Reduced Virus Production Research Articles

CK1 and PP1 regulate Rift Valley fever virus genome replication through L protein phosphorylation

Rift Valley fever virus (RVFV) is an arbovirus in the Phenuiviridae family identified initially by the large ‘abortion storms’ observed among ruminants; RVFV can also infect humans. In humans, there is a wide variation of clinical symptoms ranging from subclinical to mild febrile illness to hepatitis, retinitis, delayed-onset encephalitis, or even hemorrhagic fever. The RVFV is a tri-segmented negative-sense RNA virus consisting of S, M, and L segments. The L segment encodes the RNA-dependent RNA polymerase (RdRp), termed the L protein, which is responsible for both viral mRNA synthesis and genome replication. Phosphorylation of viral RdRps is known to regulate viral replication. This study shows that RVFV L protein is serine phosphorylated and identified Casein Kinase 1 alpha (CK1α) and protein phosphatase 1 alpha (PP1α) as L protein binding partners. Inhibition of CK1 and PP1 through small molecule inhibitor treatment, D4476 and 1E7-03, respectively, caused a change in the phosphorylated status of the L protein. Inhibition of PP1α resulted in increased L protein phosphorylation whereas inhibition of CK1α decreased L protein phosphorylation. It was also found that in RVFV infected cells, PP1α localized to the cytoplasmic compartment. Treatment of RVFV infected cells with CK1 inhibitors reduced virus production in both mammalian and mosquito cells. Lastly, inhibition of either CK1 or PP1 reduced viral genomic RNA levels. These data indicate that L protein is phosphorylated and that CK1 and PP1 play a crucial role in regulating the L protein phosphorylation cycle, which is critical to viral RNA production and viral replication.

Attenuation of neurovirulence of chikungunya virus by a single amino acid mutation in viral E2 envelope protein

BackgroundChikungunya virus (CHIKV) has reemerged as a major public health concern, causing chikungunya fever with increasing cases and neurological complications.MethodsIn the present study, we investigated a low-passage human isolate of the East/ Central/South African (ECSA) lineage of CHIKV strain LK(EH)CH6708, which exhibited a mix of small and large viral plaques. The small and large plaque variants were isolated and designated as CHIKV-SP and CHIKV-BP, respectively. CHIKV-SP and CHIKV-BP were characterized in vitro and in vivo to compare their virus production and virulence. Additionally, whole viral genome analysis and reverse genetics were employed to identify genomic virulence factors.ResultsCHIKV-SP demonstrated lower virus production in mammalian cells and attenuated virulence in a murine model. On the other hand, CHIKV-BP induced higher pro-inflammatory cytokine levels, compromised the integrity of the blood–brain barrier, and led to astrocyte infection in mouse brains. Furthermore, the CHIKV-SP variant had limited transmission potential in Aedesalbopictus mosquitoes, likely due to restricted dissemination. Whole viral genome analysis revealed multiple genetic mutations in the CHIKV-SP variant, including a Glycine (G) to Arginine (R) mutation at position 55 in the viral E2 glycoprotein. Reverse genetics experiments confirmed that the E2-G55R mutation alone was sufficient to reduce virus production in vitro and virulence in mice.ConclusionsThese findings highlight the attenuating effects of the E2-G55R mutation on CHIKV pathogenicity and neurovirulence and emphasize the importance of monitoring this mutation in natural infections.

Strain Variation Can Significantly Modulate the miRNA Response to Zika Virus Infection.

Zika virus (ZIKV) is a mosquito-transmitted virus that has emerged as a major public health concern due to its association with neurological disorders in humans, including microcephaly in fetuses. ZIKV infection has been shown to alter the miRNA profile in host cells, and these changes can contain elements that are proviral, while others can be antiviral in action. In this study, the expression of 22 miRNAs in human A549 cells infected with two different ZIKV isolates was investigated. All of the investigated miRNAs showed significant changes in expression at at least one time point examined. Markedly, 18 of the miRNAs examined showed statistically significant differences in expression between the two strains examined. Four miRNAs (miR-21, miR-34a, miR-128 and miR-155) were subsequently selected for further investigation. These four miRNAs were shown to modulate antiviral effects against ZIKV, as downregulation of their expression through anti-miRNA oligonucleotides resulted in increased virus production, whereas their overexpression through miRNA mimics reduced virus production. However, statistically significant changes were again seen when comparing the two strains investigated. Lastly, candidate targets of the miRNAs miR-34a and miR-128 were examined at the level of the mRNA and protein. HSP70 was identified as a target of miR-34a, but, again, the effects were strain type-specific. The two ZIKV strains used in this study differ by only nine amino acids, and the results highlight that consideration must be given to strain type variation when examining the roles of miRNAs in ZIKV, and probably other virus infections.

Insecticidal Traits of Variants in a Genotypically Diverse Natural Isolate of Anticarsia Gemmatalis Multiple Nucleopolyhedrovirus (AgMNPV).

Outbreaks of Anticarsia gemmatalis (Hübner, 1818) (Lepidoptera: Erebidae), a major pest of soybean, can be controlled below economic thresholds with methods that do not involve the application of synthetic insecticides. Formulations based on natural isolates of the Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) (Baculoviridae: Alphabaculovirus) played a significant role in integrated pest management programs in the early 2000s, but a new generation of chemical insecticides and transgenic soybean have displaced AgMNPV-based products over the past decade. However, the marked genotypic variability present among and within alphabaculovirus isolates suggests that highly insecticidal genotypic variants can be isolated and used to reduce virus production costs or overcome isolate-dependent host resistance. This study aimed to select novel variants of AgMNPV with suitable insecticidal traits that could complement the existing AgMNPV active ingredients. Three distinct AgMNPV isolates were compared using their restriction endonuclease profile and in terms of their occlusion body (OB) pathogenicity. One isolate was selected (AgABB51) from which eighteen genotypic variants were plaque purified and characterized in terms of their insecticidal properties. The five most pathogenic variants varied in OB pathogenicity, although none of them was faster-killing or had higher OB production characteristics than the wild-type isolate. We conclude that the AgABB51 wild-type isolates appear to be genotypically structured for fast speed of kill and high OB production, both of which would favor horizontal transmission. Interactions among the component variants are likely to influence this insecticidal phenotype.

Virucidal antiviral activity of Maytenus quadrangulata extract against Mayaro virus: Evidence for the presence of catechins.

Mayaro virus (MAYV) is an arbovirus endemic to the Amazon region, which comprises the states of the North and Midwest region of Brazil and encompasses the largest tropical forest in the world, the Amazon Forest. The confirmation of its potential transmission by Aedes aegypti and recent cases in Brazil, mainly in large centers in the northern region, led to the classification of Mayaro fever as an emerging disease. Traditional medicine is commonly used to treat various diseases, mainly by local riverside populations. Some species of the genus Maytenus, which have similar morphologies, are popularly used to treat infections and inflammations. In this context, our research group has studied and confirmed the antiviral activity of several plant-derived compounds. However, several species of this same genus have not been studied and therefore deserve attention. This study aimed to demonstrate the effects of ethyl acetate extracts of leaves (LAE) and branches (TAE) of Maytenus quadrangulata against MAYV. Mammalian cells (Vero cells) were used to evaluate the cytotoxicity of the extracts. After cell infection by MAYV and the treatment with the extracts, we evaluated the selectivity index (SI), the virucidal effect, viral adsorption and internalization, and the effect on viral gene expression. The antiviral action was confirmed by quantifying the viral genome using RT-qPCR and by analyzing the effect on virus yield in infected cells. The treatment was performed based on the effective concentration protective for 50% of the infected cells (EC50). The leaves (LAE; EC50 12.0μg/mL) and branches (TAE; EC50 101.0μg/mL) extracts showed significative selectivity against the virus, with SI values of 79.21 and 9.91, respectively, which were considered safe. Phytochemical analysis revealed that the antiviral action was associated with the presence of catechins, mainly in LAE. This extract was chosen for the subsequent studies since it reduced the viral cytopathic effect and virus production, even at high viral loads [MOI (multiplicity of infection) 1 and 5]. The effects of LAE resulted in a marked reduction in viral gene expression. The viral title was drastically reduced when LAE was added to the virus before infection or during replication stages, reducing virus production up to 5-log units compared to infected and untreated cells. Through kinetic replication, MAYV was not detected in Vero cells treated with LAE throughout the viral cycle. The virucidal effect of LAE inactivates the viral particle and can intercept the virus at the end of the cycle when it gains the extracellular environment. Therefore, LAE is a promising source of antiviral agents.

Viral subversion of selective autophagy is critical for biogenesis of virus replication organelles

Infection by many (+)RNA viruses is accompanied by ER-expansion and membrane remodelling to form viral replication organelles, followed by assembly and secretion of viral progenies. We previously identified that virus-triggered lipophagy was critical for flaviviral assembly, and is driven by the lipid droplet associated protein Ancient ubiquitin protein 1 (Aup1). A ubiquitin conjugating protein Ube2g2 that functions as a co-factor for Aup1 was identified as a host dependency factor in our study. Here we characterized its function: Ube2g2-deficient cells displayed a dramatic reduction in virus production, which could be rescued by reconstituting the wild-type but not the catalytically deficient (C89K) mutant of Ube2g2, suggesting that its enzymatic activity is necessary. Ube2g2 deficiency did not affect entry of virus particles but resulted in a profound loss in formation of replication organelles, and production of infectious progenies. This phenomenon resulted from its dual activity in (i) triggering lipophagy in conjunction with Aup1, and (ii) degradation of ER chaperones such as Herpud1, SEL1L, Hrd1, along with Sec62 to restrict ER-phagy upon Xbp1-IRE1 triggered ER expansion. Our results therefore underscore an exquisite fine-tuning of selective autophagy by flaviviruses that drive host membrane reorganization during infection to enable biogenesis of viral replication organelles.

Investigation into the use of gamma irradiated Cytodex-1 microcarriers to produce a human cytomegalovirus (HCMV) vaccine candidate in epithelial cells

V160 is a viral vaccine candidate against human cytomegalovirus (HCMV) that is manufactured using Adult Retinal Pigment Epithelial cells (ARPE-19) grown on Cytodex-1 microcarriers. The microcarriers are generally hydrated, washed, and autoclaved prior to use, which can be limiting at large production scales. To minimize microcarrier preparation and sterilization, the use of gamma irradiated Cytodex-1 was investigated. Similar ARPE-19 cell growth was observed on heat-sterilized and gamma irradiated Cytodex-1; however, significantly reduced virus production was observed in cultures exposed to gamma irradiated Cytodex-1. Additional experiments suggest that infection inhibition is not exclusive to ARPE-19 but is most directly linked to HCMV V160, as evidenced by similar inhibition of V160 with Vero cells and no inhibition of Measles virus with either cell type. These observations suggest a putative impact on HCMV infection from the presence of extractable(s)/leachable(s) in the gamma irradiated microcarriers. Thorough aseptic rinsing of gamma irradiated Cytodex-1 prior to use can mitigate this impact and enable comparable process performance to heat-sterilized Cytodex-1. Though not fully a “ready-to-use” product for the HCMV V160 production process, utilization of Cytodex-1 microcarriers was possible without requiring heat sterilization, suggesting a potential path forward for large scale production of V160.

Peroxisomal very long-chain fatty acid transport is targeted by herpesviruses and the antiviral host response

Very long-chain fatty acids (VLCFA) are critical for human cytomegalovirus replication and accumulate upon infection. Here, we used Epstein-Barr virus (EBV) infection of human B cells to elucidate how herpesviruses target VLCFA metabolism. Gene expression profiling revealed that, despite a general induction of peroxisome-related genes, EBV early infection decreased expression of the peroxisomal VLCFA transporters ABCD1 and ABCD2, thus impairing VLCFA degradation. The mechanism underlying ABCD1 and ABCD2 repression involved RNA interference by the EBV-induced microRNAs miR-9-5p and miR-155, respectively, causing significantly increased VLCFA levels. Treatment with 25-hydroxycholesterol, an antiviral innate immune modulator produced by macrophages, restored ABCD1 expression and reduced VLCFA accumulation in EBV-infected B-lymphocytes, and, upon lytic reactivation, reduced virus production in control but not ABCD1-deficient cells. Finally, also other herpesviruses and coronaviruses target ABCD1 expression. Because viral infection might trigger neuroinflammation in X-linked adrenoleukodystrophy (X-ALD, inherited ABCD1 deficiency), we explored a possible link between EBV infection and cerebral X-ALD. However, neither immunohistochemistry of post-mortem brains nor analysis of EBV seropositivity in 35 X-ALD children supported involvement of EBV in the onset of neuroinflammation. Collectively, our findings indicate a previously unrecognized, pivotal role of ABCD1 in viral infection and host defence, prompting consideration of other viral triggers in cerebral X-ALD.

Nuclear Transport of Respiratory Syncytial Virus Matrix Protein Is Regulated by Dual Phosphorylation Sites.

Respiratory syncytial virus (RSV) is a major cause of respiratory infections in infants and the elderly. Although the RSV matrix (M) protein has key roles in the nucleus early in infection, and in the cytoplasm later, the molecular basis of switching between the nuclear and cytoplasmic compartments is not known. Here, we show that protein kinase CK2 can regulate M nucleocytoplasmic distribution, whereby inhibition of CK2 using the specific inhibitor 4,5,6,7-tetrabromobenzo-triazole (TBB) increases M nuclear accumulation in infected cells as well as when ectopically expressed in transfected cells. We use truncation/mutagenic analysis for the first time to show that serine (S) 95 and threonine (T) 205 are key CK2 sites that regulate M nuclear localization. Dual alanine (A)-substitution to prevent phosphorylation abolished TBB- enhancement of nuclear accumulation, while aspartic acid (D) substitution to mimic phosphorylation at S95 increased nuclear accumulation. D95 also induced cytoplasmic aggregate formation, implying that a negative charge at S95 may modulate M oligomerization. A95/205 substitution in recombinant RSV resulted in reduced virus production compared with wild type, with D95/205 substitution resulting in an even greater level of attenuation. Our data support a model where unphosphorylated M is imported into the nucleus, followed by phosphorylation of T205 and S95 later in infection to facilitate nuclear export and cytoplasmic retention of M, respectively, as well as oligomerization/virus budding. In the absence of widely available, efficacious treatments to protect against RSV, the results raise the possibility of antiviral strategies targeted at CK2.

Differences in New Variant of Concern Replication at Physiological Temperatures In Vitro.

Using multiple cell types and isolates of Delta and Omicron variants of SARS-CoV-2, we report differences in virus production, replication, and infectivity in vitro. Ancestral and Delta SARS-CoV-2 variant exhibit reduced virus production and replication at 34°C compared to 37°C while Omicron replication is balanced between temperatures.

Signaling Pathway Reporter Screen with SARS-CoV-2 Proteins Identifies nsp5 as a Repressor of p53 Activity

The dysregulation of host signaling pathways plays a critical role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and viral pathogenesis. While a number of viral proteins that can block type I IFN signaling have been identified, a comprehensive analysis of SARS-CoV-2 proteins in the regulation of other signaling pathways that can be critical for viral infection and its pathophysiology is still lacking. Here, we screened the effect of 21 SARS-CoV-2 proteins on 10 different host signaling pathways, namely, Wnt, p53, TGFβ, c-Myc, Hypoxia, Hippo, AP-1, Notch, Oct4/Sox2, and NF-κB, using a luciferase reporter assay. As a result, we identified several SARS-CoV-2 proteins that could act as activators or inhibitors for distinct signaling pathways in the context of overexpression in HEK293T cells. We also provided evidence for p53 being an intrinsic host restriction factor of SARS-CoV-2. We found that the overexpression of p53 is capable of reducing virus production, while the main viral protease nsp5 can repress the transcriptional activity of p53, which depends on the protease function of nsp5. Taken together, our results provide a foundation for future studies, which can explore how the dysregulation of specific signaling pathways by SARS-CoV-2 proteins can control viral infection and pathogenesis.

Rapid Construction of an Infectious Clone of the Zika Virus, Strain ZKC2.

Zika virus (ZIKV) has had detrimental effects on global public health in recent years. This is because the management of the disease has been limited, in part because its pathogenic mechanisms are not yet completely understood. Infectious clones are an important tool that utilize reverse genetics; these can be used to modify the ZIKV genomic RNA at the DNA level. A homologous recombination clone was used to construct pWSK29, a low copy plasmid that contained sequences for a T7 promoter, the whole genome of ZIKV ZKC2 strain, and a hepatitis delta virus ribozyme. High fidelity PCR was then used to amplify the T7 transcription template. The transcript was then transfected into susceptible cells via lipofection to recover the ZIKV ZKC2 strain. Finally, the virulence of rZKC2 was evaluated both in vitro and in vivo. The rZKC2 was successfully obtained and it showed the same virulence as its parent, the ZIKV ZKC2 strain (pZKC2), both in vitro and in vivo. The 3730 (NS2A-D62G) mutation site was identified as being important, since it had significant impacts on rZKC2 recovery. The 4015 (NS2A, A157V) mutation may reduce virus production by increasing the interferon type I response. In this study, one of the earliest strains of ZIKV that was imported into China was used for infectious clone construction and one possible site for antiviral medication development was discovered. The use of homologous recombination clones, of PCR products as templates for T7 transcription, and of lipofection for large RNA transfection could increase the efficiency of infectious clone construction. Our infectious clone provides an effective tool which can be used to explore the life cycle and medical treatment of ZIKV.

Rottlerin inhibits La Crosse virus-induced encephalitis in mice and blocks release of replicating virus from the Golgi body in neurons.

La Crosse virus (LACV) is a mosquito-borne orthobunyavirus that causes approximately 60 to 80 hospitalized pediatric encephalitis cases in the United States yearly. The primary treatment for most viral encephalitis, including LACV, is palliative care, and specific antiviral therapeutics are needed. We screened the National Center for Advancing Translational Sciences library of 3,833 FDA-approved and bioactive small molecules for the ability to inhibit LACV-induced death in SH-SY5Y neuronal cells. The top three hits from the initial screen were validated by examining their ability to inhibit virus-induced cell death in multiple neuronal cell lines. Rottlerin consistently reduced LACV-induced death by 50% in multiple human and mouse neuronal cell lines with an effective concentration of 0.16-0.69 µg ml-1 depending on cell line. Rottlerin was effective up to 12 hours post-infection in vitro and inhibited virus particle trafficking from the Golgi apparatus to trans-Golgi vesicles. In human inducible pluripotent stem cell-derived cerebral organoids, rottlerin reduced virus production by one log and cell death by 35% compared with dimethyl sulfoxide-treated controls. Administration of rottlerin in mice by intraperitoneal or intracranial routes starting at 3 days post-infection decreased disease development by 30-50%. Furthermore, rottlerin also inhibited virus replication of other pathogenic California serogroup orthobunyaviruses (Jamestown Canyon and Tahyna virus) in neuronal cell lines.

Optimization of Large-Scale Production of Chrysodeixis includens nucleopolyhedrovirus for Its Use as a Biopesticide.

The baculovirus Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) is pathogenic to Chrysodeixis includens (Walker) (Lepidoptera: Noctuidae) larvae, known as soybean looper, which is an important pest of soybean and bean. In this study, some parameters were tested to overcome the difficulties in the in vivo production of ChinNPV aiming to increase its use as a biopesticide. First, different combinations of larval instars (3rd and 4th instars), larval incubation temperatures (23 °C and 26 °C), and rearing densities (individually and 10 larvae/cup) were compared for larval weight and the production of occlusion bodies (OBs). A positive correlation (p< 0.001) was observed for OB production and larval weight. Fourth instar larvae produced more OBs than third instar larvae (p<0.05); however, no significant differences in OBs/larva (p>0.05) were observed for larvae kept in groups or individually. Therefore, a second assay was performed using fourth instar larvae incubated at 26 °C and two larval densities (10 larvae/cup and 40 larvae/cup). The losses of insects and OB production were evaluated as well as the influence of storage temperatures post-mortem (-20 °C, 4 °C, and 15 °C) in the OB yield. As expected, insect losses due to cannibalism or microbial contamination were greater (p<0.05) with the increase in larval density, although no difference was observed in OBs/larva (p>0.05). In addition, the storage temperature post-mortem did not influence the OB yield (p>0.05). The average production of ChinNPV OBs was 3×1010 OBs/40 larvae cup. The results demonstrate the viability of rearing C. includens in groups to enhance the mass production and reduce virus production costs.

Grouper Interferon-Induced Transmembrane Protein 1 Inhibits Iridovirus and Nodavirus Replication by Regulating Virus Entry and Host Lipid Metabolism.

Interferon-induced transmembrane proteins (IFITMs) are novel viral restriction factors which inhibit numerous virus infections by impeding viral entry into target cells. To investigate the roles of IFITMs during fish virus infection, we cloned and characterized an IFITM1 homolog from orange spotted grouper (Epinephelus coioides) (EcIFITM1) in this study. EcIFITM1 encodes a 131-amino-acid polypeptide, which shares 64 and 43% identity with Seriola dumerili and Homo sapiens, respectively. The multiple sequence alignment showed that EcIFITM1 contained five domains, including NTD (aa 1–45), IMD (aa 46–67), CIL (aa 68–93), TMD (aa 94–119), and CTD (aa 120–131). In vitro, the level of EcIFITM1 mRNA expression was significantly up-regulated in response to Singapore grouper iridovirus (SGIV), or red-spotted grouper nervous necrosis virus (RGNNV) infection. EcIFITM1 encoded a cytoplasmic protein, which was partly colocalized with early endosomes, late endosomes, and lysosomes. The ectopic expression of EcIFITM1 significantly inhibited the replication of SGIV or RGNNV, which was demonstrated by the reduced virus production, as well as the levels of viral gene transcription and protein expression. In contrast, knockdown of EcIFITM1 using small interfering RNAs (siRNAs) promoted the replication of both viruses. Notably, EcIFITM1 exerted its antiviral activity in the step of viral entry into the host cells. Furthermore, the results of non-targeted lipometabolomics showed that EcIFITM1 overexpression induced lipid metabolism remodeling in vitro. All of the detected ceramides were significantly increased following EcIFITM1 overexpression, suggesting that EcIFITM1 may suppress SGIV entry by regulating the level of ceramide in the lysosomal system. In addition, EcIFITM1 overexpression positively regulated both interferon-related molecules and ceramide synthesis-related genes. Taken together, our results demonstrated that EcIFITM1 exerted a bi-functional role, including immune regulation and lipid metabolism in response to fish virus infections.

Antiviral activity of oleandrin and a defined extract of Nerium oleander against SARS-CoV-2

With continued expansion of the coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome 2 (SARS-CoV-2), both antiviral drugs as well as effective vaccines are desperately needed to treat patients at high risk of life-threatening disease. Here, we present in vitro evidence for significant inhibition of SARS-CoV-2 by oleandrin and a defined extract of N. oleander (designated as PBI-06150). Using Vero cells, we found that prophylactic (pre-infection) oleandrin (as either the pure compound or as the active principal ingredient in PBI-06150) administration at concentrations as low as 0.05 µg/ml exhibited potent antiviral activity against SARS-CoV-2, with an 800-fold reduction in virus production, and a 0.1 µg/ml concentration resulted in a greater than 3000-fold reduction in infectious virus production. The half maximal effective concentration (EC50) values were 11.98 ng/ml when virus output was measured at 24 h post-infection, and 7.07 ng/ml measured at 48 h post-infection. Therapeutic (post-infection) treatment up to 24 h after SARS-CoV-2 infection of Vero cells also reduced viral titers, with 0.1 µg/ml and 0.05 µg/ml concentrations causing greater than 100-fold reduction as measured at 48 h, and the 0.05 µg/ml concentration resulting in a 78-fold reduction. Concentrations of oleandrin up to 10 µg/ml were well tolerated in Vero cells. We also present in vivo evidence of the safety and efficacy of defined N. oleander extract (PBI-06150), which was administered to golden Syrian hamsters in a preparation containing as high as 130 µg/ml of oleandrin. In comparison to administration of control vehicle, PBI-06150 provided a statistically significant reduction of the viral titer in the nasal turbinates (nasal conchae). The potent prophylactic and therapeutic antiviral activities demonstrated here, together with initial evidence of its safety and efficacy in a relevant hamster model of COVID-19, support the further development of oleandrin and/or defined extracts containing this molecule for the treatment of SARS-CoV-2 and associated COVID-19 disease and potentially also for reduction of virus spread by persons diagnosed early after infection.

Resveratrol Inhibits Venezuelan Equine Encephalitis Virus Infection by Interfering with the AKT/GSK Pathway.

The host proteins Protein Kinase B (AKT) and glycogen synthase kinase-3 (GSK-3) are associated with multiple neurodegenerative disorders. They are also important for the replication of Venezuelan equine encephalitis virus (VEEV), thereby making the AKT/GSK-3 pathway an attractive target for developing anti-VEEV therapeutics. Resveratrol, a natural phytochemical, has been shown to substantially inhibit the AKT pathway. Therefore, we attempted to explore whether it exerts any antiviral activity against VEEV. In this study, we utilized green fluorescent protein (GFP)- and luciferase-encoding recombinant VEEV to determine the cytotoxicity and antiviral efficacy via luciferase reporter assays, flow cytometry, and immunofluorescent assays. Our results indicate that resveratrol treatment is capable of inhibiting VEEV replication, resulting in increased viability of Vero and U87MG cells as well as reduced virion production and viral RNA contents within host cells for at least 48 h with a single treatment. Furthermore, the suppression of apoptotic signaling adaptors, caspase-3, caspase-7, and annexin V may also be implicated in resveratrol-mediated antiviral activity. We found that decreased phosphorylation of the AKT/GSK-3 pathway, mediated by resveratrol, can be triggered during the early stages of VEEV infection, suggesting that resveratrol disrupts the viral replication cycle and consequently promotes cell survival. Finally, molecular docking and dynamics simulation studies revealed that resveratrol can directly bind to VEEV glycoproteins, which may interfere with virus attachment and entry. In conclusion, our results suggest that resveratrol exerts inhibitory activity against VEEV infection and upon further modification could be a useful compound to study in neuroprotective research and veterinary sciences.

Metabolic Perturbation Associated With COVID-19 Disease Severity and SARS-CoV-2 Replication

Viruses hijack host metabolic pathways for their replicative advantage. In this study, using patient-derived multiomics data and in vitro infection assays, we aimed to understand the role of key metabolic pathways that can regulate severe acute respiratory syndrome coronavirus-2 reproduction and their association with disease severity. We used multiomics platforms (targeted and untargeted proteomics and untargeted metabolomics) on patient samples and cell-line models along with immune phenotyping of metabolite transporters in patient blood cells to understand viral-induced metabolic modulations. We also modulated key metabolic pathways that were identified using multiomics data to regulate the viral reproduction in vitro. Coronavirus disease 2019 disease severity was characterized by increased plasma glucose and mannose levels. Immune phenotyping identified altered expression patterns of carbohydrate transporter, glucose transporter 1, in CD8+ T cells, intermediate and nonclassical monocytes, and amino acid transporter, xCT, in classical, intermediate, and nonclassical monocytes. In in vitro lung epithelial cell (Calu-3) infection model, we found that glycolysis and glutaminolysis are essential for virus replication, and blocking these metabolic pathways caused significant reduction in virus production. Taken together, we therefore hypothesized that severe acute respiratory syndrome coronavirus-2 utilizes and rewires pathways governing central carbon metabolism leading to the efflux of toxic metabolites and associated with disease severity. Thus, the host metabolic perturbation could be an attractive strategy to limit the viral replication and disease severity.

Antibody-independent and dependent infection of human myeloid cells with dengue virus is inhibited by carrageenan

This study demonstrated that the λ-carrageenan is a potent and selective inhibitor of the primary infection of human myeloid U937 and K562 cells with the four DENV serotypes, achieving a higher than 99 % reduction in virus production at the highest tested concentration of 20 μg/mL, without affecting cell viability at concentrations up to 1000 μg/mL. Since antibody-dependent enhancement (ADE) is thought to play a main role in the aggravation of severe DENV disease, we also evaluated the activity of carrageenan against ADE of DENV infection. The λ-carrageenan was also effective to block the antibody dependent infection mediated by Fcγ-RII in both cell lines, causing 96–99 % inhibition in virus production from cells infected with immune complexes of DENV-2 and DENV-3. Moreover, the inhibitory effectiveness of carrageenan was similar against prM-mediated ADE or E-mediated ADE. Mechanistic studies indicated that DENV-2 entry is the main antiviral target for carrageenan in DENV or DENV-Ab infected human myeloid cells since a strong inhibitory effect was observed when the carrageenan was present only during adsorption at 4 °C or internalization at 37 °C, whereas the infection was not altered when the compound was added after virus internalization. Thus, our findings have shown that carrageenan may be considered an interesting antiviral agent able to block DENV entry during both primary and antibody-dependent infection of human myeloid cells.

Crocetin Improves Dengue Virus-Induced Liver Injury

Dengue virus (DENV) infection is one of the most widespread mosquito-borne viral infections. Liver injury is commonly observed in severe DENV infection, and the present study aimed to examine the efficacy of crocetin treatment in an immunocompetent mouse model of DENV infection exhibiting liver injury. The efficacy of crocetin treatment in DENV-induced liver injury was assessed via both transaminase levels and histopathology analysis. A real-time polymerase chain reaction array was then used to describe the expression of 84 apoptosis-related genes. Using real-time RT-PCR and Western blot analysis, the gene expressions of host factors were investigated. Additionally, the effect of crocetin in NF-kB signaling during DENV infection was studied. We did not observe any significant reduction in virus production when DENV-infected mice were treated with crocetin. However, DENV-infected mice treated with crocetin showed reduced DENV-induced apoptosis. The real-time polymerase chain reaction array revealed pro-inflammatory cytokine expressions to be significantly reduced in the crocetin-treated DENV-infected mice. We also found that crocetin could effectively modulate antioxidant status in DENV-infected mice. Moreover, crocetin demonstrated the ability to reduce the nuclear translocation of NF-kB in DENV-infected mice. Our results suggest that crocetin treatment does not inhibit DENV replication in the liver of DENV-infected mice; however, we did find that crocetin improves host responses that reduce liver injury.