Construction and evaluation of a self-replicative RNA vaccine against SARS-CoV-2 using yellow fever virus replicon.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a global threat. To forestall the pandemic, developing safe and effective vaccines is necessary. Because of the rapid production and little effect on the host genome, mRNA vaccines are attractive, but they have a relatively low immune response after a single dose. Replicon RNA (repRNA) is a promising vaccine platform for safety and efficacy. RepRNA vaccine encodes not only antigen genes but also the genes necessary for RNA replication. Thus, repRNA is self-replicative and can play the role of an adjuvant by itself, which elicits robust immunity. This study constructed and evaluated a repRNA vaccine in which the gene encoding the spike (S) protein of SARS-CoV-2 was inserted into a replicon of yellow fever virus 17D strain. Upon electroporation of this repRNA into baby hamster kidney cells, the S protein and yellow fever virus protein were co-expressed. Additionally, the self-replication ability of repRNA vaccine was confirmed using qRT-PCR, demonstrating its potency as a vaccine. Immunization of C57BL/6 mice with 1 µg of the repRNA vaccine induced specific T-cell responses but not antibody responses. Notably, the T-cell response induced by the repRNA vaccine was significantly higher than that induced by the nonreplicative RNA vaccine in our experimental model. In the future, it is of the essence to optimize vaccine administration methods and improve S protein expression, like protection of repRNA by nanoparticles and evasion of innate immunity of the host to enhance the immune-inducing ability of the repRNA vaccine.

Inactivation of SARS-CoV-2 and influenza A virus by dry fogging hypochlorous acid solution and hydrogen peroxide solution.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is transmitted mainly by droplet or aerosol infection; however, it may also be transmitted by contact infection. SARS-CoV-2 that adheres to environmental surfaces remains infectious for several days. We herein attempted to inactivate SARS-CoV-2 and influenza A virus adhering to an environmental surface by dry fogging hypochlorous acid solution and hydrogen peroxide solution. SARS-CoV-2 and influenza virus were air-dried on plastic plates and placed into a test chamber for inactivation by the dry fogging of these disinfectants. The results obtained showed that the dry fogging of hypochlorous acid solution and hydrogen peroxide solution inactivated SARS-CoV-2 and influenza A virus in CT value (the product of the disinfectant concentration and contact time)-dependent manners. SARS-CoV-2 was more resistant to the virucidal effects of aerosolized hypochlorous acid solution and hydrogen peroxide solution than influenza A virus; therefore, higher concentrations of disinfectants or longer contact times were required to inactivate SARS-CoV-2 than influenza A virus. The present results provide important information for the development of a strategy that inactivates SARS-CoV-2 and influenza A virus on environmental surfaces by spatial fogging.

MARCH8 Targets Cytoplasmic Lysine Residues of Various Viral Envelope Glycoproteins.

The host transmembrane protein MARCH8 is a RING finger E3 ubiquitin ligase that downregulates various host transmembrane proteins, such as MHC-II. We have recently reported that MARCH8 expression in virus-producing cells impairs viral infectivity by reducing virion incorporation of not only HIV-1 envelope glycoprotein but also vesicular stomatitis virus G-glycoprotein through two different pathways. However, the MARCH8 inhibition spectrum remains largely unknown. Here, we show the antiviral spectrum of MARCH8 using viruses pseudotyped with a variety of viral envelope glycoproteins. Infection experiments revealed that viral envelope glycoproteins derived from the rhabdovirus, arenavirus, coronavirus, and togavirus (alphavirus) families were sensitive to MARCH8-mediated inhibition. Lysine mutations at the cytoplasmic tails of rabies virus-G, lymphocytic choriomeningitis virus glycoproteins, SARS-CoV and SARS-CoV-2 spike proteins, and Chikungunya virus and Ross River virus E2 proteins conferred resistance to MARCH8. Immunofluorescence showed impaired downregulation of the mutants of these viral envelope glycoproteins by MARCH8, followed by lysosomal degradation, suggesting that MARCH8-mediated ubiquitination leads to intracellular degradation of these envelopes. Indeed, rabies virus-G and Chikungunya virus E2 proteins proved to be clearly ubiquitinated. We conclude that MARCH8 has inhibitory activity on a variety of viral envelope glycoproteins whose cytoplasmic lysine residues are targeted by this antiviral factor. IMPORTANCE A member of the MARCH E3 ubiquitin ligase family, MARCH8, downregulates many different kinds of host transmembrane proteins, resulting in the regulation of cellular homeostasis. On the other hands, MARCH8 acts as an antiviral factor when it binds to and downregulates HIV-1 envelope glycoprotein and vesicular stomatitis virus G-glycoprotein that are viral transmembrane proteins. This study reveals that, as in the case of cellular membrane proteins, MARCH8 shows broad-spectrum inhibition against various viral envelope glycoproteins by recognizing their cytoplasmic lysine residues, resulting in lysosomal degradation.

A PCR amplicon-based SARS-CoV-2 replicon for antiviral evaluation.

The development of specific antiviral compounds to SARS-CoV-2 is an urgent task. One of the obstacles for the antiviral development is the requirement of biocontainment because infectious SARS-CoV-2 must be handled in a biosafety level-3 laboratory. Replicon, a non-infectious self-replicative viral RNA, could be a safe and effective tool for antiviral evaluation. Herein, we generated a PCR-based SARS-CoV-2 replicon. Eight fragments covering the entire SARS-CoV-2 genome except S, E, and M genes were amplified with HiBiT-tag sequence by PCR. The amplicons were ligated and in vitro transcribed to RNA. The cells electroporated with the replicon RNA showed more than 3000 times higher luminescence than MOCK control cells at 24 h post-electroporation, indicating robust translation and RNA replication of the replicon. The replication was drastically inhibited by remdesivir, an RNA polymerase inhibitor for SARS-CoV-2. The IC50 of remdesivir in this study was 0.29 µM, generally consistent to the IC50 obtained using infectious SARS-CoV-2 in a previous study (0.77 µM). Taken together, this system could be applied to the safe and effective antiviral evaluation without using infectious SARS-CoV-2. Because this is a PCR-based and transient replicon system, further improvement including the establishment of stable cell line must be achieved.

Characterization of JC Polyomavirus Derived from COS-IMRb Cells.

JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system affecting immunocompromised patients. The study of PML-type JCPyV in vitro has been limited owing to the inefficient propagation of the virus in cultured cells. In this study, we carried out long-term culture of COS-7 cells (designated as COS-IMRb cells) transfected with PML-type M1-IMRb, an adapted viral DNA with a rearranged non-coding control region (NCCR). The JCPyV derived from COS-IMRb cells were characterized by analyzing the viral replication, amount of virus by hemagglutination (HA), production of viral protein 1 (VP1), and structure of the NCCR. HA assays indicated the presence of high amounts of PML-type JCPyV in COS-IMRb cells. Immunostaining showed only a small population of JCPyV carrying COS-IMRb cells to be VP1-positive. Sequencing analysis of the NCCR of JCPyV after long-term culture revealed that the NCCR of M1-IMRb was conserved in COS-IMRb cells without any point mutation. The JCPyV genomic DNA derived from a clone of COS-IMRb-3 cells was detected, via Southern blotting, as a single band of approximately 5.1 kbp without deletion. These findings suggest the potential of using COS-IMRb-3 cells as a useful tool for screening anti-JCPyV drugs.