Usama Ashraf

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.

MicroRNA-15b Modulates Japanese Encephalitis Virus–Mediated Inflammation via Targeting RNF125

Japanese encephalitis virus (JEV) can target CNS and cause neuroinflammation that is characterized by profound neuronal damage and concomitant microgliosis/astrogliosis. Although microRNAs (miRNAs) have emerged as a major regulatory network with profound effects on inflammatory response, it is less clear how they regulate JEV-induced inflammation. In this study, we found that miR-15b is involved in modulating the JEV-induced inflammatory response. The data demonstrate that miR-15b is upregulated during JEV infection of glial cells and mouse brains. In vitro overexpression of miR-15b enhances the JEV-induced inflammatory response, whereas inhibition of miR-15b decreases it. Mechanistically, ring finger protein 125 (RNF125), a negative regulator of RIG-I signaling, is identified as a direct target of miR-15b in the context of JEV infection. Furthermore, inhibition of RNF125 by miR-15b results in an elevation in RIG-I levels, which, in turn, leads to a higher production of proinflammatory cytokines and type I IFN. In vivo knockdown of virus-induced miR-15b by antagomir-15b restores the expression of RNF125, reduces the production of inflammatory cytokines, attenuates glial activation and neuronal damage, decreases viral burden in the brain, and improves survival in the mouse model. Taken together, our results indicate that miR-15b modulates the inflammatory response during JEV infection by negative regulation of RNF125 expression. Therefore, miR-15b targeting may constitute an interesting and promising approach to control viral-induced neuroinflammation.

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

ABSTRACT 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 response in vitro and in 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-1β, 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 vivo silencing 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

ABSTRACT 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.

Quantitative Label-Free Phosphoproteomics Reveals Differentially Regulated Protein Phosphorylation Involved in West Nile Virus-Induced Host Inflammatory Response.

West Nile virus (WNV) can cause neuro-invasive and febrile illness that may be fatal to humans. The production of inflammatory cytokines is key to mediating WNV-induced immunopathology in the central nervous system. Elucidating the host factors utilized by WNV for productive infection would provide valuable insights into the evasion strategies used by this virus. Although attempts have been made to determine these host factors, proteomic data depicting WNV-host protein interactions are limited. We applied liquid chromatography-tandem mass spectrometry for label-free, quantitative phosphoproteomics to systematically investigate the global phosphorylation events induced by WNV infection. Quantifiable changes to 1,657 phosphoproteins were found; of these, 626 were significantly upregulated and 227 were downregulated at 12 h postinfection. The phosphoproteomic data were subjected to gene ontology enrichment analysis, which returned the inflammation-related spliceosome, ErbB, mitogen-activated protein kinase, nuclear factor kappa B, and mechanistic target of rapamycin signaling pathways. We used short interfering RNAs to decrease the levels of glycogen synthase kinase-3 beta, bifunctional polynucleotide phosphatase/kinase, and retinoblastoma 1 and found that the activity of nuclear factor kappa B (p65) is significantly decreased in WNV-infected U251 cells, which in turn led to markedly reduced inflammatory cytokine production. Our results provide a better understanding of the host response to WNV infection and highlight multiple targets for the development of antiviral and anti-inflammatory therapies.

Transcriptional regulation of miR-15b by c-Rel and CREB in Japanese encephalitis virus infection

MicroRNAs (miRNAs) have been well known to play diverse roles in viral infection at the level of posttranscriptional repression. However, much less is understood about the mechanism by which miRNAs are regulated during viral infection. It is likely that both host and virus contain factors to modulate miRNA expression. Here we report the up-regulation of microRNA-15b (miR-15b) in vitro upon infection with Japanese encephalitis virus (JEV). Analysis of miR-15b precursor, pri-miR-15b and pre-miR-15b, suggest that the regulation occurs transcriptionally. Further, we identified the transcriptional regulatory region of miR-15b that contains consensus binding motif for NF-κB subunit c-Rel and cAMP-response element binding protein (CREB), which are known as transcription factor to regulate gene expression. By promoter fusion and mutational analyses, we demonstrated that c-Rel and CREB bind directly to the promoter elements of miR-15b, which are responsible for miR-15b transcription in response to JEV infection. Finally, we showed that pharmacological inhibition of ERK and NF-κB signaling pathway blocked induction of miR-15b in JEV infection, suggesting important roles of ERK and NF-κB pathway in the regulation of miR-15b gene. Therefore, our observations indicate that induced expression of miR-15b is modulated by c-Rel and CREB in response to JEV infection.

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-β and inflammatory cytokines, including tumor necrosis factor-α, interleukin-1β, 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.

Quantitative phosphoproteomic analysis identifies the critical role of JNK1 in neuroinflammation induced by Japanese encephalitis virus

Blocking JNK signaling halts neuroinflammation caused by infection with Japanese encephalitis virus. Halting viral encephalitis Japanese encephalitis virus (JEV) is a member of the mosquito-borne flavivirus family, which includes hepatitis C virus, West Nile virus, yellow fever virus, Zika virus, and dengue virus. Common in Asia and Australia, JEV crosses the blood-brain barrier and produces massive neuroinflammation, which causes neuronal damage and death, such that even people who survive the infection have long-term neurological impairment. Ye et al. took a phosphoproteomic approach and identified several kinase pathways induced by JEV infection of cultured human glial cells. Substrates of the JNK pathway were the most overrepresented in the data set, and treating glial cells in culture with a JNK inhibitor reduced the JEV infection–induced stimulation of inflammatory cytokines. This same inhibitor prevented JEV lethality in mice, which was associated with a decrease in neuroinflammation (reduced astrocytosis and microgliosis), as well as less neuronal injury (reduced apoptotic neurons). These results may prove useful in treating JEV and other neurotropic viruses of this or other viral families. Japanese encephalitis virus (JEV) is the leading cause of epidemic encephalitis worldwide. The pathogenesis of JEV is linked to a robust inflammatory response in the central nervous system (CNS). Glial cells are the resident immune cells in the CNS and represent critical effectors of CNS inflammation. To obtain a global overview of signaling events in glial cells during JEV infection, we conducted phosphoproteomics profiling of a JEV-infected glial cell line. We identified 1816 phosphopeptides, corresponding to 1264 proteins, that exhibited a change in phosphorylation status upon JEV infection. Bioinformatics analysis revealed that these proteins were predominantly related to transcription regulation, signal transduction, the cell cycle, and the cytoskeleton. Kinase substrate motif revealed that substrates for c-Jun N-terminal kinase 1 (JNK1) were the most overrepresented, along with evidence of increased AKT1 and protein kinase A (PKA) signaling. Pharmacological inhibition of JNK, AKT, or PKA reduced the inflammatory response of cultured glial cells infected with JEV, as did knockdown of JNK1 or its target JUN. JEV genomic RNA was sufficient to activate JNK1 signaling in cultured glial cells. Of potential clinical relevance, we showed that inhibition of JNK signaling significantly attenuated the production of inflammatory cytokines in the brain and reduced lethality in JEV-infected mice, thereby suggesting that JNK signaling is a potential therapeutic target for the management of Japanese encephalitis.

Microarray Analysis Identifies the Potential Role of Long Non-Coding RNA in Regulating Neuroinflammation during Japanese Encephalitis Virus Infection

Japanese encephalitis virus (JEV) is the leading cause of epidemic encephalitis worldwide. JEV-induced neuroinflammation is characterized by profound neuronal cells damage accompanied by activation of glial cells. Albeit long non-coding RNAs (lncRNAs) have been emerged as important regulatory RNAs with profound effects on various biological processes, it is unknown how lncRNAs regulate JEV-induced inflammation. Here, using microarray approach, we identified 618 lncRNAs and 1,007 mRNAs differentially expressed in JEV-infected mice brain. The functional annotation analysis revealed that differentially regulated transcripts were predominantly involved in various signaling pathways related to host immune and inflammatory responses. The lncRNAs with their potential to regulate JEV-induced inflammatory response were identified by constructing the lncRNA-mRNA coexpression network. Furthermore, silencing of the two selected lncRNAs (E52329 and N54010) resulted in reducing the phosphorylation of JNK and MKK4, which are known to be involved during inflammatory response. Collectively, we first demonstrated the transcriptomic landscape of lncRNAs in mice brain infected with JEV and analyzed the coexpression network of differentially regulated lncRNAs and mRNAs during JEV infection. Our results provide a better understanding of the host response to JEV infection and suggest that the identified lncRNAs may be used as potential therapeutic targets for the management of Japanese encephalitis.

p21-Activated Kinase 4 Signaling Promotes Japanese Encephalitis Virus-Mediated Inflammation in Astrocytes

Japanese encephalitis virus (JEV) targets central nervous system, resulting in neuroinflammation with typical features of neuronal death along with hyper activation of glial cells. Exploring the mechanisms responsible for the JEV-caused inflammatory response remains a pivotal area of research. In the present study, we have explored the function of p21-activated kinase 4 (PAK4) in JEV-mediated inflammatory response in human astrocytes. The results showed that JEV infection enhances the phosphorylation of PAK4 in U251 cells and mouse brain. Knockdown of PAK4 resulted in decreased expression of inflammatory cytokines that include tumor necrosis factor alpha, interleukin-6, interleukin-1β, and chemokine (C-C motif) ligand 5 and interferon β upon JEV infection, suggesting that PAK4 signaling promotes JEV-mediated inflammation. In addition, we found that knockdown of PAK4 led to the inhibition of MAPK signaling including ERK, p38 MAPK and JNK, and also resulted in the reduced nuclear translocation of NF-κB and phosphorylation of AP-1. These results demonstrate that PAK4 signaling actively promotes JEV-mediated inflammation in human astrocytes via MAPK-NF-κB/AP-1 pathway, which will provide a new insight into the molecular mechanism of the JEV-induced inflammatory response.