and molnupiravir (20 mg/kg) in lethally infected mice showed profound inhibition of SARS-CoV-2 replication in both the lung and brain and synergistically improved survival rates up to 80% compared to those with nirmatrelvir (36%, P < 0.001) and molnupiravir (43%, P < 0.001) administered alone. This combination therapy effectively reduced clinical severity score, virus-induced tissue damage, and viral distribution compared to those in animals treated with these monotherapies. Furthermore, all these assessments associated with this combination were also significantly higher than that of mice receiving remdesivir monotherapy (P < 0.001) and the nirmatrelvir (20 mg/kg) and remdesivir (20 mg/kg) combination (P < 0.001), underscored the clinical significance of this combination. By contrast, the nirmatrelvir and remdesivir combination showed less antiviral efficacy, with lower survival compared to nirmatrelvir monotherapy due to the insufficient plasma exposure of the remdesivir, demonstrating the inefficient therapeutic effect of this combination in the mouse model. The combination therapy with nirmatrelvir and molnupiravir contributes to alleviated morbidity and mortality, which can serve as a basis for the design of clinical studies of this combination in the treatment of COVID-19 patients.