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1.
Asian Pac J Cancer Prev ; 23(9): 3113-3123, 2022 Sep 01.
Article in English | MEDLINE | ID: covidwho-2057002

ABSTRACT

BACKGROUND: This study was carried out to synthesize a new complex of Fe(II) with isoleucine dithiocarbamate ligand and to determine its potential as an anticancer and antiviral agent for SARSCOV-2. METHODS: The synthesized complexes were then characterized by UV-vis and FT-IR spectroscopy and their melting points. The value of the conductivity of the complex compound is also determined. Anti-cancer activity was tested in vitro and molecular docking. Its potential as an antiviral against SARSCOV-2 was also carried out by molecular docking. Pharmacokinetics/ADMET properties were also carried out on the complex. RESULT: Spectral results showed the successful synthesis of Fe(II) isoleucine dithiocarbamate complex. The complex produced UV-vis spectra at 268 and 575 nm, and the IR data at 399-599 cm-1 showed the coordination between the Fe(II) atoms with sulphur, nitrogen and oxygen of the isoleucine dithiocarbamate ligand. Fe(II) isoleucine dithiocarbamate had a cytotoxicity effect on the MCF-7 cell line (IC50 =613 µg/mL). The complex significantly caused morphological changes in the breast cancer cell line, finally leading to cell apoptosis. CONCLUSION: Cytotoxic test of Fe(II) isoleucine dithiocarbamate showed moderate anticancer activity on MCF-7 cancer cells and showed antiviral activity against SARSCOV-2 by interfering with spike glycoprotein -ACE2 receptors, and inhibiting major proteases and 3Clpro.


Subject(s)
Antineoplastic Agents , COVID-19 , Coordination Complexes , Angiotensin-Converting Enzyme 2 , Antineoplastic Agents/chemistry , Antiviral Agents/pharmacology , COVID-19/drug therapy , Coordination Complexes/pharmacology , Ferrous Compounds , Humans , Isoleucine , Ligands , Molecular Docking Simulation , Nitrogen , Oxygen , Spectroscopy, Fourier Transform Infrared , Sulfur
2.
Journal of Tropical Medicine ; 21(9):1119-1124, 2021.
Article in Chinese | CAB Abstracts | ID: covidwho-2047145

ABSTRACT

Objective: To investigate the effects of miR-221-3p on the proliferation and apoptosis of vascular smooth muscle cells (VSMC) in abdominal aortic aneurysm (AAA) by targeting tissue inhibitor of metalloproteinase- 2 (TIMP-2).

3.
Zhongguo Bingyuan Shengwuxue Zazhi / Journal of Pathogen Biology ; 15(9):997-1004, 2020.
Article in Chinese | CAB Abstracts | ID: covidwho-2040442

ABSTRACT

Objective: To investigate the molecular mechanism of the action by which the MERS-CoV E proxein induces autophagy in 293T cells.

4.
Journal of South China Agricultural University ; 41(5):27-35, 2020.
Article in Chinese | CAB Abstracts | ID: covidwho-2040361

ABSTRACT

Objective: To prepare monoclonal antibodies against porcine epidemic diarrhea virus (PEDV) N protein, and develop an indirect immuno-fluorescence assay method used for detecting PEDV. Method: The expressed recombinantly PEDV N protein was used as an immunogen and 8-week-old female BALB/c mice were immunized. Then their spleen cells with high antibody titer were isolated and fused with SP2/0 cells. The hybridoma cell lines secreting monoclonal antibodies against PEDV N protein were screened. In Vero cells infected with PEDV, monoclonal antibody of anti-PEDV N protein was used as the primary antibody and FITC-goat-anti-mouse IgG was used as the secondary antibody to develop indirect immuno-fluorescence assay method used for detecting PEDV. Result: The prepared hybridoma cell lines could stably secrete anti-PEDV N protein antibodies, ELISA antibody titer in cell supernatant was above 1:3 200, and in mouse ascites above 1:1 000 000. While monoclonal antibodies were applied in established indirect immuno-fluorescence assay, the optimal conditions were that cells were fixed with 80% () acetone at -20 degrees C for 30 min;The primary antibody was diluted 1 000 times by PBS buffer solution and incubated at 4 degrees C overnight;The secondary antibody was diluted 100 times by PBS buffer solution and incubated at 37 degrees C for 1 h. Transmissible gastroenteritis virus (TGEV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine reproductive virus (PRV), porcine enteric a corone virus (PEAV), porcine rotavirus (PoRV) and PEDV were detected by established indirect immuno-fluorescence assay method, only PEDV showed positive, all the else viruses showed negative.

5.
Acta Veterinaria et Zootechnica Sinica ; 53(6):2024-2028, 2022.
Article in Chinese | CAB Abstracts | ID: covidwho-2025545

ABSTRACT

This study aimed to analyze the proliferation characteristics of porcine deltacoronavirus (PDCoV) in suspension cultured porcine kidney cells LLC-PK1, so as to provide Candidate cell for large-scale production of PDCoV inactivated vaccine. LLC-PK1 cells were suspended by gradually decreasing serum method. PDCoV adaptive monoclonal cell lines were screened by limited dilution method. Indirect immunofluorescence method was used to identify the infectivity of PDCoV. The initial cell density, MOI, time of receiving virus collection and TPCK pancreatin concentration were screened to determine the best suspension culture conditions. The suspension cell strain LLC-PK1Sa which can proliferate PDCoV efficiently was screened out;PDCoV can specifically infect LLC-PK1 cells;PDCoV inoculated LLC-PK1Sa cells with a density of 2 x 106 cells.mL-1 according to the MOI of 10-3, When the final concentration of TPCK pancreatin reached 7.5 g.mL-1, the titer of virus solution harvested 48 h after inoculation was the highest. In this study, the efficient proliferation of PDCoV in LLC-PK1Sa suspension cells was realized for the first time, and the suspension culture conditions were preliminarily optimized, which could provide theoretical reference for large-scale production of PDCoV inactivated vaccine.

6.
Life Sci ; 308: 120930, 2022 Nov 01.
Article in English | MEDLINE | ID: covidwho-2007929

ABSTRACT

AIMS: This study evaluated SARS-CoV-2 replication in human cell lines derived from various tissues and investigated molecular mechanisms related to viral infection susceptibility and replication. MAIN METHODS: SARS-CoV-2 replication in BEAS-2B and A549 (respiratory tract), HEK-293 T (kidney), HuH7 (liver), SH-SY5Y (brain), MCF7 (breast), Huvec (endothelial) and Caco-2 (intestine) was evaluated by RT-qPCR. Concomitantly, expression levels of ACE2 (Angiotensin Converting Enzyme) and TMPRSS2 were assessed through RT-qPCR and western blot. Proteins related to autophagy and mitochondrial metabolism were monitored in uninfected cells to characterize the cellular metabolism of each cell line. The effect of ACE2 overexpression on viral replication in pulmonary cells was also investigated. KEY FINDINGS: Our data show that HuH7, Caco-2 and MCF7 presented a higher viral load compared to the other cell lines. The increased susceptibility to SARS-CoV-2 infection seems to be associated not only with the differential levels of proteins intrinsically related to energetic metabolism, such as ATP synthase, citrate synthase, COX and NDUFS2 but also with the considerably higher TMPRSS2 mRNA expression. The two least susceptible cell types, BEAS-2B and A549, showed drastically increased SARS-CoV-2 replication capacity when ACE2 was overexpressed. These modified cell lines are relevant for studying SARS-CoV-2 replication in vitro. SIGNIFICANCE: Our data not only reinforce that TMPRSS2 expression and cellular energy metabolism are important molecular mechanisms for SARS-CoV-2 infection and replication, but also indicate that HuH7, MCF7 and Caco-2 are suitable models for mechanistic studies of COVID-19. Moreover, pulmonary cells overexpressing ACE2 can be used to understand mechanisms associated with SARS-CoV-2 replication.


Subject(s)
COVID-19 , Neuroblastoma , Adenosine Triphosphate , Angiotensin-Converting Enzyme 2/genetics , Autophagy , Caco-2 Cells , Citrate (si)-Synthase , HEK293 Cells , Humans , Peptidyl-Dipeptidase A/metabolism , RNA, Messenger/genetics , SARS-CoV-2
7.
Genetics & Applications ; 5(2):1-9, 2021.
Article in English | CAB Abstracts | ID: covidwho-1994923

ABSTRACT

The genus Artemisia (fam. Asteraceae) is one of the largest and widely distributed with around 500 species, majority used as aromatic and medicinal plants. Artemisia annua L. is widely used as a dietary spice, herbal tea, as a supplement, and in a non-pharmaceutical form for treatment of malaria and fever. It is orally consumed as capsules, extracts and tinctures and topically applied as an essential oil diluted in lotions and ointments. Artemisinin is the main constituent of Artemisia annua L. extracts. Since the discovery that the artemisinin is efficient in malaria treatment, there is also a growth in consumption of A. annua extracts for antitumour and even recently for antiviral treatments against SARS-CoV-2 infections. This study aimed to investigate genotoxic effect in peripheral blood culture and cytotoxic effects in cancer and normal cell lines, of commercially available A. annua L. tincture in series of dilutions. Both comet and neutral red uptake assays revealed dose-dependent genotoxicity and cytotoxicity of A. annua tincture dilutions. Comet assay revealed significantly increased DNA damage in peripheral blood cells while neutral-red assays showed increase in cytotoxicity (p<0.001) in both normal and cancer cell cultures treated with the lowest extract dilution compared to the highest one applied. Obtained results indicate caution needed in A. annua L. tincture use, especially when poorly diluted.

8.
Chinese Veterinary Science / Zhongguo Shouyi Kexue ; 50(5):603-609, 2020.
Article in Chinese | CAB Abstracts | ID: covidwho-1994656

ABSTRACT

To establish a J2-KD (knockdown) cell line stably expressing interfered IFITM1 and study the effect of interference with IFITMI gene on the infection of PCV2, PRV and TGEV. Gene cloning tech- niques were used to constructed pLKO. l-EGFP-Puro-IFITMI recombinant vector, which was co-transfected into 293 FT cells with lentiviral packaging plasmids psPAXZ and pMDZ. G to produce green fluorescent protein labeled lentiviruses expression IFITMlshRNA, the viral supernatant was collected at 48 hours after post transfection. J2 cells were infected with the harvested lentiviruses, screened by puromycin and cloned via cell limited dilution. Real-time PCR identify that the cell lines with stable interference with IFITMl gene were obtained, and via MTT method verify that interference with IFITMI expression had no effect on the growth of J2 cells, the successfully constructed J2 stable cell line interfere with IFITMl expression was named as JZ-KD. PRV, PCV2 and TGEV infected J2-KD cells, respectively. Using real-time fluorescence quantitative PCR detect virus replication. The results showed that J2-KD cell line was successfully generated with interfered IFITMl expression;the copy number of PCV2 and TGEV were in- creased, while PRV was decreased in J'Z-KD cell. Indicating that the interference of IFITMI gene expression markedly inhibited the replication of PRV while promoted that. of TGEV and PCV2, providing a basis for further study on the function of porcine IFITMI protein and elucidates its antiviral mechanism.

9.
Chinese Veterinary Science / Zhongguo Shouyi Kexue ; 50(5):537-544, 2020.
Article in Chinese | CAB Abstracts | ID: covidwho-1994651

ABSTRACT

Long noncoding RNA (lncRNA) is a type of non-coding RNA molecule longer than 200 nt, which plays vital roles in biological events. Our previous results demonstrated that the host's lncRNA expression profile was significantly changed after porcine epidemic diarrhea virus (PEDV) infection. In this study, one of the lncRNAs, lncRNA9606, was selected to investigate its impact on PEDV replication. First, the kinetics of lncRNA9606 expression in IPEC-J2 cells were examined at different time points after PEDV infection. The results confirmed that PEDV infection significantly upregulated the expression of lncRNA9606. The lncRNA9606 expression levels in different cells or tissues were evaluated and the results showed that the amount of lncRNA9606 in Peyer's patches and peripheral blood mononuclear cells were significantly higher than that in small intestinal epithelial cell lines. It was mainly localized in the nucleus. Further investigations indicated that over expression of lncRNA in LLC-PK1 cells significantly inhibited PEDV replication. In conclusion, lncRNA9606 can suppress the PEDV replication in LLC-PK1 cells.

10.
Acta Veterinaria et Zootechnica Sinica ; 53(5):1587-1597, 2022.
Article in Chinese | CAB Abstracts | ID: covidwho-1994513

ABSTRACT

HEK293 cells were used as the cell model to investigate the role of human aminopeptidase N (hAPN) in the invasion of porcine deltacoronavirus (PDCoV) into human cells. The proliferation of PDCoV on HEK293 cells was firstly identified by RT-qPCR/RT-PCR. And then, hAPN knockout cell line was constructed by CRISPR/Cas9 technology and cell viability of HEK293 hAPN knockout and wild-type cells was verified by CCK-8 assay. Effect of hAPN knockout and overexpression on PDCoV replication was detected by RT-qPCR and Western blot. Meanwhile, interaction of PDCoV S protein and hAPN protein was analyzed by homology modeling and molecular docking. Results showed that PDCoV virus copies rapidly increased at 12-36 h and reached peak level at 36 h, it could propagate at least for passage 2 on HEK293 cells. There was no significant difference in cell viability between hAPN knockout cells and wild-type cells. Knockout of hAPN inhibit PDCoV replication and overexpression of hAPN enhance PDCoV replication. Homology modeling and molecular docking analysis showed S1 protein could bind hAPN domain II. Residues TYR92, THR51, THR48, PHE16 and MET14of S1 protein receptor binding motif 1 (RBM1) can form hydrogen bonds with residues PHE490, GLN531, ARG528 and SER529 of hAPN. This study indicates that hAPN plays a critical role in HEK293 cells during PDCoV infection, which provides new theoretical evidence for further studies on the mechanism of PDCoV entry into host cells and cross-species transmission.

11.
Chinese Journal of Virology ; 36(2):155-159, 2020.
Article in Chinese | CAB Abstracts | ID: covidwho-1975405

ABSTRACT

In January 2020, Guangdong Province, China imported several suspected cases with SARS-CoV-2 from Wuhan City, Hubei Province. China, which were detected as SARS-CoV-2 positive in laboratory. To further understand the SARS-CoV-2 virulence, as well as drug development and epidemic prevention and control needs, we established a SARS-CoV-2 isolation procedure. Vero-E6 cells were infected with the positive bronchoalveolar-lavage sample. The cells were monitored daily for cytopathic effects using light microscopy. The presence of viral nucleic acid in the supernatant was detected by RT-PCR. RNA extracted from culture supernatants were used as a template to clone and sequence the genome. We used Illumina sequencing to characterize the virus genome and results showed that the isolated virus was SARS-CoV-2.

12.
Indian J Med Res ; 155(1): 129-135, 2022 01.
Article in English | MEDLINE | ID: covidwho-1924409

ABSTRACT

Background & objectives: Polio, measles, rubella, influenza and rotavirus surveillance programmes are of great public health importance globally. Virus isolation using cell culture is an integral part of such programmes. Possibility of unintended isolation of SARS-CoV-2 from clinical specimens processed in biosafety level-2 (BSL-2) laboratories during the above-mentioned surveillance programmes, cannot be ruled out. The present study was conducted to assess the susceptibility of different cell lines to SARS-CoV-2 used in these programmes. Methods: Replication of SARS-CoV-2 was studied in RD and L20B, Vero/hSLAM, MA-104 and Madin-Darby Canine Kidney (MDCK) cell lines, used for the isolation of polio, measles, rubella, rotavirus and influenza viruses, respectively. SARS-CoV-2 at 0.01 multiplicity of infection was inoculated and the viral growth was assessed by observation of cytopathic effects followed by real-time reverse transcription-polymerase chain reaction (qRT-PCR). Vero CCL-81 cell line was used as a positive control. Results: SARS-CoV-2 replicated in Vero/hSLAM, and MA-104 cells, whereas it did not replicate in L20B, RD and MDCK cells. Vero/hSLAM, and Vero CCL-81 showed rounding, degeneration and detachment of cells; MA-104 cells also showed syncytia formation. In qRT-PCR, Vero/hSLAM and MA-104 showed 106 and Vero CCL-81 showed 107 viral RNA copies per µl. The 50 per cent tissue culture infectious dose titres of Vero/hSLAM, MA-104 and Vero CCL-81 were 105.54, 105.29 and 106.45/ml, respectively. Interpretation & conclusions: Replication of SARS-CoV-2 in Vero/hSLAM and MA-104 underscores the possibility of its unintended isolation during surveillance procedures aiming to isolate measles, rubella and rotavirus. This could result in accidental exposure to high titres of SARS-CoV-2, which can result in laboratory acquired infections and community risk, highlighting the need for revisiting biosafety measures in public health laboratories.


Subject(s)
COVID-19 , Measles , Poliomyelitis , Rubella , Animals , Cell Line , Chlorocebus aethiops , Containment of Biohazards , Dogs , Public Health Surveillance , SARS-CoV-2 , Vero Cells
13.
Molecules ; 27(10)2022 May 23.
Article in English | MEDLINE | ID: covidwho-1875718

ABSTRACT

(1) Background: A novel bioreactor platform of neuronal cell cultures using low-magnitude, low-frequency (LMLF) vibrational stimulation was designed to discover vibration influence and mimic the dynamic environment of the in vivo state. To better understand the impact of 40 Hz and 100 Hz vibration on cell differentiation, we join biotechnology and advanced medical technology to design the nano-vibration system. The influence of vibration on the development of nervous tissue on the selected cell line SH-SY5Y (experimental research model in Alzheimer's and Parkinson's) was investigated. (2) Methods: The vibration stimulation of cell differentiation and elongation of their neuritis were monitored. We measured how vibrations affect the morphology and differentiation of nerve cells in vitro. (3) Results: The highest average length of neurites was observed in response to the 40 Hz vibration on the collagen surface in the differentiating medium, but cells response did not increase with vibration frequency. Also, vibrations at a frequency of 40 Hz or 100 Hz did not affect the average density of neurites. 100 Hz vibration increased the neurites density significantly with time for cultures on collagen and non-collagen surfaces. The exposure of neuronal cells to 40 Hz and 100 Hz vibration enhanced cell differentiation. The 40 Hz vibration has the best impact on neuronal-like cell growth and differentiation. (4) Conclusions: The data demonstrated that exposure to neuronal cells to 40 Hz and 100 Hz vibration enhanced cell differentiation and proliferation. This positive impact of vibration can be used in tissue engineering and regenerative medicine. It is planned to optimize the processes and study its molecular mechanisms concerning carrying out the research.


Subject(s)
Neurons , Vibration , Cell Cycle , Cell Differentiation , Cell Proliferation
14.
Marine Drugs ; 20(5):304, 2022.
Article in English | ProQuest Central | ID: covidwho-1871726

ABSTRACT

Four new cytotoxic indole-diterpenoids (penerpenes K-N), along with twelve other known compounds, have been discovered by Dai et al. from the fermentation broth produced by adding L-tryptophan to the culture medium of Penicillium sp. Three compounds (penerpene N, epipaxilline, emindole SB) were found to be cytotoxic to cancer cell lines, of which the known compound, epipaxilline, was the most active and showed cytotoxic activity against the human liver cancer cell line BeL-7402 with an IC50 value of 5.3 μM. Moreover, six compounds, namely paxilline, 7-hydroxyl-13-dehydroxypaxilline, 7-hydroxypaxilline-13-ene, 4a-demethylpaspaline-4a-carboxylic acid, PC-M6 and emindole SB, showed antibacterial activities against Staphylococcus aureus ATCC 6538 and Bacillus subtilis ATCC 6633 [3]. [...]the authors of this manuscript have also reported, for the first time, a putative lysosomal acid lipase produced by a green microalgae [7]. In this review, the authors have summarized the list of 145 natural products isolated from microorganisms associated with sea cucumbers between 2000 and 2021, which include polyketides, alkaloids and terpenoids as well as their reported biological activities [8].

15.
Molecules ; 27(11)2022 May 26.
Article in English | MEDLINE | ID: covidwho-1869715

ABSTRACT

Impaired autophagy, responsible for increased inflammation, constitutes a risk factor for the more severe COVID-19 outcomes. Spermidine (SPD) is a known autophagy modulator and supplementation for COVID-19 risk groups (including the elderly) is recommended. However, information on the modulatory effects of eugenol (EUG) is scarce. Therefore, the effects of SPD and EUG, both singularly and in combination, on autophagy were investigated using different cell lines (HBEpiC, SHSY5Y, HUVEC, Caco-2, L929 and U937). SPD (0.3 mM), EUG (0.2 mM) and 0.3 mM SPD + 0.2 mM EUG, significantly increased autophagy using the hallmark measure of LC3-II protein accumulation in the cell lines without cytotoxic effects. Using Caco-2 cells as a model, several crucial autophagy proteins were upregulated at all stages of autophagic flux in response to the treatments. This effect was verified by the activation/differentiation and migration of U937 monocytes in a three-dimensional reconstituted intestinal model (Caco-2, L929 and U937 cells). Comparable benefits of SPD, EUG and SPD + EUG in inducing autophagy were shown by the protection of Caco-2 and L929 cells against lipopolysaccharide-induced inflammation. SPD + EUG is an innovative dual therapy capable of stimulating autophagy and reducing inflammation in vitro and could show promise for COVID-19 risk groups.


Subject(s)
COVID-19 , Syzygium , Aged , Autophagy , COVID-19/drug therapy , Caco-2 Cells , Eugenol/pharmacology , Humans , Inflammation , Monocytes , Plant Oils , Spermidine/pharmacology , Triticum
16.
Pakistan Journal of Pharmaceutical Sciences ; 35(3):859-864, 2022.
Article in English | CAB Abstracts | ID: covidwho-1865773

ABSTRACT

SARS-CoV-2 pandemic, drawn attention to the need of virus culture. In vitro SARS-COV-2 culture was performed to carry out therapeutic, environmental and virus genome studies. Isolation of virus from nasopharyngeal swab was performed by inoculating virus positive samples in available cell lines. SARS-CoV-2 topography was observed by using Scanning Electron Microscopy (SEM). Virus specificity was defined by serological confirmation through neutralization assay with COVID 19 convalescent sera. The SARS-COV -2 virus replicated successfully in Vero cell lines (both in E6 and CCL-81). The TCID50 and PFUs of isolated virus were defined as 107 TCID50/mL and 1.4 x 106 pfu/mL respectively. The virus particles with the SARS-CoV morphology was 150 engM size. Virus inhibition in presence of convalescent sera of COVID-19 patients was observed. Sisybrium irio (Khaksi) was found cytotoxic on Vero E6 cell line and its antiviral activity could not be established against SARS-COV-2 virus in vitro. Successful isolated and archived native SARS-COV-2 may be utilized further for therapeutic, environmental and virus genome sequencing studies.

17.
PLoS Computational Biology ; 18(4), 2022.
Article in English | ProQuest Central | ID: covidwho-1843149

ABSTRACT

Comparing SARS-CoV-2 infection-induced gene expression signatures to drug treatment-induced gene expression signatures is a promising bioinformatic tool to repurpose existing drugs against SARS-CoV-2. The general hypothesis of signature-based drug repurposing is that drugs with inverse similarity to a disease signature can reverse disease phenotype and thus be effective against it. However, in the case of viral infection diseases, like SARS-CoV-2, infected cells also activate adaptive, antiviral pathways, so that the relationship between effective drug and disease signature can be more ambiguous. To address this question, we analysed gene expression data from in vitro SARS-CoV-2 infected cell lines, and gene expression signatures of drugs showing anti-SARS-CoV-2 activity. Our extensive functional genomic analysis showed that both infection and treatment with in vitro effective drugs leads to activation of antiviral pathways like NFkB and JAK-STAT. Based on the similarity—and not inverse similarity—between drug and infection-induced gene expression signatures, we were able to predict the in vitro antiviral activity of drugs. We also identified SREBF1/2, key regulators of lipid metabolising enzymes, as the most activated transcription factors by several in vitro effective antiviral drugs. Using a fluorescently labeled cholesterol sensor, we showed that these drugs decrease the cholesterol levels of plasma-membrane. Supplementing drug-treated cells with cholesterol reversed the in vitro antiviral effect, suggesting the depleting plasma-membrane cholesterol plays a key role in virus inhibitory mechanism. Our results can help to more effectively repurpose approved drugs against SARS-CoV-2, and also highlights key mechanisms behind their antiviral effect.

18.
Acta Microbiologica Sinica ; 2:672-685, 2022.
Article in Chinese | CAB Abstracts | ID: covidwho-1841702

ABSTRACT

[Objective] To explore whether porcine deltacoronavirus (PDCoV) can infect and proliferate in different animal species-derived cell lines. [Methods] The Sichuan isolate CHN-SC2015of PDCoV was inoculated in twelve cell lines derived from hamster,poultry,monkey, human and swine. After at least five blindly passages in each cell line, the virus was identified by RT-PCR,RT-q PCR, indirect immunofluorescence assay (IFA), and sequencing. [Results] PDCoV caused distinct cytopathic effect (CPE) in Vero,PAM,PK15,ST, and LLC-PK1 cells at the 1st passage (P1) and proliferated to various degrees in PAM,PK15,ST, and LLC-PK1 cells, while the CPE gradually disappeared during subsequent passages in Vero and PAM cells. Except that in the three susceptible cell lines (PK15,LLC-PK1, and ST), the viral copies of the infected cell lines gradually decreased with the increase in passages, and PDCoV could not be detected at P4 or P5 of DEF,Marc-145,HEK-293,ZYM-SIEC02, and PAM cells. PCR results showed that PDCoV could be detected only in CEF and Vero cells at P5. The IFA results showed that PDCoV could infect other cell lines except BHK-21 and ZYM-SIEC02, and specific immunofluorescence was observed in PK15,LLC-PK1, and ST cells at P1,P3, and P9. Therefore, only three cell lines (PK15,LLC-PK1, and ST) were suitable for serial passage, with the virus titers up to 107.11,107.00, and 107.37 TCID50/mL at P9,respectively. After passage in different cell lines,CHN-SC2015 accumulated 14 nucleotide mutations corresponding to 12 amino acid mutations. [Conclusion] This study indicates that PDCoV can infect a variety of cells in vitro, suggesting that it may have the potential of cross-species transmission.

19.
Front Genet ; 13: 801382, 2022.
Article in English | MEDLINE | ID: covidwho-1785331

ABSTRACT

The Vero cell line is an immortalized cell line established from kidney epithelial cells of the African green monkey. A variety of Vero sublines have been developed and can be classified into four major cell lineages. In this study, we determined the whole-genome sequence of Vero E6 (VERO C1008), which is one of the most widely used cell lines for the proliferation and isolation of severe acute respiratory syndrome coronaviruses (SARS-CoVs), and performed comparative analysis among Vero JCRB0111, Vero CCL-81, Vero 76, and Vero E6. Analysis of the copy number changes and loss of heterozygosity revealed that these four sublines share a large deletion and loss of heterozygosity on chromosome 12, which harbors type I interferon and CDKN2 gene clusters. We identified a substantial number of genetic differences among the sublines including single nucleotide variants, indels, and copy number variations. The spectrum of single nucleotide variants indicated a close genetic relationship between Vero JCRB0111 and Vero CCL-81, and between Vero 76 and Vero E6, and a considerable genetic gap between the former two and the latter two lines. In contrast, we confirmed the pattern of genomic integration sites of simian endogenous retroviral sequences, which was consistent among the sublines. We identified subline-specific/enriched loss of function and missense variants, which potentially contribute to the differences in response to viral infection among the Vero sublines. In particular, we identified four genes (IL1RAP, TRIM25, RB1CC1, and ATG2A) that contained missense variants specific or enriched in Vero E6. In addition, we found that V739I variants of ACE2, which functions as the receptor for SARS-CoVs, were heterozygous in Vero JCRB0111, Vero CCL-81, and Vero 76; however, Vero E6 harbored only the allele with isoleucine, resulting from the loss of one of the X chromosomes.

20.
Journal of Research in Pharmacy ; 25(6):944-952, 2021.
Article in English | CAB Abstracts | ID: covidwho-1761620

ABSTRACT

Favipiravir (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) is a purine nucleic acid analog, which is an antiviral agent used in the treatment of influenza. Since the recent outbreak caused by 2019-novel coronavirus (nCoV), there has been a seek for effective antiviral agents to be used in the treatment of coronavirus disease 2019 (COVID-19), and favipiravir has been one of the options which provides a broad-spectrum therapy. Herein, we studied the aqueous solubility and in vitro permeability characteristics of favipiravir in order to shed light on the BCS classification of this antiviral agent used in COVID-19 therapy. The in vitro solubility was assessed using saturated solution of favipiravir in four different aqueous media and the solubility values were evaluated during 72 h at 37..C. The solubility of favipiravir was between 4.48 to 8.5 mg/ml, which is 5.85 to 10.63 times of calculated solubility limit. Caco-2 cell monolayers were utilized for the permeability assessment, and the drug solutions in three different concentrations including the highest dose required for bioequivalence exemption of the immediate release dosage form were applied. The effect of efflux transporters on the permeability of favipiravir was also determined using a P-gp inhibitor, Verapamil HCl. According to the data obtained from the in vitro studies, favipiravir can be considered as a representative of class I compound.

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