scholarly journals Low-pH Endocytic Entry of the Porcine Alphaherpesvirus Pseudorabies Virus

2018 ◽  
Vol 93 (2) ◽  
Author(s):  
Jonathan L. Miller ◽  
Darin J. Weed ◽  
Becky H. Lee ◽  
Suzanne M. Pritchard ◽  
Anthony V. Nicola
Keyword(s):  
Cell Lines ◽  
Pseudorabies Virus ◽  
Vero Cells ◽  
Low Ph ◽  
Host Cells ◽  
Multiple Model ◽  
Trade Restrictions ◽  
African Green Monkey Kidney ◽  
Entry Pathway ◽  
Model Cell

ABSTRACTThe alphaherpesvirus pseudorabies virus (PRV) is the causative agent of pseudorabies, a disease of great economic and welfare importance in swine. Other alphaherpesviruses, including herpes simplex virus (HSV), utilize low-pH-mediated endocytosis to enter a subset of cell types. We investigated whether PRV used this entry pathway in multiple laboratory model cell lines. Inhibition of receptor-mediated endocytosis by treatment with hypertonic medium prevented PRV entry. PRV entry into several cell lines, including porcine kidney (PK15) cells and African green monkey kidney (Vero) cells, was inhibited by noncytotoxic concentrations of the lysosomotropic agents ammonium chloride and monensin, which block the acidification of endosomes. Inactivation of virions by acid pretreatment is a hallmark of viruses that utilize a low-pH-mediated entry pathway. Exposure of PRV virions to pH 5.0 in the absence of host cell membranes reduced entry into PK15 and Vero cells by >80%. Together, these findings suggest that endocytosis followed by fusion with host membranes triggered by low endosomal pH is an important route of entry for PRV.IMPORTANCEPRV is a pathogen of great economic and animal welfare importance in many parts of the world. PRV causes neurological, respiratory, and reproductive disorders, often resulting in mortality of young and immunocompromised animals. Mortality, decreased production, and trade restrictions result in significant financial losses for the agricultural industry. Understanding the molecular mechanisms utilized by PRV to enter host cells is an important step in identifying novel strategies to prevent infection and spread. A thorough understanding of these mechanisms will contribute to a broader understanding of alphaherpesvirus entry. Here, we demonstrate PRV entry into multiple model cell lines via a low-pH endocytosis pathway. Together, these results provide a framework for elucidating the early events of the PRV replicative cycle.

scholarly journals Specificity of Legionella pneumophila and Coxiella burnetii Vacuoles and Versatility of Legionella pneumophila Revealed by Coinfection

2005 ◽  
Vol 73 (8) ◽  
pp. 4494-4504 ◽  
Author(s):  
John-Demian Sauer ◽  
Jeffrey G. Shannon ◽  
Dale Howe ◽  
Stanley F. Hayes ◽  
Michele S. Swanson ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Coxiella Burnetii ◽  
Legionella Pneumophila ◽  
Close Association ◽  
Cell Types ◽  
Vero Cells ◽  
Host Cells ◽  
Mouse Bone Marrow ◽  
Wild Type ◽  
African Green Monkey Kidney

ABSTRACT Legionella pneumophila and Coxiella burnetii are phylogenetically related intracellular bacteria that cause aerosol-transmitted lung infections. In host cells both pathogens proliferate in vacuoles whose biogenesis displays some common features. To test the functional similarity of their respective intracellular niches, African green monkey kidney epithelial (Vero) cells, A/J mouse bone marrow-derived macrophages, human macrophages, and human dendritic cells (DC) containing mature C. burnetii replication vacuoles were superinfected with L. pneumophila, and then the acidity, lysosome-associated membrane protein (LAMP) content, and cohabitation of mature replication vacuoles was assessed. In all cell types, wild-type L. pneumophila occupied distinct vacuoles in close association with acidic, LAMP-positive C. burnetii replication vacuoles. In murine macrophages, but not primate macrophages, DC, or epithelial cells, L. pneumophila replication vacuoles were acidic and LAMP positive. Unlike wild-type L. pneumophila, type IV secretion-deficient dotA mutants trafficked to lysosome-like C. burnetii vacuoles in Vero cells where they survived but failed to replicate. In primate macrophages, DC, or epithelial cells, growth of L. pneumophila was as robust in superinfected cell cultures as in those singly infected. Thus, despite their noted similarities, L. pneumophila and C. burnetii are exquisitely adapted for replication in unique replication vacuoles, and factors that maintain the C. burnetii replication vacuole do not alter biogenesis of an adjacent L. pneumophila replication vacuole. Moreover, L. pneumophila can replicate efficiently in either lysosomal vacuoles of A/J mouse cells or in nonlysosomal vacuoles of primate cells.

scholarly journals Attachment and invasion ofToxoplasma gondiiandNeospora caninumto epithelial and fibroblast cell linesin vitro

Parasitology ◽  
2005 ◽  
Vol 131 (5) ◽  
pp. 583-590 ◽  
Author(s):  
YING LEI ◽  
M. DAVEY ◽  
J. T. ELLIS
Keyword(s):  
Cell Lines ◽  
Neospora Caninum ◽  
Fibroblast Cell ◽  
Cell Types ◽  
Vero Cells ◽  
Host Cells ◽  
Trypsin Treatment ◽  
Epithelial Cell Lines ◽  
Pre Treatment

Attachment and invasion ofToxoplasma gondiiandNeospora caninumto a cat and a dog fibroblast cell line and 2 epithelial cell lines (a cat kidney and Vero) were comparedin vitrousing fluorescence antibody methodology. In addition, trypsin treatment of tachyzoites was used to determine whether protein molecules were essential to the process of invasion. The results show that bothT. gondiiandN. caninuminvaded all 4 cell lines, and that pre-treatment ofT. gondiitachyzoites with trypsin caused an increase in the ability of the parasite to invade these host cells. FurthermoreT. gondii, in comparison toN. caninum, invaded all 4 cell lines at greater levels. The results here support the conclusion that bothT. gondiiandN. caninumhave the ability to invade a variety of cell types including both dog and cat cells, and questions the utility of Vero cells as an appropriate host cell forin vitrostudies on the biology of these taxa.

Chemical constituents and antiproliferative effects of cultured Mougeotia nummuloides and Spirulina major against cancerous cell lines

2016 ◽  
Vol 71 (3-4) ◽  
pp. 87-92 ◽  
Author(s):  
Ramazan Erenler ◽  
Koksal Pabuccu ◽  
Ayse Sahin Yaglioglu ◽  
Ibrahim Demirtas ◽  
Fatih Gul
Keyword(s):  
Cell Lines ◽  
Chemical Constituents ◽  
Vero Cells ◽  
C6 Cells ◽  
African Green Monkey Kidney ◽  
Cancerous Cell ◽  
Brain Tumor Cells ◽  
Rat Brain Tumor ◽  
High Concentrations

AbstractIn this study, the effect ofMougeotia nummuloidesandSpirulina majoron Vero cells (African green monkey kidney), C6 cells (rat brain tumor cells) and HeLa cells (human uterus carcinoma) was investigatedin vitro. The antiproliferative effect of the methanol extract ofM. nummuloidesandS. majorcompared with 5-fluorourasil (5-FU) and cisplatin was tested at various concentrations using the BrdU Cell Proliferation ELISA. BothM. nummuloidesandS. majorextracts significantly inhibited the proliferation of Vero, HeLa and C6 cancer cell lines with IC50and IC75values. TheM. nummuloidesextract exhibited higher activity than 5-FU and cisplatin on Vero and C6 cells at high concentrations. TheS. majorextract revealed better antifproliferative activity than standards against Vero cells at 500 μg/mL. The compounds of methanol extracts were determined by GC-MS after the silylation process. Trehalose, monostearin and 1-monopalmitin were detected as major products in theM. nummuloidesextract where as in theS. majorextract; monostearin, 1-monopalmitin and hexyl alcohol were the main constituents.

scholarly journals Roles for Endocytosis and Low pH in Herpes Simplex Virus Entry into HeLa and Chinese Hamster Ovary Cells

2003 ◽  
Vol 77 (9) ◽  
pp. 5324-5332 ◽  
Author(s):  
Anthony V. Nicola ◽  
Anna M. McEvoy ◽  
Stephen E. Straus
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Surface ◽  
Vero Cell ◽  
Cell Lines ◽  
Vero Cells ◽  
Low Ph ◽  
Dependent Manner ◽  
Cho Cell ◽  
Simplex Virus

ABSTRACT Herpes simplex virus (HSV) infection of many cultured cells, e.g., Vero cells, can be initiated by receptor binding and pH-neutral fusion with the cell surface. Here we report that a major pathway for HSV entry into the HeLa and CHO-K1 cell lines is dependent on endocytosis and exposure to a low pH. Enveloped virions were readily detected in HeLa or receptor-expressing CHO cell vesicles by electron microscopy at <30 min postinfection. As expected, images of virus fusion with the Vero cell surface were prevalent. Treatment with energy depletion or hypertonic medium, which inhibits endocytosis, prevented uptake of HSV from the HeLa and CHO cell surface relative to uptake from the Vero cell surface. Incubation of HeLa and CHO cells with the weak base ammonium chloride or the ionophore monensin, which elevate the low pH of organelles, blocked HSV entry in a dose-dependent manner. Noncytotoxic concentrations of these agents acted at an early step during infection by HSV type 1 and 2 strains. Entry mediated by the HSV receptor HveA, nectin-1, or nectin-2 was also blocked. As analyzed by fluorescence microscopy, lysosomotropic agents such as the vacuolar H+-ATPase inhibitor bafilomycin A1 blocked the delivery of virus capsids to the nuclei of the HeLa and CHO cell lines but had no effect on capsid transport in Vero cells. The results suggest that HSV can utilize two distinct entry pathways, depending on the type of cell encountered.

scholarly journals Bovine Herpesvirus 1 Entry by a Low-pH Endosomal Pathway

2018 ◽  
Vol 92 (20) ◽  
Author(s):  
Gabrielle Pastenkos ◽  
Becky Lee ◽  
Suzanne M. Pritchard ◽  
Anthony V. Nicola
Keyword(s):  
Bovine Herpesvirus ◽  
Low Ph ◽  
Dependent Manner ◽  
Bovine Herpesvirus 1 ◽  
African Green Monkey Kidney ◽  
Effective Prevention ◽  
Entry Pathway ◽  
Lysosomotropic Agents ◽  
Mdbk Cells ◽  
Herpesvirus 1

ABSTRACTBovine herpesvirus 1 (BoHV-1) is an alphaherpesvirus that poses a significant challenge to health and welfare in the cattle industry. We investigated the cellular entry route utilized by BoHV-1. We report that BoHV-1 enters Madin Darby bovine kidney (MDBK) cells, bovine turbinate cells, and African green monkey kidney (Vero) cells via a low-pH-mediated endocytosis pathway. Treatment of MDBK cells with hypertonic medium, which inhibits receptor-mediated endocytosis, prevented infection as measured by a beta-galactosidase reporter assay. Treatment of cells with noncytotoxic concentrations of the lysosomotropic agents ammonium chloride and monensin, which block the acidification of endosomes, inhibited BoHV-1 entry in a concentration-dependent fashion. The kinetics of endocytic uptake of BoHV-1 from the cell surface was rapid (50% uptake by ∼5 min). Time-of-addition experiments indicated that the lysosomotropic agents acted at early times postinfection, consistent with entry. Inactivation of virions by pretreatment with mildly acidic pH is a hallmark characteristic of viruses that utilize a low-pH-activated entry pathway. When BoHV-1 particles were exposed to pH 5.0 in the absence of target membrane, infectivity was markedly reduced. Lastly, treatment of cells with the proteasome inhibitor MG132 inhibited BoHV-1 entry in a concentration-dependent manner. Together, these results support a model of BoHV-1 infection in which low endosomal pH is a critical host trigger for fusion of the viral envelope with an endocytic membrane and necessary for successful infection of the target cell.IMPORTANCEBoHV-1 is a ubiquitous pathogen affecting cattle populations worldwide. Infection can result in complicated, polymicrobial infections due to the immunosuppressive properties of the virus. While there are vaccines on the market, they only limit disease severity and spread but do not prevent infection. The financial and animal welfare ramifications of this virus are significant, and in order to develop more effective prevention and treatment regimens, a more complete understanding of the initial steps in viral infection is necessary. This research establishes the initial entry pathway of BoHV-1, which provides a foundation for future development of effective treatments and preventative vaccines. Additionally, it allows comparisons to the entry pathways of other alphaherpesviruses, such as HSV-1.

scholarly journals Bioactive Volatile Content of the Stem and Root ofCentaurea carduiformisDC. subsp.carduiformisvar.carduiformis

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Ibrahim Demirtas ◽  
Ayse Sahin
Keyword(s):  
Cell Lines ◽  
Vero Cells ◽  
Hexane Extract ◽  
African Green Monkey Kidney ◽  
Brain Tumor Cells ◽  
Green Monkey ◽  
Rat Brain Tumor ◽  
Medicinal Properties ◽  
Asteraceae Family

Several species ofCentaurea, biennial (or more rarely perennial) plants which belong to the Asteraceae family, possess medicinal properties and are currently used in phytotherapy. In the present study, antiproliferative activity of hexane extract from stems and roots ofCentaurea carduiformisDC. subsp.carduiformisvar.carduiformis(CCS and CCR) on Vero cells (African green monkey kidney), C6 cells (Rat Brain tumor cells), and HeLa cells (human uterus carcinoma) was investigatedin vitro. Antiproliferative effect of the extract was tested at 500 μg/mL and 1000 μg/mL using BrDu Cell Proliferation ELISA. The hexane extract was significantly inhibited proliferation of Vero, HeLa, and C6 cancer cell lines with absorbance values. The extract of CCS and CCR showed the highest activity against the Vero, HeLa, and C6 cell lines at 500 μg/mL and 1000 μg/mL.

scholarly journals Avian Flavivirus Enters BHK-21 Cells by a Low pH-Dependent Endosomal Pathway

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1112 ◽  
Author(s):  
Abdul Sattar Baloch ◽  
Chunchun Liu ◽  
Xiaodong Liang ◽  
Yayun Liu ◽  
Jing Chen ◽  
...  
Keyword(s):  
Eastern China ◽  
Low Ph ◽  
Host Cells ◽  
Bafilomycin A1 ◽  
Acid Pretreatment ◽  
Entry Pathway ◽  
Endosomal Pathway ◽  
Endosomal Ph ◽  
Host Cell Membranes ◽  
Endocytic Pathways

Duck Tembusu virus (DTMUV), a pathogenic member of the Flavivirus family, was first discovered in the coastal provinces of South-Eastern China in 2010. Many previous reports have clearly shown that some Flaviviruses utilize several endocytic pathways to enter the host cells, however, the detailed mechanism of DTMUV entry into BHK-21 cells, which is usually employed to produce commercial veterinary vaccines for DTMUV, as well as of other Flaviviruses by serial passages, is still unknown. In this study, DTMUV entry into BHK-21 cells was found to be inhibited by noncytotoxic concentrations of the agents chloroquine, NH4Cl, and Bafilomycin A1, which blocked the acidification of the endosomes. Inactivation of virions by acid pretreatment is a hallmark of viruses that utilize a low-pH-mediated entry pathway. Exposure of DTMUV virions to pH 5.0 in the absence of host cell membranes decreased entry into cells by 65%. Furthermore, DTMUV infection was significantly decreased by chlorpromazine treatment, or by knockdown of the clathrin heavy chain (CHC) through RNA interference, which suggested that DTMUV entry depends on clathrin. Taken together, these findings highlight that a low endosomal pH is an important route of entry for DTMUV.

Long-read RNA sequencing identifies polyadenylation elongation and differential transcript usage of host transcripts during SARS-CoV-2 in vitro infection

2021 ◽  
Author(s):  
Jessie J-Y Chang ◽  
Josie Gleeson ◽  
Daniel Rawlinson ◽  
Miranda E Pitt ◽  
Ricardo De Paoli-Iseppi ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Ribosomal Protein ◽  
Rna Sequencing ◽  
Cell Lines ◽  
Human Lung ◽  
Vero Cells ◽  
Late Time ◽  
Ribosomal Protein Genes ◽  
African Green Monkey Kidney ◽  
Long Read

Better methods to interrogate host-pathogen interactions during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections are imperative to help understand and prevent this disease. Here we implemented RNA-sequencing (RNA-seq) combined with the Oxford Nanopore Technologies (ONT) long-reads to measure differential host gene expression, transcript polyadenylation and isoform usage within various epithelial cell lines permissive and non-permissive for SARS-CoV-2 infection. SARS-CoV-2-infected and mock-infected Vero (African green monkey kidney epithelial cells), Calu-3 (human lung adenocarcinoma epithelial cells), Caco-2 (human colorectal adenocarcinoma epithelial cells) and A549 (human lung carcinoma epithelial cells) were analysed over time (0, 2, 24, 48 hours). Differential polyadenylation was found to occur in both infected Calu-3 and Vero cells during a late time point (48 hpi), with Gene Ontology (GO) terms such as viral transcription and translation shown to be significantly enriched in Calu-3 data. Poly(A) tails showed increased lengths in the majority of the differentially polyadenylated transcripts in Calu-3 and Vero cell lines (up to ~136 nt in mean poly(A) length, padj = 0.029). Of these genes, ribosomal protein genes such as RPS4X and RPS6 also showed downregulation in expression levels, suggesting the importance of ribosomal protein genes during infection. Furthermore, differential transcript usage was identified in Caco-2, Calu-3 and Vero cells, including transcripts of genes such as GSDMB and KPNA2, which have previously been implicated in SARS-CoV-2 infections. Overall, these results highlight the potential role of differential polyadenylation and transcript usage in host immune response or viral manipulation of host mechanisms during infection, and therefore, showcase the value of long-read sequencing in identifying less-explored host responses to disease.

La3+ and pH sensitivity of Ca2+ entry and intracellular store filling in gastric parietal cells

1995 ◽  
Vol 269 (5) ◽  
pp. G770-G778 ◽  
Author(s):  
P. A. Negulescu ◽  
T. E. Machen
Keyword(s):  
Positive Feedback ◽  
Low Ph ◽  
Parietal Cells ◽  
Additional Increase ◽  
Cholinergic Agonist ◽  
Intracellular Store ◽  
Entry Pathway ◽  
Signaling Function ◽  
Previously Treated ◽  
Gastric Parietal Cells

The fluorescent Ca2+ indicator fura 2 was used to measure cytosolic free [Ca2+] ([Ca2+]i) in order to obtain information about relative rates of Ca2+ influx into parietal cells during treatment with carbachol (a cholinergic agonist) or thapsigargin (TG, a Ca(2+)-mobilizing agent) or during reloading of the internal Ca2+ stores. In Ca(2+)-containing solutions, carbachol-, TG-, and reloading-stimulated Ca2+ entry exhibited nearly identical sensitivity to La3+ [inhibition constant (Ki) approximately 10 microM] or low pH (pKi approximately 7.0). In experiments in which carbachol and TG were used, there was no additional increase in [Ca2+]i when TG was added to carbachol-treated cells or when carbachol was added to cells previously treated with TG. Thus it is likely that a single Ca2+ entry pathway serves a signaling function as well as a role in refilling the Ca2+ store during reloading. Because the Ca2+ pathway is exquisitely sensitive to pH and serosal pH increases during stimulant-induced H+ secretion (which is activated by increases in [Ca2+]i), this mechanism will exert positive feedback on parietal cells in the intact stomach. When parietal cells were pretreated with carbachol in Ca(2+)-free solutions, reloading was independent of pH and La3+, suggesting that Ca(2+)-containing solutions should be used to determine the properties of the influx pathway.

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