A booster of Delta-Omicron RBD-dimer protein subunit vaccine augments sera neutralization of Omicron sub-variants BA.1/BA.2/BA.2.12.1/BA.4/BA.5.

The SARS-CoV-2 Omicron variants of concern (VOCs) showed severe resistance to the early-approved COVID-19 vaccines-induced immune responses. The breakthrough infections by the Omicron VOCs are currently the major challenge for pandemic control. Therefore, booster vaccination is crucial to enhance immune responses and protective efficacy. Previously, we developed a protein subunit COVID-19 vaccine ZF2001, based on the immunogen of receptor-binding domain (RBD) homodimer, which was approved in China and other countries. To adapt SARS-CoV-2 variants, we further developed chimeric Delta-Omicron BA.1 RBD-dimer immunogen which induced broad immune responses against SARS-CoV-2 variants. In this study, we tested the boosting effect of this chimeric RBD-dimer vaccine in mice after priming with two doses of inactivated vaccines, compared with a booster of inactivated vaccine or ZF2001. The results demonstrated that boosting with bivalent Delta-Omicron BA.1 vaccine greatly promoted the neutralizing activity of the sera to all tested SARS-CoV-2 variants. Therefore, the Delta-Omicron chimeric RBD-dimer vaccine is a feasible booster for those with prior vaccination of COVID-19 inactivated vaccines.

Regulation Networks of Non-Coding RNA-Associated ceRNAs in Cisplatin-Induced Acute Kidney Injury.

Cisplatin is widely used as a chemotherapeutic drug to treat various solid tumors. However, it often induces severe side effects, including nephrotoxicity, which limits its application in clinical settings. Furthermore, the underlying mechanisms of action are unclear. Here, we applied whole-transcriptome RNA sequencing to a cisplatin-induced acute kidney injury (CP-AKI) mouse model to evaluate competing endogenous RNA (ceRNA) networks. We found 4460 mRNAs, 1851 long non-coding RNAs, 101 circular RNAs, and 102 microRNAs significantly differentially expressed between CP-AKI and control mice. We performed gene set enrichment analysis to reveal the biological functions of the mRNAs and constructed non-coding RNA-associated ceRNA networks in CP-AKI mice. Two ceRNA regulatory pathways, Lhx1os-203/mmu-miR-21a-3p/Slc7a13 and circular RNA_3907/mmu-miR-185-3p/Ptprn, were validated using quantitative real-time PCR. The protein-protein interaction network indicated that Il6, Cxcl1, Cxcl2, and Plk1 serve as hub genes and are highly connected with the inflammatory response or DNA damage. Transcription factors, such as Stat3, Cebpb, and Foxm1, regulate gene expression levels in CP-AKI. Our study provides insight into non-coding RNA-associated ceRNA networks and mRNAs in CP-AKI and identifies potential treatment targets.

Effect of intrarenal renin-angiotensin-aldosterone system on renal function in patients after cardiac surgery.

ABSTRACT: The aim of the study was to investigate the influence of intrarenal RAS on the decrease of renal function in patients undergoing cardiac surgery with cardiopulmonary bypass. This observational study investigated the activation of intrarenal RAS in 24 patients with AKI after cardiac surgery with cardiopulmonary bypass. The activation of intrarenal RAS was determined by urinary angiotensinogen (uAGT), which was measured at 12 hours before surgery, 0 and12 hours after surgery. The results were compared with those of 21 patients without AKI after cardiac surgery with cardiopulmonary bypass. Clinical and laboratory data were collected. Compared with baseline, all patients with cardiac surgery had activation of intrarenal RAS at 0 and 12 hours after surgery. The activation of intrarenal RAS was found significantly higher at both 0 and 12 hours after surgery in AKI group versus non AKI group (6.18 ±â€Š1.93 ng/mL vs 3.49 ±â€Š1.71 ng/mL, 16.38 ±â€Š7.50 ng/mL vs 6.04 ±â€Š2.59 ng/mL, respectively). There was a positive correlation between the activation of RAS at 0 hour after surgery and the decrease of renal function at 48 hours after surgery (r = 0.654, P = .001). These findings suggest that uAGT might be a suitable biomarker for prediction of the occurrence and severity of AKI after cardiac surgery. Inhibition of intrarenal RAS activation might be one the path of future treatment for this type of disease.

Screening of novel synthetic derivatives of dehydroepiandrosterone for antivirals against flaviviruses infections.

Flaviviruses are important arthropod-borne pathogens that represent an immense global health problem. Their unprecedented epidemic rate and unpredictable clinical features underscore an urgent need for antiviral interventions. Dehydroepiandrosterone (DHEA) is a natural occurring adrenal-derived steroid in the human body that has been associated in protection against various infections. In the present study, the plaque assay based primary screening was conducted on 32 synthetic derivatives of DHEA against Japanese encephalitis virus (JEV) to identify potent anti-flaviviral compounds. Based on primary screening, HAAS-AV3026 and HAAS-AV3027 were selected as hits from DHEA derivatives that exhibited strong antiviral activity against JEV (IC50 â€‹= â€‹2.13 and 1.98 â€‹µmol/L, respectively) and Zika virus (ZIKV) (IC50 â€‹= â€‹3.73 and 3.42 â€‹µmol/L, respectively). Mechanism study indicates that HAAS-AV3026 and HAAS-AV3027 do not exhibit inhibitory effect on flavivirus binding and entry process, while significantly inhibit flavivirus infection at the replication stage. Moreover, indirect immunofluorescence assay, Western blot analyses, and quantitative reverse transcription-PCR (qRT-PCR) revealed a potent antiviral activity of DHEA derivatives hits against JEV and ZIKV in terms of inhibition of viral infection, protein production, and viral RNA synthesis in Vero cells. Taken together, our results may provide a basis for the development of new antivirals against flaviviruses.

Evaluation of Vertebrate-Specific Replication-Defective Zika Virus, a Novel Single-Cycle Arbovirus Vaccine, in a Mouse Model.

The flavivirus Zika (ZIKV) has emerged as a global threat, making the development of a ZIKV vaccine a priority. While live-attenuated vaccines are known to induce long-term immunity but reduced safety, inactivated vaccines exhibit a weaker immune response as a trade-off for increased safety margins. To overcome the trade-off between immunogenicity and safety, the concept of a third-generation flavivirus vaccine based on single-cycle flaviviruses has been developed. These third-generation flavivirus vaccines have demonstrated extreme potency with a high level of safety in animal models. However, the production of these single-cycle, encapsidation-defective flaviviruses requires a complicated virion packaging system. Here, we investigated a new single-cycle flavivirus vaccine, a vertebrate-specific replication-defective ZIKV (VSRD-ZIKV), in a mouse model. VSRD-ZIKV replicates to high titers in insect cells but can only initiate a single-round infection in vertebrate cells. During a single round of infection, VSRD-ZIKV can express all the authentic viral antigens in vertebrate hosts. VSRD-ZIKV immunization elicited a robust cellular and humoral immune response that protected against a lethal ZIKV challenge in AG129 mice. Additionally, VSRD-ZIKV-immunized pregnant mice were protected against vertically transferring a lethal ZIKV infection to their offspring. Immunized male mice were protected and prevented viral accumulation in the testes after being challenged with lethal ZIKV. Overall, our results indicate that VSRD-ZIKV induces a potent protective immunity against ZIKV in a mouse model and represents a promising approach to develop novel single-cycle arbovirus vaccines.

Advances in understanding the innate immune-associated diabetic kidney disease.

Millions of human deaths occur annually due to chronic kidney disease, caused by diabetic kidney disease (DKD). Despite having effective drugs controlling the hyperglycemia and high blood pressure, the incidence of DKD is increasing, which indicates the need for the development of novel therapies to control DKD. In this article, we discussed the recent advancements in the basic innate immune mechanisms in renal tissues triggered under the diabetes environment, leading to the pathogenesis and progression of DKD. We also summarized the currently available innate immune molecules-targeting therapies tested against DKD in clinical and preclinical settings, and highlighted additional drug targets that could potentially be employed for the treatment of DKD. The improved understanding of the disease pathogenesis may open avenues for the development of novel therapies to rein in DKD, which consequently, can reduce morbidity and mortality in humans in the future.

Identification of fenoldopam as a novel LSD1 inhibitor to abrogate the proliferation of renal cell carcinoma using drug repurposing strategy.

Although targeted therapy for renal cell carcinoma (RCC) has achieved good therapeutic effects in clinic, a considerable number of patients develop drug resistance over time. So, there is still an urgent need to develop new drugs for RCC treatment. As LSD1 is considered as a promising drug target in diverse cancers, including RCC, we tried to find new LSD1 inhibitor using drug repurposing strategy from a compound library, and fenoldopam, an FDA-approved drug, was identified as a potent LSD1 inhibitor with IC50 = 0.8974 µM in a reversible manner. Molecular docking predicted that fenoldopam occupied the FAD cavity of LSD1, forming hydrogen bonds with surrounding residues. Moreover, fenoldopam inactivated LSD1 and performed antiproliferative activity against ACHN cells and promoted cells apoptosis in vitro. Taken together, fenoldopam was identified as a novel LSD1 inhibitor firstly, and may serve as a new skeleton for RCC therapy.

Generation and preliminary characterization of vertebrate-specific replication-defective Zika virus.

Zika virus (ZIKV) is a mosquito-borne flavivirus that replicates in both vertebrate and insect cells, whereas insect-specific flaviviruses (ISF) replicate only in insect cells. We sought to convert ZIKV, from a dual-tropic flavivirus, into an insect-specific virus for the eventual development of a safe ZIKV vaccine. Reverse genetics was used to introduce specific mutations into the furin cleavage motif within the ZIKV pre-membrane protein (prM). Mutant clones were selected, which replicated well in C6/36 insect cells but exhibited reduced replication in non-human primate (Vero) cells. Further characterization of the furin cleavage site mutants indicated they replicated poorly in both human (HeLa, U251), and baby hamster kidney (BHK-21) cells. One clone with the induced mutation in the prM protein and at positions 291and 452 within the NS3 protein was totally and stably replication-defective in vertebrate cells (VSRD-ZIKV). Preliminary studies in ZIKV sensitive, immunodeficient mice demonstrated that VSRD-ZIKV-infected mice survived and were virus-negative. Our study indicates that a reverse genetic approach targeting the furin cleavage site in prM can be used to select an insect-specific ZIKV with the potential utility as a vaccine strain.

The Japanese Encephalitis Virus NS1' Protein Inhibits Type I IFN Production by Targeting MAVS.

Japanese encephalitis virus (JEV) is a mosquito-borne Flavivirus that causes severe neurologic disease in humans. NS1' is a NS1-related protein only reported in the Japanese encephalitis serogroup members of Flavivirus It is produced through programmed -1 ribosomal frameshift in NS2A. Our previous study demonstrated that JEV NS1' could antagonize type I IFN (IFN-I) production, but the mechanism is still unclear. In the current study, we found that JEV NS1' inhibits the expression of MAVS, and knockdown of MAVS hampers inhibition of IFN-ß induction by NS1', suggesting that JEV NS1' inhibits IFN-I production by targeting MAVS. This finding is further supported by the result of the in vivo assay that showed the similar mortality caused by NS1'-deficient virus and its wild type virus in MAVS-deficient mice. Based on our previous sequencing results of noncoding RNA in JEV-infected cells, microRNA-22 (miR-22) was identified to be a key regulator for MAVS expression during JEV infection. Furthermore, we demonstrated that JEV NS1' could induce the expression of miR-22 by increasing the binding of transcriptional factors, CREB and c-Rel, to the promoter elements of miR-22. Taken together, our results reveal a novel mechanism by which JEV NS1' antagonizes host MAVS by regulating miR-22, thereby inhibiting the IFN-I production and facilitating viral replication.

Development and application of a monoclonal-antibody-based blocking ELISA for detection of Japanese encephalitis virus NS1 antibodies in swine.

Japanese encephalitis virus (JEV) is a zoonotic pathogen transmitted by Culex mosquitoes and is the leading cause of viral encephalitis in humans. JEV infection of swine, which are the main amplifying hosts for JEV, can cause reproductive failure in sows; in boars it can cause testitis and infertility. The prevalence of JEV in swine is a continuous threat to human health. A practical diagnostic method for monitoring JEV infection in swine herds is essential for control of the disease in both swine and humans. Here, we have identified a high-affinity anti-JEV NS1 monoclonal antibody (mAb) by indirect ELISA and utilized it for the development of a blocking ELISA (bELISA). The optimal NS1 protein coating concentration (2 µg/mL) and mAb working concentration (1 µg/mL) were determined by checkerboard titration. One hundred ten JEV-antibody-negative serum samples were used to establish 34.03% inhibition as the cutoff value for a negative result. By the bELISA, seroconversion in 80% of newly JEV-vaccinated pigs was detected by 7 days post-immunization, while by the commercial envelope-protein-based iELISA, seroconversion was detected in 20% of the newly vaccinated pigs. We found 98.7% agreement between the bELISA and the commercial iELISA when we tested 157 field samples using both methods. From an epidemiological survey of swine serum collected between 2014 and 2016, we found that the average JEV seropositive rate in unvaccinated commodity pigs was 8.1%, and in vaccinated boars and sows, it was 67.6%.

Benzoxazine monomer derived carbon dots as a broad-spectrum agent to block viral infectivity.

Multiple viruses can cause infection and death of millions annually. Of these, flaviviruses are found to be highly prevalent in recent years with no distinctive antiviral therapies. Therefore, there is a desperate need for broad-spectrum antiviral drugs that can be active against a large number of existing and emerging viruses. Herein, we prepared a kind of benzoxazine monomer derived carbon dots (BZM-CDs) and demonstrated their infection-blocking ability against life-threatening flaviviruses (Japanese encephalitis, Zika, and dengue viruses) and non-enveloped viruses (porcine parvovirus and adenovirus-associated virus). It was found that BZM-CDs could directly bind to the surface of the virion, and eventually the first step of virus-cell interaction was impeded. The developed nanoparticles are active against both flaviviruses and non-enveloped viruses in vitro. Thus, the application of BZM-CDs may constitute an intriguing broad-spectrum approach to rein in viral infections.

MicroRNA-22 negatively regulates poly(I:C)-triggered type I interferon and inflammatory cytokine production via targeting mitochondrial antiviral signaling protein (MAVS).

MicroRNAs (miRNAs) are small non-coding RNAs that play important roles in regulating the host immune response. Here we found that miR-22 is induced in glial cells upon stimulation with poly(I:C). Overexpression of miR-22 in the cultured cells resulted in decreased activity of interferon regulatory factor-3 and nuclear factor-kappa B, which in turn led to reduced expression of interferon-ß and inflammatory cytokines, including tumor necrosis factor-α, interleukin-1ß, interleukin-6, and chemokine (C-C motif) ligand 5, upon stimulation with poly(I:C), whereas knockdown of miR-22 had the opposite effect. We used a combination of bioinformatics and experimental techniques to demonstrate that mitochondrial antiviral signaling protein (MAVS), which positively regulates type I interferon production, is a novel target of miR-22. Overexpression of miR-22 decreased the activity of a luciferase reporter containing the MAVS 3'-untranslated region and led to decreased MAVS mRNA and protein levels. In contrast, ectopic expression of miR-22 inhibitor led to elevated MAVS expression. Collectively, our results demonstrate that miR-22 negatively regulates poly(I:C)-induced production of type I interferon and inflammatory cytokines via targeting MAVS.

MicroRNA-19b-3p Modulates Japanese Encephalitis Virus-Mediated Inflammation via Targeting RNF11.

UNLABELLED: Japanese encephalitis virus (JEV) can invade the central nervous system and consequently induce neuroinflammation, which is characterized by profound neuronal cell damage accompanied by astrogliosis and microgliosis. Albeit microRNAs (miRNAs) have emerged as major regulatory noncoding RNAs with profound effects on inflammatory response, it is unknown how astrocytic miRNAs regulate JEV-induced inflammation. Here, we found the involvement of miR-19b-3p in regulating the JEV-induced inflammatory responsein vitroandin vivo The data demonstrated that miR-19b-3p is upregulated in cultured cells and mouse brain tissues during JEV infection. Overexpression of miR-19b-3p led to increased production of inflammatory cytokines, including tumor necrosis factor alpha, interleukin-6, interleukin-1ß, and chemokine (C-C motif) ligand 5, after JEV infection, whereas knockdown of miR-19b-3p had completely opposite effects. Mechanistically, miR-19b-3p modulated the JEV-induced inflammatory response via targeting ring finger protein 11, a negative regulator of nuclear factor kappa B signaling. We also found that inhibition of ring finger protein 11 by miR-19b-3p resulted in accumulation of nuclear factor kappa B in the nucleus, which in turn led to higher production of inflammatory cytokines.In vivosilencing of miR-19b-3p by a specific antagomir reinvigorates the expression level of RNF11, which in turn reduces the production of inflammatory cytokines, abrogates gliosis and neuronal cell death, and eventually improves the survival rate in the mouse model. Collectively, our results demonstrate that miR-19b-3p positively regulates the JEV-induced inflammatory response. Thus, miR-19b-3p targeting may constitute a thought-provoking approach to rein in JEV-induced inflammation. IMPORTANCE: Japanese encephalitis virus (JEV) is one of the major causes of acute encephalitis in humans worldwide. The pathological features of JEV-induced encephalitis are inflammatory reactions and neurological diseases resulting from glia activation. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally. Accumulating data indicate that miRNAs regulate a variety of cellular processes, including the host inflammatory response under pathological conditions. Recently, a few studies demonstrated the role of miRNAs in a JEV-induced inflammatory response in microglia; however, their role in an astrocyte-derived inflammatory response is largely unknown. The present study reveals that miR-19b-3p targets ring finger protein 11 in glia and promotes inflammatory cytokine production by enhancing nuclear factor kappa B activity in these cells. Moreover, administration of an miR-19b-3p-specific antagomir in JEV-infected mice reduces neuroinflammation and lethality. These findings suggest a new insight into the molecular mechanism of the JEV-induced inflammatory response and provide a possible therapeutic entry point for treating viral encephalitis.

MicroRNA-33a-5p Modulates Japanese Encephalitis Virus Replication by Targeting Eukaryotic Translation Elongation Factor 1A1.

UNLABELLED: Japanese encephalitis virus (JEV) is a typical mosquito-borne flavivirus responsible for acute encephalitis and meningitis in humans. However, the molecular mechanism for JEV pathogenesis is still unclear. MicroRNAs (miRNAs) are small noncoding RNAs that act as gene regulators. They are directly or indirectly involved in many cellular functions owing to their ability to target mRNAs for degradation or translational repression. However, how cellular miRNAs are regulated and their functions during JEV infection are largely unknown. In the present study, we found that JEV infection downregulated the expression of endogenous cellular miR-33a-5p. Notably, artificially transfecting with miR-33a-5p mimics led to a significant decrease in viral replication, suggesting that miR-33a-5p acts as a negative regulator of JEV replication. A dual-luciferase reporter assay identified eukaryotic translation elongation factor 1A1 (EEF1A1) as one of the miR-33a-5p target genes. Our study further demonstrated that EEF1A1 can interact with the JEV proteins NS3 and NS5 in replicase complex. Through this interaction, EEF1A1 can stabilize the components of viral replicase complex and thus facilitates viral replication during JEV infection. Taken together, these results suggest that miR-33a-5p is downregulated during JEV infection, which contributes to viral replication by increasing the intracellular level of EEF1A1, an interaction partner of JEV NS3 and NS5. This study provides a better understanding of the molecular mechanisms of JEV pathogenesis. IMPORTANCE: MiRNAs are critical regulators of gene expression that utilize sequence complementarity to bind to and modulate the stability or translation efficiency of target mRNAs. Accumulating data suggest that miRNAs regulate a wide variety of molecular mechanisms in the host cells during viral infections. JEV, a neurotropic flavivirus, is one of the major causes of acute encephalitis in humans worldwide. The roles of cellular miRNAs during JEV infections are widely unexplored. The present study explores a novel role of miR-33a-5p as a negative regulator of JEV replication. We found EEF1A1 as a direct target of miR-33a-5p. We also demonstrated that EEF1A1 interacts with and stabilize the components of JEV replicase complex, which positively regulates JEV replication. These findings suggest a new insight into the molecular mechanism of JEV pathogenesis and provide a possible therapeutic entry point for viral encephalitis.

The Yin and Yang of Antiviral Innate Immunity in Central Nervous System.

The innate immune system provides protection against invading neurotropic viruses. It acts as the first line of defense against invading viruses and plays an elementary role in their pathogenesis. The list of viruses capable of infecting human central nervous system (CNS) is quite long, most important of them are Japanese Encephalitis virus (JEV), rabies virus, West Nile virus (WNV), herpes simplex virus (HSV), St. Louis encephalitis virus (SLEV), La Crosse virus, tick borne encephalitis virus (TEBE) and polio virus. Germ line pattern recognition receptors (PRRs) such as Toll like receptors (TLRs), nucleotide binding oligomerization domain (NOD) - like receptors (NLRs), retinoic acid-inducible gene I (RIG-I) -like helicases or RIG-I-like receptors (RLRs) and cytosolic DNA sensors recognize the pathogen associated molecular patterns (PAMPs) and initiate an immune response against invading pathogen. Although PRRs were originally characterized in peripheral immune cells but accumulating evidence also suggest their crucial roles in CNS to combat against neurotropic viruses. In this review, we will highlight the recent developments in our understating of the mechanisms by which PRRs in resident brain cells provide protection against invading neurotropic viruses.

The Role of Ubiquitination in Regulation of Innate Immune Signaling.

Ubiquitination, also denoted ubiquitylation, is a posttranslational modification that has been implicated in the regulation of both innate and adaptive immune responses. Ubiquitination plays crucial roles in innate immune signaling by ensuring the proper orchestration of several signaling mediators that constitute a functional immune response. Herein, we briefly summarize the latest discoveries concerning the molecular ubiquitination-related machinery that senses, assembles, and disassembles innate immune signaling mediators.

Usutu virus: an emerging flavivirus in Europe.

Usutu virus (USUV) is an African mosquito-borne flavivirus belonging to the Japanese encephalitis virus serocomplex. USUV is closely related to Murray Valley encephalitis virus, Japanese encephalitis virus, and West Nile virus. USUV was discovered in South Africa in 1959. In Europe, the first true demonstration of circulation of USUV was reported in Austria in 2001 with a significant die-off of Eurasian blackbirds. In the subsequent years, USUV expanded to neighboring countries, including Italy, Germany, Spain, Hungary, Switzerland, Poland, England, Czech Republic, Greece, and Belgium, where it caused unusual mortality in birds. In 2009, the first two human cases of USUV infection in Europe have been reported in Italy, causing meningoencephalitis in immunocompromised patients. This review describes USUV in terms of its life cycle, USUV surveillance from Africa to Europe, human cases, its cellular tropism and pathogenesis, its genetic relationship with other flaviviruses, genetic diversity among USUV strains, its diagnosis, and a discussion of the potential future threat to Asian countries.

miR-206 modulates lipopolysaccharide-mediated inflammatory cytokine production in human astrocytes.

Astrocyte-derived inflammation is a common component of acute or chronic injury in the central nervous system. MicroRNAs (miRNAs) are small non-coding RNAs that play important regulatory roles in the inflammatory response. In this study, we found that miR-206 is induced upon stimulation with lipopolysaccharide. Overexpression of miR-206 in astrocytes led to increased expression of inflammatory cytokines (interleukin-6, interleukin-1ß, CCL5) upon exposure to lipopolysaccharide, whereas knockdown of miR-206 had completely opposite effects. We used a combination of bioinformatics and experimental techniques to demonstrate that NR4A2, which belongs to the nuclear receptor (NR) 4 family of orphan nuclear receptors, is a direct target of miR-206. Overexpression of miR-206 mimics decreased the activity of a luciferase reporter containing the NR4A2 3'-untranslated region and led to decreased NR4A2 mRNA and protein levels. In contrast, ectopic expression of an miR-206 inhibitor led to elevated NR4A2 expression. We also found that miR-206 modulated the lipopolysaccharide-induced proinflammatory response by targeting NR4A2 and activating nuclear factor-kappa B activity. Finally, we demonstrated that the transcription factor AP-1 plays a critical role in lipopolysaccharide-induced expression of miR-206 and that the extracellular signal-regulated kinase signaling pathway contributes to the regulation of miR-206 level in astrocytes. These data demonstrate that miR-206 positively regulates the lipopolysaccharide-induced inflammatory response in human astrocytes.