Vero Cells Research Articles

Isolation of Orientia tsutsugamushi in Culture from Vellore, South India.

Background: Orientia tsutsugamushi, causative agent of scrub typhus is an obligate intracellular parasite. We present information on isolation of this pathogen at a tertiary care centre in Vellore, Southern India. Materials & Methods: PBMCs (peripheral blood mononuclear cells) collected from suspected scrub typhus patients were inoculated into Vero and L929 cell lines and incubated at 37°C with 5% CO2 for 30 days. They were examined for presence of Orientia tsutsugamushi on 10, 15, 20 days post-inoculation and everyday thereafter for a maximum of 30 days post inoculation. The scrapings were subjected to Giemsa staining, IFA, 47kDa qPCR and transmission electron microscopy (TEM). The isolates were passaged 3-4 times to ensure viability and then stored in DMEM with 10% FBS (-80). Genotyping of the isolates was performed by amplifying a 650 bp segment of the TSA 56 (type specific antigen 56) gene. Results: Amongst the 50 samples inoculated, three were culture positive as confirmed by 47 kDa qPCR on 24th day post inoculation. This was further confirmed by Giemsa, IFA staining and TEM. The 650bp amplicons showed 99.5 to 100% homology with Orientia tsutsugamushi MW604716, MH003839, MW604718, MW604717, MH922787 and MH003838 strains. Phylogenetic analysis revealed that 2 isolates belong to TA763 genotype and one belongs to Gilliam genotype. Conclusion: Orientia tsutsugamushi has been isolated for the first time at Vellore, South India from PBMCs. Complete genomic analysis will give more information.

Isolation and characterization of a subtype G2c variant of porcine epidemic diarrhea virus that adapts well to cell culture.

Porcine epidemic diarrhea virus (PEDV) causes the third most important disease in the pig industry, after African swine fever and porcine reproductive and respiratory syndrome, and leads to illness or death of the entire litter, causing significant economic losses. In this study, three PEDV strains (HN-1, HN-2, and SC2023) were isolated from swine farms with suspected PEDV infections in Sichuan and Henan provinces. Phylogenetic analysis based on complete S gene sequences showed that all three strains belonged to the G2c subgroup. HN-1 adapted readily to cell culture, grew to a viral titer as high as 2 × 108 TCID50/mL in Vero cells, and caused the formation of large syncytia. We analyzed the amino acid sequence of the HN-1 isolate and found that its S1 subunit contained a three-amino-acid insertion (355KRL358). A seven-amino-acid-deletion (1377FEKVHVQ1383) in the S2 subunit resulted in the partial deletion of the endocytosis signal YxxΦ and the complete deletion of the endoplasmic reticulum retrieval signal (ERRS) KVHVQ in the cytoplasmic tail of the S protein. Consequently, HN-1 is predicted to be less pathogenic than its parent strain, an attribute that facilitates rapid cell-to-cell spread by enhancing syncytium formation. In addition, strain HN-1 was found to have the mutation 884-885SG→RR, which may favor adaptation to cell culture by providing new trypsin cleavage sites. These results suggest that HN-1 is a G2c subtype variant that adapts well to cell culture and can be used to study the adaptive mechanisms of PEDV and develop attenuated vaccines.

Assessment of genetic diversity, tissue tropism, and antigenic properties of Grimsö betacoronavirus in Swedish bank voles (Clethrionomys glareolus)

Zoonotic coronaviruses can transmit over species barriers and infect humans. To understand the zoonotic potential of a betacoronavirus, Grimsö virus (GRIV), we investigated the geographic distribution and tissue tropism of GRIV in Swedish bank voles (Clethrionomys glareolus), and the antigenicity of the nucleocapsid (N) protein. We screened the lung tissues from animals collected in the southern Sweden by RT-PCR with primers targeting the spike gene. Seven out of 74 animals were found to be positive. They are genetically close to GRIV from Grimsö, central Sweden. Positive rodents were studied for the tissue distribution of GRIV and GRIV RNA was mainly found in the respiratory tract. After three attempts of virus isolation were failed, we successfully established a Vero E6 cell line that stably expressed GRIV N protein, which has no cross-reactivity with patient serum containing antibodies against SARS-CoV-2, or with MERS-CoV. However, a low level of cross-reactivity to common cold coronaviruses was found, likely HCoV-OC43 or HCoV-HKU1, probably due to shared linear epitopes. With the high prevalence and the suggested respiratory transmission route, GRIV may have a high potential for spillover and cross-species transmission, and future serological screening of GRIV infections in domestic animals or humans will be needed.

Cell-based high-content approach for SARS-CoV-2 neutralization identifies unique monoclonal antibodies and PI3K pathway inhibitors.

The sudden rise of the SARS-CoV-2 virus and the delay in the development of effective therapeutics to mitigate it made evident a need for ways to screen for compounds that can block infection and prevent further pathogenesis and spread. Yet, identifying effective drugs efficacious against viral infection and replication with minimal toxicity for the patient can be difficult. Monoclonal antibodies were shown to be effective, yet as the SARS-CoV-2 mutated, these antibodies became ineffective. Small molecule antivirals were identified using pseudovirus constructs to recapitulate infection in non-human cells, such as Vero E6 cells. However, the impact was limited due to poor translation of these compounds in the clinical setting. This is partly due to the lack of similarity of screening platforms to the in vivo physiology of the patient and partly because drugs effective in vitro showed dose-limiting toxicities. In this study, we performed two high-throughput screens in human lung adenocarcinoma cells with authentic SARS-CoV-2 virus to identify both monoclonal antibodies that neutralize the virus and clinically useful kinase inhibitors to block the virus and prioritize minimal host toxicity. Using high-content imaging combined with single-cell and multidimensional analysis, we identified antibodies and kinase inhibitors that reduce virus infection without affecting the host. Our screening technique uncovered novel antibodies and overlooked kinase inhibitors (i.e. PIK3i, mTORi, multiple RTKi) that could be effective against SARS-CoV-2 virus. Further characterization of these molecules will streamline the repurposing of compounds for the treatment of future pandemics and uncover novel mechanisms viruses use to hijack and infect host cells.

Organotin(IV) complexes of tridentate (ONO) hydrazone ligands: synthesis, spectral characterization, antituberculosis, antimicrobial, anti-inflammatory, molecular docking and cytotoxicity studies.

Infectious diseases have a significant impact in the historical trajectory of humanity, exerting profound influence on societies, driving advancements in medical science, and significantly impacting individuals on a worldwide scale. Consequently, this research endeavours to identify potent agents combatting tuberculosis, inflammation, and microbial deformities. The investigation focuses on hydrazones (1,2) endowed eight organotin(IV) complexes, where hydrazones were derived from 2-acetyl-1H-indene-1,3(2H)-dione and 2-phenoxypropanehydrazide/2-(2,4-dichlorophenoxy)propanehydrazide. All compounds underwent thorough characterization utilizing a variety of spectral and analytical techniques including, multinuclear NMR, FT-IR, HRMS, UV-Vis, SEM-EDAX, TGA, XRD, molar conductance measurements, establishing the pentacoordinated environment around tin(IV) ion with tridentate (ONO) mode of chelation of hydrazones. Powder XRD revealed the ligand's crystalline and complexes' semi-crystalline nature, while thermal analysis indicated two-step decomposition leaving tin oxide residue. In vitro evaluations utilize microplate alamar blue assay for assessing anti-tuberculosis activity, serial dilution technique for antimicrobial efficacy, and bovine serum albumin method for evaluating anti-inflammatory properties. The complexes exhibited higher biological activities than their respective ligands and the activity of the complexes follow the order: Ph2SnL1-2 > Bu2SnL1-2 > Et2SnL1-2 > Me2SnL1-2. Among them, phenyl complex (10) [Ph2SnL2] displays superior efficacy against TB dysfunction (MIC: 0.0180 ± 0.009μmol/mL) and also demonstrates exceptional potency in combating inflammation (IC50: 7.27 ± 0.04μM), and microbial (MIC: 0.0045μmol/mL) infections, comparable to standard drugs. Additionally, cytotoxicity testing against vero cell line revealed decreased toxicity at lower concentrations, and attenuated by chelation. Phenyl complex (10) [Ph2SnL2] shows promising cytotoxicity at 3.12µg/mL (19.29 ± 0.09%). Further, The diphenyltin(IV) complex (10), identified as the most effective against TB, shows stronger binding to key 3PTY protein residues (-42.2kJ/mol) compared to ligand (2) (-33.4kJ/mol), correlating with its superior anti-tuberculosis potency in biological assays. This comprehensive approach aims to actively contribute to ongoing initiatives addressing infectious diseases, thereby advancing global health and overall well-being.

3,5-disubstituted pyridines with potent activity against drug-resistant Mycobacterium tuberculosis clinical isolates.

Aim: We designed and synthesized a series of compounds with a 3,5-disubstituted pyridine moiety and evaluated them against Mycobacterium tuberculosis (Mtb) and drug-resistant Mtb clinical isolates.Methodology: A library of 3,5-disubstituted pyridine was synthesized. The compounds were screened for activity against M. tuberculosis H37Rv. The optimal substitutions needed for the activity were identified through structure-activity relationship (SAR) studies.Results: From the screening studies, compounds 24 and 26 were identified as potent members of this series with Minimum Inhibitory Concentration (MIC) of 1.56μg/ml against M. tuberculosis H37Rv. These compounds did not show any inhibition against a panel of ESKAPE pathogens at >50μg/ml indicating their selective killing of M. tuberculosis H37Rv. Importantly, compound 24 showed a selectivity index of 54.64 against CHO-K1 and 78.26 against VERO cell lines, while compound 26 showed a selectivity index of 108.5 against CHO-K1 and 63.2 against VERO cell lines, respectively. Compound 24 formed a stable complex with the target protein DprE1 with predicted binding energy -8.73kcal/mol and inhibited multidrug-resistant clinical isolate of M. tuberculosis at 6.25μg/ml.Conclusion: This study identified the 3,5-disubstituted pyridine derivative 24 with potent antituberculosis activity and can be taken forward to generate new preclinical candidate.

B-215 Evaluating RT-qPCR for Dengue Virus Detection: Outcomes of a Brazilian External Quality Assessment Program

Abstract Background Dengue virus (DENV), responsible for a prevalent arthropod-borne viral illness, poses a substantial public health challenge worldwide. Clinically, DENV's manifestation often resembles that of other arboviruses, resulting in frequent healthcare visits and complicating the early diagnosis, which is critical for patient management. While virus isolation is a viable method, RT-qPCR assays yield significantly quicker results. Rapid immunochromatographic tests are available; yet, RT-qPCR not only matches their sensitivity but also provides distinct advantages in clinical laboratory settings. These include higher throughput and integrated, traceable quality control within each routine, offering less subjective and more dependable results. Maintaining high diagnostic standards for DENV is imperative. External quality assessment programs (EQAP) play a key role in enhancing laboratory diagnostic capabilities by monitoring and assessing diagnostic methods. This study evaluates the diagnostic efficacy of RT-qPCR for DENV using data from an EQAP conducted by a Brazilian provider, accredited by ABNT NBR ISO/IEC 17043:2011. Methods The quality control samples comprised lyophilized suspensions containing viable DENV particles, grown on vero cells (BCRJ 0245/ATCC CCL-81) under BSL-3 conditions. From February 2017 to November 2022, twenty-four EQAP survey rounds were carried out, each encompassing three samples, culminating in 1,153 data points. The evaluation metrics included participant numbers, adequacy percentage, sensitivity, specificity, and the incidence of false positives and negatives. The Mann-Kendall test assessed trends in these metrics over the survey rounds. The RT-qPCR kits employed by participants were classified either as in-vitro diagnostic (IVD) or as laboratory-developed tests (LDT). Kits that demonstrated high performance in terms of adequacy, sensitivity, and specificity were identified and listed. Results The number of laboratories participating has increased. Starting with four in 2017, the program expanded to 28 by 2022. Laboratory accuracy was high during the evaluation period, with a median of 99% (97.8% in 2017 and 99.2% in 2022). The Mann-Kendall test indicated a significant trend of increased accuracy (p<0.001). Specificity remained stable at 98.8%, while sensitivity improved, with a median of 99.4%—rising from 93.3% in 2017 to 100% in 2022 (p<0.001). Only three false negatives were reported over three separate rounds, and six false positives were reported across four rounds. Both LDT and IVD RT-qPCR kits demonstrated high accuracy (99.4% and 98.4%, respectively). Biomanguinhos ZDC, Viasure ZDC, and ZDC Multiplex kits all showed 100% adequacy, sensitivity, and specificity. Conclusions This study highlights the increasing adoption of RT-qPCR for DENV detection, as evidenced by the growing number of laboratories in the EQAP program and the consistently high adequacy rates from 2017 to 2022. Nonetheless, the number of participating laboratories remains relatively small for a vast tropical country like Brazil. The high sensitivity and specificity, along with the infrequency of false results, underscore its effectiveness. The superior performance of certain kits suggests possible advancements in DENV diagnosis, contributing to global public health initiatives against this arboviral disease.

Inhibitory activity of Euterpe oleracea Mart. fruit extract in West Nile virus infection

West Nile virus (WNV) is a neurovirulent arbovirus whose epidemic capacity is enhanced by the wide occurrence of competent vectors and susceptible avian amplifying hosts. In this study, we investigated the antiviral potential of Euterpe oleracea Mart. fruit extract (EoFE) in WNV infection of monkey kidney (Vero) cell cultures. A chromatographic authentication of the extract revealed a typical two-peak fingerprint attributable to the major anthocyanins of the fruit. As assessed by plaque assays in Vero cells, the extract showed a significant concentration-dependent antiviral effect when present throughout the infection procedure, reaching a maximum inhibition of 66.8% at 2 mg/mL without significant cytotoxicity or direct action on virus particles. A time-of-addition assay revealed that this anti-WNV effect was mostly exerted after virus entry, as incubation of Vero cells with EoFE before or during virus addition resulted in a nonsignificant decrease of infection efficiency. These results demonstrated a promising potential of EoFE in inhibiting WNV infection that can be further explored as an antiviral strategy.

Synthesis and evaluation of anticancer activity of new 4,5,6,7-tetrabromo-1H-benzimidazole derivatives

An efficient method for the synthesis of new 4,5,6,7-tetrabromo-1H-benzimidazole derivatives has been developed. New ketones were obtained by N-alkylation of TBBi or 2-Me-TBBi with various phenacyl halides and then reduced to the corresponding alcohols. All compounds were obtained with satisfactory yields in the range of 40–91 %. The synthesized compounds appeared a weak CK2 and PIM-1 inhibitors but exhibit an interesting cytotoxic activity against cancer cell lines, i.e. MCF-7, PC-3, CCRF-CEM, K-562. 1-Phenyl-2-(4,5,6,7-tetrabromo-1H-benzimidzol-1-yl)ethanone 3aA exhibits the highest cytotoxic activity with IC50 value of 5.30 µM for MCF-7 and 6.80 µM for CCRF-CEM. Moreover, this compound shows the highest selectivity against both MCF-7 and CCRF-CEM with SI selectivity coefficients (against MRC-5 and Vero cells) equal 5.45 and 4.30 for MCF-7 and 4.25 and 3.35 for CCRF-CEM, respectively. Furthermore, it was shown that compound 3aA exhibits very good pro-apoptotic properties, through induction of the mitochondrial apoptotic pathway in CCRF-CEM cells. These results correlate with data showing the effect of 3aA on intracellular level of CK2α protein and CK2-mediated phosphorylation of Ser529 in NF-κBp65. Study of the effect of compound 3aA on mRNA levels of CK2α and CK2α’ showed no significant differences in gene expression levels in control CCRF-CEM and cells treated with 3aA, indicating 3aA action at the protein level.

Lithospermic acid inhibits dengue virus infection through binding with envelope proteins

The dengue virus has emerged as a global pandemic, highlighting the urgent need for the immediate development of antiviral therapeutics. Lithospermum erythrorhizon, a medicinal plant commonly used in China for various ailments including viral infections, inflammation, rheumatism, and cancer, showed promising antiviral properties in our research. Specifically, both the ethanol extract of Lithospermum erythrorhizon and its active component, lithospermic acid, demonstrated significant inhibitory effects on Dengue virus (DENV) replication in Vero cells, with EC50 values of 6.50 μg/mL(95% CI: 2.25 to 18.98)and 15.00 μM(95% CI: 12.13 to 18.07), respectively. Notably, lithospermic acid exhibited potent antiviral activity across multiple cell lines against DENV, impeding virus replication and specifically impeding the expression of viral E and NS3 proteins during the early stages of DENV infection. Experimental assays involving RNase digestion and sucrose density gradient analysis confirmed that lithospermic acid did not directly inactivate DENV but rather interfered with viral processes. Furthermore, the compound was found to bind to the E protein of DENV, effectively inhibiting viral infection and mitigating the cytopathic effects induced by DENV. Collectively, these findings underscore the potential of lithospermic acid as a promising candidate for the development of therapeutic strategies targeting DENV infection.

Investigating the in vitro antibacterial, antibiofilm, antioxidant, anticancer and antiviral activities of zinc oxide nanoparticles biofabricated from Cassia javanica.

It is thought to be risk-free, environmentally benign, and safe for biological processes to produce zinc oxide nanoparticles from renewable resources. This study examined Cassia javanica's ability to create ZnONPs. The generated ZnONPs were analyzed using a variety of techniques, such as TEM, FTIR spectroscopy, UV-Vis spectroscopy, and XRD analysis. The antibacterial potential of ZnONPs has been investigated using both Agar well diffusion and microtitreplate (MTP) methods. One method used to evaluate ZnONPs' capacity to scavenge free radicals at different concentrations was the DPPH method. The permanent zinc oxide (ZnO) shape and the naturally occurring crystal structure of ZnONPs were validated by the XRD data. ZnONPs showed antibacterial activity with MICs of 31.7 μg/mL toward Bacillus subtilis, 62.5 μg/mL for Salmonella typhimurium, Escherichia coli while Clostridium sporogenes and Bacillus pumilus was 125μg/mL. Furthermore, ZnONPs demonstrated a range of antibiofilm activities toward Staphylococcus aureus (MRSA). ZnONPs showed an intriguing antioxidant capacity, achieving IC50 of 109.3 μg/ml μg/mL. Additionally, ZnONPs demonstrated low toxic effect on Vero cell with IC50 154.01 μg/mL as well as possible anticancer action when applied to the carcinoma cell lines HepG2 with IC50 of 47.48 μg/mL. Furthermore, ZnONPs at 62.5 μg/mL had a promising antiviral impact against HSV1 and COX B4, with antiviral activities of 75.4% and 65.8%, respectively.

Benzyl/phenyl‐1,2,3‐Triazole Tethered 3‐Acetyl Coumarins as Potential Drug‐Resistant Antitubercular Agents: Synthesis, Biology, and in Silico Investigations as Mtb DNA Gyrase Inhibitors

AbstractOwing to the emergence of multi‐drug resistant tuberculosis, there is a need for the exploration of new antitubercular agents. In this context, new coumarin‐based 1,2,3‐triazole hybrids were developed and evaluated for their antimicrobial activity against ESKAPE pathogens and the Mtb H37Rv strain. Among them, compounds 9 c and 12 showed MICs of 1 and 2 μg/mL, respectively, against the Mtb strain. The lead compounds exhibited a good selectivity index against Vero cells and were equally effective against ETB‐resistant and RIF‐resistant Mtb strains. Time‐kill kinetic studies revealed the bacteriostatic properties of the lead compounds, while combination studies using FDA‐approved antibiotics showed no drug interactions. Based on the structural similarity, it was envisaged that they might inhibit the DNA gyrase, which was further proved by the DNA supercoiling inhibition assay. Additionally, in silico docking studies, binding energy calculations, and ADME/T studies for the synthesized conjugates showed favourable pharmacokinetic and physicochemical characteristics. Hence, these molecules could further pave the way for discovering new potent antitubercular agents to combat AMR.

The antiviral effect and potential mechanism of Houttuynia cordata thunb. (HC) against coxsackievirus A4

Ethnopharmacological relevanceHand, foot, and mouth disease (HFMD) is mainly caused by various of enteroviruses such as enterovirus 71 (EVA71), coxsackievirus A16 (CVA16), CVA6, and CVA10 in infants and children under 5 years old. During the past 5 years, CVA4 has become the dominant pathogen resulting in HFMD in China. However, there are no effective vaccines and antiviral drugs available. Houttuynia cordata Thunb (HC). is a Chinese herbal medicine eaten as vegetables for treating viral infection diseases, but whether HC has anti-CVA4 effect remains unclear. Aim of the studyIn this study, we want to investigate the antiviral activity of HC against CVA4 in vitro and in vivo and elucidate the potential mechanism of HC against CVA4. Materials and methodsMTT assay were used to evaluate the cytotoxicity of HC. Virus titers assay, CPE assay, violet staining and immunofluorescence were used to investigate the antiviral effect of HC against CVA4. A 13-day-old suckling mice model was established to evaluate the therapeutic efficacy of HC against CVA4 infection. Western blot, qRT-PCR and time-of-drug addition assay were performed to elucidate the potential mechanism of HC against CVA4 infection. ResultsMTT assay indicated the cytotoxicity concentration of HC on Vero cells and RD cells were more than 1 mg/ml, suggesting that the low cytotoxicity of HC. In vitro antiviral assay revealed that HC could dose-dependently prevent the CPE, suppress the release of newborn virus, and inhibit the replication of CVA4 by decreasing viral RNA transcription and protein expression with IC50 of 88.96 μg/mL. A time-of-addition assay showed that HC mainly exerted anti-CVA4 effect by inhibiting virus replication at the post-entry stage. In vivo results further demonstrated that HC could effectively prevent the lethal infection of CVA4 by promoting survival, improving clinical symptoms, prolonging the survival time, inhibiting excessive inflammatory responses, and reducing pathological injury in vivo. Mechanistic studies revealed inhibition of p38 MAPK and JNK pathway over-activation may be the primary mechanism of HC against CVA4 infection. ConclusionIn summary, our results for the first time demonstrated that HC not only effectively inhibited CVA4 replication, but also partially protected the lethal infection of CVA4 in vivo. Furthermore, pharmacological mechanism studies revealed that the primary mechanism of HC against CVA4 infection may be associated with its effect of inhibiting over-activation of p38 MAPK and JNK signaling pathways caused by enteroviruses. Our finding indicated that HC might be a potential innovative medicine for treating HFMD.

RpIFN-λ1 alleviates the clinical symptoms of porcine epidemic diarrhea

Porcine epidemic diarrhea (PED), caused by the porcine epidemic diarrhea virus (PEDV), primarily affects the jejunum and ileum of pigs. Interferons, glycoproteins with high species specificity and potent antiviral activity, are crucial in defending against viral infections. Unlike other interferons, interferon-lambda (IFN-λ) mainly acts on mucosal epithelial cells and exhibits robust antiviral activity at mucosal surfaces. However, the high cost limits the use of naturally extracted interferons in farming. In this study, we expressed recombinant porcine interferon-lambda 1 (rpIFN-λ1) in eukaryotic cells, demonstrating effective antiviral activity against PEDV in Vero E6 and IPI-FX cells. In vivo, rpIFN-λ1 alleviated clinical symptoms and intestinal damage, enhanced antioxidant capacity, reduced inflammation, and significantly improved the survival rate of piglets following PEDV infection. Both in vitro and in vivo studies confirmed that rpIFN-λ1 upregulated interferon-stimulated genes (ISGs) via the JAK-STAT pathway, thereby exerting antiviral effects. In conclusion, rpIFN-λ1 significantly inhibited PEDV replication and alleviated clinical symptoms. The selectivity of rpIFN-λ1 for intestinal cells and its ability to reduce viral shedding suggest that this agent is a promising antiviral for enteric viruses such as PEDV. Our findings highlight rpIFN-λ1 as a cost-effective, efficient, and novel strategy for antiviral treatment of PEDV.

Identification of nitrile-containing isoquinoline-related natural product derivatives as coronavirus entry inhibitors in silico and in vitro

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of infections and deaths worldwide since its emergence in Wuhan, China, in late 2019. Natural product inhibitors targeting the interaction between the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and human angiotensin-converting enzyme 2 (ACE2), crucial for viral attachment and cellular entry, are of significant interest as potential antiviral agents. In this study a library of nitrile- and sulfur-containing natural product derived compounds were used for virtual drug screening against the RBD of the SARS-CoV-2 spike protein. The top 18 compounds from docking were tested for their efficacy to inhibit virus entry. In vitro experiments revealed that compounds 9, 14, and 15 inhibited SARS-CoV-2 pseudovirus and live virus entry in HEK-ACE2 and Vero E6 host cells at low micromolar IC50 values. Cell viability assays showed these compounds exerted low cytotoxicity towards MRC5, Vero E6, and HEK-ACE2 cell lines. Microscale thermophoresis revealed all three compounds strongly bound to the RBDs of SARS-CoV-2, SARS-CoV-2 XBB, SARS-CoV-1, MERS-CoV, and HCoV-HKU1, with their Kd values increasing as RBD sequence similarity decreased. Molecular docking studies indicated compounds 9, 14, and 15 bound to the SARS-CoV-2 spike protein RBD and interacted with hotspot amino acid residues required for the RBD-ACE2 interaction and cellular infection. These three nitrile-containing candidates, particularly compound 15, should be considered for further development as potential pan-coronavirus entry inhibitors.

In vitro cytotoxicity evaluation of green synthesized alumina nanoscales on different mammalian cell lines

Nanoscale research is gaining interest in the biomedical, engineering, and environmental fields. Current expensive traditional chemical methods for synthesizing nanoparticles (NPs) inevitably lead to the synthesis of NPs with potentially less or no toxic effects on living cells. To overcome these challenges, in this study, we use a simple, inexpensive, and less toxic one-pot green chemistry approach instead of a chemical method to synthesize alumina nanoparticles (AlNPs) from Carica papaya extract. Nano-alumina has been widely studied due to its remarkable biological and physiochemical properties at nanoscale. However, to date, its biomedical application is limited due to the lack of sufficient data on cytotoxicity in living cells. The physicochemical properties of nano-alumina were determined by FT-IR, DLS, SEM and HRTEM. The cytotoxic effects of the synthesized nano-alumina were studied in cell lines LT and VERO at concentrations of 10–480 µg/mL in vitro. The cell viability of nano-alumina was evaluated using the MTT assay and the AO /EB double staining technique. Our results based on DLS and HRTEM analyzes confirmed spherical AlNPs with a zeta potential and average particle size of − 25 to 5 mV and 52 nm, respectively. The nano-alumina tested showed low toxicity to both cell lines after 28- and 48-h exposure. Furthermore, cell viability statistically decreased with increasing incubation time and concentration of AlNPs up to 480 μg/mL (p < 0.001). However, a minimal increase in cytotoxicity was observed at threshold levels in the range of 120–480 µg/mL. The half-maximal inhibitory concentration (IC50) of AlNPs in the VERO and LT cell lines were 153.3, 252.0 µg/mL and 186.6, 395.3 µg/mL, respectively, after 24- and 48-h exposure to AlNPs. Thus, we conclude that the cytotoxic effect of AlNPs depends on the concentration, exposure time and cell type. The result suggests that the concentration used in this study may be useful for biomedical applications.

Polypurine Reverse Hoogsteen hairpins as a therapeutic tool for SARS-CoV-2 infection

Although COVID-19 pandemic was declared no longer a global emergency by the World Health Organization in May 2023, SARS-CoV-2 is still infecting people across the world. Many therapeutic oligonucleotides such as ASOs, siRNAs or CRISPR-based systems emerged as promising antiviral strategies for the treatment of SARS-CoV-2. In this work we explored the inhibitory potential on SARS-CoV-2 replication of Polypurine Reverse Hoogsteen Hairpins (PPRHs), CC1-PPRH and CC3-PPRH, targeting specific polypyrimidine sequences within the replicase and Spike regions, respectively, and previously validated for COVID-19 diagnosis. Both PPRHs bound to their target sequences in the viral genome with high affinity in the order of nM. In vitro, both PPRHs reduced viral replication by more than 92% when transfected into VERO-E6 cells 24 hours prior infection with SARS-CoV-2. In vivo intranasal administration of CC1-PPRH in K18-hACE2 mice expressing the human ACE receptor protected all the animals from SARS-CoV-2 infection. The properties of PPRHs position them as promising candidates for the development of novel therapeutics against SARS-CoV-2 and other viral infections.

New Acetophenone Scaffolds: Synthesis, Antifungal Activities, Biocompatibility, Molecular Docking, ADMET Analysis, and Dynamic Simulations

In this work, the chemical reactivity of 2-cyano-N'-(1-phenylethylidene)acetohydrazide (3) towards different aldehydes is utilized to synthesize binary and fused heterocyclic compounds, including arylidenes 6, 7, 12, 13, 14, 18a-c, 19a,b, 20, chromone 23, and chromene derivatives 24, 26a-c, 29, and 30. The capability of newly synthesized compounds to inhibit fungal growth of Aspergillus niger ATCC 16404, A. flavus ATCC 9643, Penicillium chrysogenum ATCC 10106, and Rhizopus oryazae ATCC 96382 are assessed. It is observed that compound 29 had strong antifungal activity against all fungi with MFIC values varying from 250 to 1000 μg/ml, while compound 18a demonstrated potent antifungal effect against A. niger and A. flavus. In addition, it is found that compound 29 was cytotoxic to Vero cells at doses of 1000-500 μg/ml; however, no discernible variation was observed between the concentrations of 250-31.25 μg/ml. Moreover, the interactions between the promising compounds 18a, 19b, and 29 and antifungal target proteins are assessed using molecular docking. The results of the docking simulations demonstrated that these compounds demonstrated optimal binding energies and effectively engaged with the active sites of FDC1 protein in A. niger, UDP-N-acetylglucosamine in A. fumigatus, Adenosine 5′-phosphosulfate kinase in P. chrysogenum, and protease in Rhizopus oryazae. These interactions involved various types of molecular interactions, indicating that these compounds have the ability to impede enzymes and exhibit strong antimicrobial effects. Furthermore, the in-silico ADMET profiles of compounds 18a, 19b, and 29 reveal that they adhere to the Lipinski rules, suggesting favorable physicochemical properties. Also, MD simulations revealed stable complexes of 29 with anti-fungal receptors including fdc1, UDP-N-acetylglucosamine, APS kinase, and protease with RMSD (0.1-0.8, 0.19-0.25, 0.20-0.30, and 0.20-0.35 nm), RMSF (0.1-0.8 nm), SASA (140-155, 130-140, 135-145, and 120-130 nm2), and Rg (1.90-2.00, 1.80-1.90, 2.40-2.50, and 2.10-2.20 nm) respectively. Our results provide potential and promising compounds for fungal infection diseases.

Enhanced Expression of the L1R Gene of Vaccinia Virus by the tPA Signal Sequence Inserted in a Fowlpox-Based Recombinant Vaccine.

The use of Vaccinia virus (VACV) as a preventive vaccine against variola, the etiological agent of smallpox, led to the eradication of smallpox as a human disease. The L1 protein, a myristylated transmembrane protein present on the surface of mature virions, plays a significant role in infection and morphogenesis, is well-conserved in all orthopoxviruses, and is the target of neutralizing antibodies. DNA recombinant vaccines expressing this protein were successfully used, but they showed lower efficacy in non-human and human primates when used alone, and viral-vectored fowlpox vaccines were already proved to increase immunogenicity when used as a boost. Here, we constructed a novel fowlpox-based recombinant (FPtPA-L1R), in which the tissue plasminogen activator signal sequence was linked to the 5' end of the L1R gene to drive the L1 protein into the cellular secretion pathway. FPtPA-L1R expresses a functional heterologous protein that can be immunoprecipitated by hyperimmune rabbit serum. The protein shows cytoplasmic and membrane subcellular localizations and long-lasting expression in CEF, non-human primate Vero and human MRC-5 cells. The tissue plasminogen activator signal sequence can thus contribute significantly to the expression of the L1 protein and may enhance the immunogenicity of a putative DNA/FP prime-boost vaccine.

The epidemiology and phylogenetic trends of Omicron subvariants from BA.5 to XBB.1 in Taiwan

BackgroundOmicron, a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant, entered Taiwan at the end of 2021. The Taiwanese government ended its "zero-COVID" policy in March 2022. Multiple coronavirus disease 2019 (COVID-19) outbreaks began in April 2022. We monitored the replacement of Omicron subvariants after BA.1/BA.2 and analyzed their correlation with COVID-19 outbreaks. MethodsWe collected SARS-CoV-2 real-time qRTPCR-positive nasopharyngeal swabs from Kaohsiung Medical University Hospital (KMUH), Kaohsiung City, Taiwan, and performed sequencing for specimens exhibiting a cytopathic effect in Vero E6 cells to determine their clades and lineages. We analyzed the medical records of COVID-19 patients and identified hospitalization risk factor(s). We retrieved SARS-CoV-2 sequences identified in Taiwan from GISAID and analyzed their correlation with COVID-19 data from the Taiwan Centers for Disease Control. ResultsWe analyzed the phylogenesis of KMUH-47 to KMUH-104 (SARS-CoV-2 isolates identified herein) and all of the Omicron subvariants from BA.5 to XBB.1 (n = 1930). Age and comorbidities were hospitalization risk factors. Men generally exhibited a greater fatality rate than women. COVID-19-related deaths predominantly occurred in individuals over 70 years old. The COVID-19-related case fatality rate increased as nucleotide (NT) and amino acid (AA) substitutions increased. The number of COVID-19-related cases and deaths progressively decreased with each outbreak between August 2022 and October 2023. ConclusionHospitalization was associated with age and the presence of comorbidities. COVID-19-related fatality was linked to sex, age, and the accumulation of NT and AA substitutions in emerging Omicron subvariants.