Distinctive Combinations of RBD Mutations Contribute to Antibody Evasion in the Case of the SARS-CoV-2 Beta Variant.

Since its first report in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a grave threat to public health. Virus-specific countermeasures, such as vaccines and therapeutics, have been developed and have contributed to the control of the viral pandemic, which has become endemic. Nonetheless, new variants continue to emerge and could cause a new pandemic. Consequently, it is important to comprehensively understand viral evolution and the roles of mutations in viral infectivity and transmission. SARS-CoV-2 beta variant encode mutations (D614G, N501Y, E484K, and K417N) in the spike which are frequently found in other variants as well. While their individual role in viral infectivity has been elucidated against various therapeutic antibodies, it still remains unclear whether those mutations may act additively or synergistically when combined. Here, we report that N501Y mutation shows differential effect on two therapeutic antibodies tested. Interestingly, the relative importance of E484K and K417N mutations in antibody evasion varies depending on the antibody type. Collectively, these findings suggest that continuous efforts to develop effective antibody therapeutics and combinatorial treatment with multiple antibodies are more rational and effective forms of treatment.

Lupeol alleviates atopic dermatitis-like skin inflammation in 2,4-dinitrochlorobenzene/Dermatophagoides farinae extract-induced mice.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease that affects from children to adults widely, presenting symptoms such as pruritus, erythema, scaling, and dryness. Lupeol, a pentacyclic triterpenoid, has anti-inflammatory and antimicrobial activities. Based on these properties, the therapeutic effects of lupeol on skin disorders have been actively studied. In the present study, we aimed to determine the effectiveness of lupeol on AD. METHODS: We utilized tumor necrosis factor (TNF)-α/interferon (IFN)-γ-stimulated keratinocytes and 2, 4-dinitrochlorobenzene/Dermatophagoides farinae extract (DFE)-induced AD mice to confirm the action. RESULTS: Lupeol inhibited TNF-α/IFN-γ-stimulated keratinocytes activation by reducing the expressions of pro-inflammatory cytokines and chemokines which are mediated by the activation of signaling molecules such as signal transducer and activator of transcription 1, mitogen-activated protein kinases (p38 and ERK), and nuclear factor-κB. Oral administration of lupeol suppressed epidermal and dermal thickening and immune cell infiltration in ear tissue. Immunoglobulin (Ig) E (total and DFE-specific) and IgG2a levels in serum were also reduced by lupeol. The gene expression and protein secretion of T helper (Th) 2 cytokines, Th1 cytokines, and pro-inflammatory cytokine in ear tissue were decreased by lupeol. CONCLUSIONS: These results suggest that lupeol has inhibitory effects on AD-related responses. Therefore, lupeol could be a promising therapeutic agent for AD.

Chikungunya Virus nsP2 Impairs MDA5/RIG-I-Mediated Induction of NF-κB Promoter Activation: A Potential Target for Virus-Specific Therapeutics.

Chikungunya virus (CHIKV) was first identified in 1952 as a causative agent of outbreaks. CHIKV is transmitted by two mosquito species, Aedes aegypti and A. albopictus. Symptoms after CHIKV infection in human are typically fever and joint pain, but can also include headache, muscle pain, joint swelling, polyarthralgia, and rash. CHIKV is an enveloped single-stranded, positive-sense RNA virus with a diameter of approximately 70 nm. The pathogenesis of CHIKV infection and the mechanism by which the virus evades the innate immune system remain poorly understood. Moreover, little is known about the roles of CHIKV-encoded genes in the viral evasion of host immune responses, especially type I interferon (IFN) responses. Therefore, in the present study, we screened CHIKV-encoded genes for their regulatory effect on the activation of nuclear factor kappa B (NF-κB), a critical transcription factor for the optimal activation of IFN-ß. Among others, nonstructural protein 2 (nsP2) strongly inhibited melanoma differentiation-associated protein 5 (MDA5)-mediated induction of the NF-κB pathway in a dose-dependent manner. Elucidation of the detailed mechanisms of nsP2-mediated inhibition of the MDA5/RIG-I signaling pathway is anticipated to contribute to the development of virus-specific therapeutics against CHIKV infection.

Chikungunya Virus-Encoded nsP2, E2 and E1 Strongly Antagonize the Interferon-ß Signaling Pathway.

Chikungunya virus (CHIKV) is a single-stranded positive-sense RNA virus, belonging to the genus Alphavirus of the Togaviridae family. It causes multiple symptoms, including headache, fever, severe joint and muscle pain, and arthralgia. Since CHIKV was first isolated in Tanzania in 1952, there have been multiple outbreaks of chikungunya fever. However, its pathogenesis and mechanisms of viral immune evasion have been poorly understood. In addition, the exact roles of individual CHIKV genes on the host innate immune response remain largely unknown. To investigate if CHIKV-encoded genes modulate the type I interferon (IFN) response, each and every CHIKV gene was screened for its effects on the induction of the IFN-ß promoter. Here we report that CHIKV nsP2, E2 and E1 strongly suppressed activation of the IFN-ß promoter induced by the MDA5/RIG-I receptor signaling pathway, suggesting that nsP2, E2, and E1 are the major antagonists against induction of IFN-ß. Delineation of underlying mechanisms of CHIKV-mediated inhibition of the IFN-ß pathway may help develop virus-specific therapeutics and vaccines.

Middle East respiratory syndrome coronavirus-encoded ORF8b strongly antagonizes IFN-ß promoter activation: its implication for vaccine design.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a causative agent of severe-to-fatal pneumonia especially in patients with pre-existing conditions, such as smoking and chronic obstructive pulmonary disease (COPD). MERS-CoV transmission continues to be reported in the Saudi Arabian Peninsula since its discovery in 2012. However, it has rarely been epidemic outside the area except one large outbreak in South Korea in May 2015. The genome of the epidemic MERS-CoV isolated from a Korean patient revealed its homology to previously reported strains. MERS-CoV encodes 5 accessory proteins and generally, they do not participate in the genome transcription and replication but rather are involved in viral evasion of the host innate immune responses. Here we report that ORF8b, an accessory protein of MERS-CoV, strongly inhibits both MDA5- and RIG-I-mediated activation of interferon beta promoter activity while downstream signaling molecules were left largely unaffected. Of note, MDA5 protein levels were significantly down-regulated by ORF8b and co-expression of ORF4a and ORF4b. These novel findings will facilitate elucidation of mechanisms of virus-encoded evasion strategies, thus helping design rationale antiviral countermeasures against deadly MERS-CoV infection.

Middle East Respiratory Syndrome Coronavirus-Encoded Accessory Proteins Impair MDA5-and TBK1-Mediated Activation of NF-κB.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly emerging coronavirus which is zoonotic from bats and camels. Its infection in humans can be fatal especially in patients with preexisting conditions due to smoking and chronic obstructive pulmonary disease (COPD). Among the 25 proteins encoded by MERS-CoV, 5 accessory proteins seem to be involved in viral evasion of the host immune responses. Here we report that ORF4a, ORF4b, and ORF8b proteins, alone or in combination, effectively antagonize nuclear factor kappa B (NF-κB) activation. Interestingly, the inhibition of NF-κB by MERS-CoV accessory proteins was mostly at the level of pattern recognition receptors: melanoma differentiationassociated gene 5 (MDA5). ORF4a and ORF4b additively inhibit MDA5-mediated activation of NF-κB while that of retinoic acid-inducible gene 1 (RIG-I) is largely not perturbed. Of note, ORF8b was found to be a novel antagonist of MDA5-mediated NF-kκB activation. In addition, ORF8b also strongly inhibits Tank-binding kinase 1 (TBK1)-mediated induction of NF-κB signaling. Taken together, MERS-CoV accessory proteins are involved in viral escape of NF-κB-mediated antiviral immune responses.

Generation of Full-Length Infectious cDNA Clones of Middle East Respiratory Syndrome Coronavirus.

Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in Saudi Arabia in 2012 and related infection cases have been reported in over 20 countries. Roughly 10,000 human cases have so far been reported in total with fatality rates at up to 40%. The majority of cases have occurred in Saudi Arabia with mostly sporadic outbreaks outside the country except for the one in South Korea in 2015. The Korean MERS-CoV strain was isolated from the second Korean patient and its genome was fully sequenced and deposited. To develop virusspecific protective and therapeutic agents against the Korean isolate and to investigate molecular determinants of virus-host interactions, it is of paramount importance to generate its full-length cDNA. Here we report that two full-length cDNAs from a Korean patientisolated MERS-CoV strain were generated by a combination of conventional cloning techniques and efficient Gibson assembly reactions. The full-length cDNAs were validated by restriction analysis and their sequence was verified by Sanger method. The resulting cDNA was efficiently transcribed in vitro and the T7 promoter-driven expression was robust. The resulting reverse genetic system will add to the published list of MERS-CoV cDNAs and facilitate the development of Korean isolate-specific antiviral measures.

Flavisolibacter tropicus sp. nov., isolated from tropical soil.

A Gram-stain-negative, non-motile, deep yellow, rod-shaped bacterium, designated strain LCS9T, was isolated from a soil sample at the tropical zone within the Ecorium of the National Institute of Ecology in Seocheon, central-western Korea. 16S rRNA gene sequence analysis showed that strain LCS9T clustered with members of the genus Flavisolibacter of the family Chitinophagaceae, phylum Bacteroidetes. Sequence similarities between strain LCS9T and the type strains of the genus Flavisolibacter ranged from 94.6 to 94.9 %. Strain LCS9T grew at 10-37 °C (optimum, 25 °C) and at pH 6.0-10.0 (optimum, pH 7); was positive for catalase and oxidase; and negative for nitrate reduction and production of indole. Cells showed pigment absorbance peaks at 451 and 479 nm, and had 0.03 % survival following exposure to 3 kGy gamma radiation. Strain LCS9T had the following chemotaxonomic characteristics: the major quinone was menaquinone-7 (MK-7); the major fatty acids were iso-C15 : 0 and iso-C17 : 0 3-OH; polar lipids included phosphoatidylethanolamine, an unidentified aminophospholipid, unidentified aminolipidsand unidentified lipids. The DNA G+C content was 39.4 mol%. Based on polyphasic analysis, the type strain LCS9T (=KCTC 42070T=JCM 19972T) represents a novel species for which the name Flavisolibacter tropicus sp. nov. is proposed. Radiation resistance in the genus Flavisolibacter has not been reported to date, and so this is the first report of low-level radiation resistance of a member of the genus.