Multi-cohort study on cytokine and chemokine profiles in the progression of COVID-19.

Various substances in the blood plasma serve as prognostic indicators of the progression of COVID-19. Consequently, multi-omics studies, such as proteomic and metabolomics, are ongoing to identify accurate biomarkers. Cytokines and chemokines, which are crucial components of immune and inflammatory responses, play pivotal roles in the transition from mild to severe illness. To determine the relationship between plasma cytokines and the progression of COVID-19, we used four study cohorts to perform a systematic study of cytokine levels in patients with different disease stages. We observed differential cytokine expression between patients with persistent-mild disease and patients with mild-to-severe transformation. For instance, IL-4 and IL-17 levels significantly increased in patients with mild-to-severe transformation, indicating differences within the mild disease group. Subsequently, we analysed the changes in cytokine and chemokine expression in the plasma of patients undergoing two opposing processes: the transition from mild to severe illness and the transition from severe to mild illness. We identified several factors, such as reduced expression of IL-16 and IL-18 during the severe phase of the disease and up-regulated expression of IL-10, IP-10, and SCGF-ß during the same period, indicative of the deterioration or improvement of patients' conditions. These factors obtained from fine-tuned research cohorts could provide auxiliary indications for changes in the condition of COVID-19 patients.

PLX8394, a RAF inhibitor, inhibits enterovirus 71 replication by blocking RAF/MEK/ERK signaling.

Enterovirus 71 (EV71) poses a serious threat to human health, with scattered outbreaks worldwide. There are several vaccines against a few EV71 strains but no efficient drug for the treatment of EV71 infection. Therefore, it is urgent and of significance to develop anti-EV71 drugs. Here, we found that PLX8394, a RAF inhibitor, possesses high antiviral activity against EV71 in vitro, being superior to the traditional clinical drug ribavirin. Moreover, PLX8394 exhibits broad-spectrum antiviral activity against enteroviruses. Notably, in a suckling mouse model, PLX8394 provided a 70% protection rate for EV71-infected mice, reduced the viral load in liver and heart tissues, and relieved the inflammatory response. A mechanistic study showed that PLX8394 inhibited EV71 by suppressing the RAF/MEK/ERK signaling pathway. Thus, PLX8394 lays a foundation for the development of new drugs against EV71.

ML390 inhibits enterovirus 71 replication by targeting de novo pyrimidine biosynthesis pathway.

Enterovirus 71 (EV71), a small, single-stranded, positive-sense RNA virus belonging to the enterovirus genus in the family Picornaviridae, causes hand, foot, and mouth disease. Although EV71 seriously threatens to public health, no effective antiviral drugs are available for treating this disease. In this study, we found that ML390, a dihydroorotate dehydrogenase inhibitor, has potential anti-EV71 activity. ML390 dose-dependently inhibited EV71 replication with IC50 and selectivity index values of 0.06601 µM and 156.5, respectively. Supplementation with the downstream product orotate significantly suppressed the ability of ML390 to inhibit EV71 replication. Moreover, an adequate supply of exogenous uridine and cytosine suppressed the anti-EV71 activity of ML390. Thus, the antiviral activity of ML390 is mediated by the inhibition of the pyrimidine synthesis pathway. In an EV71-infected mouse model, ML390 reduced the load of EV71 in the brain, liver, heart, spleen, front legs, and hind legs, and significantly increased the survival rate of the mice infected by EV71. ML390 shows potential for the treatment of hand, foot, and mouth disease caused by EV71 infection.

Interactome profiling of Crimean-Congo hemorrhagic fever virus glycoproteins.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a biosafety level-4 pathogen requiring urgent research and development efforts. The glycoproteins of CCHFV, Gn and Gc, are considered to play multiple roles in the viral life cycle by interactions with host cells; however, these interactions remain largely unclear to date. Here, we analyzed the cellular interactomes of CCHFV glycoproteins and identified 45 host proteins as high-confidence Gn/Gc interactors. These host molecules are involved in multiple cellular biological processes potentially associated with the physiological actions of the viral glycoproteins. Then, we elucidated the role of a representative cellular protein, HAX1. HAX1 interacts with Gn by its C-terminus, while its N-terminal region leads to mitochondrial localization. By the strong interaction, HAX1 sequestrates Gn to mitochondria, thus depriving Gn of its normal Golgi localization that is required for functional glycoprotein-mediated progeny virion packaging. Consistently, the inhibitory activity of HAX1 against viral packaging and hence propagation was further elucidated in the contexts of pseudotyped and authentic CCHFV infections in cellular and animal models. Together, the findings provide a systematic CCHFV Gn/Gc-cell protein-protein interaction map, but also unravel a HAX1/mitochondrion-associated host antiviral mechanism, which may facilitate further studies on CCHFV biology and therapeutic approaches.

A simple method for the preparation of positive samples to preliminarily determine the quality of phosphorylation-specific antibody.

Protein phosphorylation is one of the most common and important post-translational modifications and is involved in many biological processes, including DNA damage repair, transcriptional regulation, signal transduction, and apoptosis regulation. The use of antibodies targeting phosphorylated protein is a convenient method to detect protein phosphorylation. Therefore, high-quality antibodies are essential, and uniform and effective standards are urgently needed to evaluate the quality of these phosphorylation-specific antibodies. In this study, we established a simple, broad-spectrum system for the preparation of phosphorylation-positive samples. The positive samples for evaluation of phosphorylation-specific antibodies were then validated in cells from different species and tissues, and also been proven effectively in western blot, enzyme-linked immunosorbent assays, LC-MS/MS and immunofluorescence analysis. Overall, our findings established a novel approach for evaluation of the quality of phosphorylation-specific antibodies and may have applications in various biomedical fields.

Recent Advances in Bunyavirus Reverse Genetics Research: Systems Development, Applications, and Future Perspectives.

Bunyaviruses are members of the Bunyavirales order, which is the largest group of RNA viruses, comprising 12 families, including a large group of emerging and re-emerging viruses. These viruses can infect a wide variety of species worldwide, such as arthropods, protozoans, plants, animals, and humans, and pose substantial threats to the public. In view of the fact that a better understanding of the life cycle of a highly pathogenic virus is often a precondition for developing vaccines and antivirals, it is urgent to develop powerful tools to unravel the molecular basis of the pathogenesis. However, biosafety level -3 or even -4 containment laboratory is considered as a necessary condition for working with a number of bunyaviruses, which has hampered various studies. Reverse genetics systems, including minigenome (MG), infectious virus-like particle (iVLP), and infectious full-length clone (IFLC) systems, are capable of recapitulating some or all steps of the viral replication cycle; among these, the MG and iVLP systems have been very convenient and effective tools, allowing researchers to manipulate the genome segments of pathogenic viruses at lower biocontainment to investigate the viral genome transcription, replication, virus entry, and budding. The IFLC system is generally developed based on the MG or iVLP systems, which have facilitated the generation of recombinant infectious viruses. The MG, iVLP, and IFLC systems have been successfully developed for some important bunyaviruses and have been widely employed as powerful tools to investigate the viral replication cycle, virus-host interactions, virus pathogenesis, and virus evolutionary process. The majority of bunyaviruses is generally enveloped negative-strand RNA viruses with two to six genome segments, of which the viruses with bipartite and tripartite genome segments have mostly been characterized. This review aimed to summarize current knowledge on reverse genetic studies of representative bunyaviruses causing severe diseases in humans and animals, which will contribute to the better understanding of the bunyavirus replication cycle and provide some hints for developing designed antivirals.

Non-structural Proteins of Severe Fever With Thrombocytopenia Syndrome Virus Suppress RNA Synthesis in a Transcriptionally Active cDNA-Derived Viral RNA Synthesis System.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by the tick-borne SFTS bunyavirus (SFTSV) resulting in a high fatality rate up to 30%. SFTSV is a negative-strand RNA virus containing three single-stranded RNA genome segments designated as L, M, and S, which respectively, encode the RNA-dependent RNA polymerase (RdRp), glycoproteins Gn and Gc, and nucleoprotein (N) and non-structural proteins (NSs). NSs can form inclusion bodies (IBs) in infected and transfected cells. A previous study has provided a clue that SFTSV NSs may be involved in virus-like or viral RNA synthesis; however, the details remain unclear. Our work described here reveals that SFTSV NSs can downregulate virus-like RNA synthesis in a dose-dependent manner within a cDNA-derived viral RNA synthesis system, i.e., minigenome (-) and minigenome (+) systems based on transfection, superinfection, and luciferase reporter activity determination; meanwhile, NSs also show a weak inhibitory effect on virus replication. By using co-immunoprecipitation (Co-IP) and RT-PCR combined with site-directed mutagenesis, we found that NSs suppress virus-like RNA or virus replication through interacting with N but not with RdRp, and the negative regulatory effect correlates closely with the IB structure it formed but is not associated with its role of antagonizing host innate immune responses. When the cytoplasmic structure of IB formed by SFTSV NSs was deprived, the inhibitory effect of NSs on virus-like RNA synthesis would weaken and even disappear. Similarly, we also evaluated other bandavirus NSs that cannot form IB in neither infected nor transfected cells, and the results showed that the NSs of Heartland bandavirus (HRTV) did not show a significant inhibitory effect on virus-like RNA synthesis within a minigenome system. Our findings provide experimental evidence that SFTSV NSs participate in regulating virus-like or viral RNA synthesis and the negative effect may be due to the NSs-N interaction.

Combinatorial Minigenome Systems for Emerging Banyangviruses Reveal Viral Reassortment Potential and Importance of a Protruding Nucleotide in Genome "Panhandle" for Promoter Activity and Reassortment.

Banyangvirus is a new genus (Phenuiviridae family, Bunyavirales order) that comprises a group of emerging tick-borne viruses with severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV) as virulent representatives. As segmented RNA viruses, bunyaviruses may have genome reassortment potential, increasing the concern about new life-threatening bunyavirus emergence. Using a series of combinatory minigenome reporter assays based on transfection and superinfection, we showed that replication machinery proteins of designated banyangviruses can recognize genomic untranslated regions (UTRs) of other banyangviruses and assemble heterogenous minigenomes into functional ribonucleoproteins (RNPs). Moreover, both heterogenous and heterozygous RNPs were efficiently packaged by viral glycoproteins into infectious virus-like particles, manifesting remarkable reassortment potential of banyangviruses. Meanwhile, UTR promoter strength of the three banyangvirus segments appeared to be M > L > S. Secondary structure analysis revealed a conservative non-basepairing protruding nucleotide in the terminal UTR panhandles of M and L (but not S) segments of all banyangviruses and some related phleboviruses (Phlebovirus genus). Furthermore, not only a conserved panhandle region but also the protruding nucleotide proved important for UTR function. Removal of the protruding nucleotide abated M and L UTR activities and compatibilities with heterogenous viral proteins, and introduction of a protruding nucleotide into S panhandle, conversely, enhanced UTR promoter strength and compatibility, revealing the significance of the protruding nucleotide as a new signature of the genomic panhandle structure in both UTR activity and reassortment potential. The study demonstrates not only banyangvirus reassortment potential but also the notable role of the protruding nucleotide in UTR function and reassortment, providing clues to viral evolution and replication mechanisms and perhaps benefiting disease control and prevention in the future.

Phenotypic and proteomic characteristics of sorghum (Sorghum bicolor) albino lethal mutant sbe6-a1.

Leaf color mutants are ideal materials for chloroplast development and photosynthetic mechanism research. Here, we characterized an EMS (ethyl methane sulfonate)-mutagenized sorghum (Sorghum bicolor) mutant, sbe6-a1, in which the severe disruption in chloroplast structure and a chlorophyll deficiency promote an albino leaf phenotype and lead to premature death. The proteomic analyses of mutant and its progenitor wild-type (WT) were performed using a Q Exactive plus Orbitrap mass spectrometer and 4,233 proteins were accurately quantitated. The function analysis showed that most of up-regulated proteins in mutant sbe6-a1 had not been well characterized. GO-enrichment analysis of the differentially abundant proteins (DAPs) showed that up-regulated DAPs were significantly enriched in catabolic process and located in mitochondria, while down regulated DAPs were located in chloroplasts and participated in photosynthesis and some other processes. KEGG pathway-enrichment analyses indicated that the degradation and metabolic pathways of fatty acids, as well as some amino acids and secondary metabolites, were significantly enhanced in the mutant sbe6-a1, while photosynthesis-related pathways, some secondary metabolites' biosynthesis and ribosomal pathways were significantly inhibited. Analysis also shows that some DAPs, such as FBAs, MDHs, PEPC, ATP synthase, CABs, CHLM, PRPs, pathogenesis-related protein, sHSP, ACP2 and AOX may be closely associated with the albino phenotype. Our analysis will promote the understanding of the molecular phenomena that result in plant albino phenotypes.