scholarly journals In vitro and in vivo replication of influenza A H1N1 WSN33 viruses with different M1 proteins

2013 ◽  
Vol 94 (4) ◽  
pp. 884-895 ◽  
Author(s):  
Zhiguang Ran ◽  
Ying Chen ◽  
Huigang Shen ◽  
Xiaoxiao Xiang ◽  
Qinfang Liu ◽  
...  
Keyword(s):  
Influenza A ◽  
Cultured Cells ◽  
Important Determinant ◽  
Clinical Manifestations ◽  
Structural Protein ◽  
M1 Protein ◽  
Influenza A H1n1

The M1 protein is a major structural protein that has multiple functions in various steps within the life cycle of the influenza A virus (IAV). However, little is currently known about the role of M1 in IAV replication in vivo and the associated pathogenesis. In this study, six isogenic H1N1 WSN33 viruses, constructed to express unique M1 proteins derived from various strains, subtypes or WSN33 itself, were tested to determine in vitro and in vivo functional exchangeability of M1 proteins in the replication and pathogenesis of the WSN33 virus. Despite five chimeric M1 viruses replicating to levels similar to those of the parental WSN33 virus in cell cultures, all M1 chimeras exhibited improved replication and enhanced virulence in mice when compared with the WSN33 virus. Interestingly, M1 proteins derived from swine viruses caused more severe clinical diseases than those from human or quail. These data indicate that the M1 protein is an important determinant of viral replication and pathogenic properties in mice, although the functions of M1 observed in vivo are not adequately reflected in simple infections of cultured cells. Chimeric M1 viruses that are variable in their clinical manifestations described here will aid future understanding of the role of M1 in IAV pathogenesis.

Abstract 237: PRMT1-p53-Slug Axis Regulates Epicardial EMT and Ventricular Development

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Olan Jackson-Weaver ◽  
Jian Wu ◽  
Yongchao Gou ◽  
Yibu Chen ◽  
Meng Li ◽  
...  
Keyword(s):  
Blood Vessels ◽  
Heart Development ◽  
Cultured Cells ◽  
Migration And Invasion ◽  
P53 Expression ◽  
Epicardial Cells ◽  
Coronary Blood Vessels

Rationale: Epicardial epithelial-to-mesenchymal trasition (EMT) is a vital process in embryonic heart development. During EMT, epicardial cells acquire migratory and invasive properties, and differentiate into new cell types, including cardiac fibroblasts and coronary smooth muscle cells. Non-histone protein methylation is an emerging modulator of cell signaling. We have recently established a role for protein arginine methyltransferase-1 (PRMT1) in TGF-β-induced EMT in cultured cells. Objective: To determine the role of PRMT1 in epicardial EMT. Methods and Results: We investigated the role of PRMT1 in epicardial EMT in mouse epicardial cells. Embryonic day 9.5 (E9.5) tamoxifen administration of WT1-Cre ERT ;PRMT1 fl/fl ;ROSA-YFP fl/fl mouse embryos was used to delete PRMT1 in the epicardium. Epicardial PRMT1 deletion led to reduced epicardial migration into the myocardium, a thinner compact myocardial layer, and dilated coronary blood vessels at E15.5. Using the epicardial cell line MEC1, we found that PRMT1 siRNA prevented the increase in mesenchymal proteins Slug and Fibronectin and the decrease in epithelial protein E-Cadherin during TGF-β treatment-induced EMT. PRMT1 siRNA also reduced the migration and invasion of MEC1 cells. We further identified that PRMT1 siRNA also increased the expression of p53, a key regulator of the Slug degradation pathway. PRMT1 siRNA increases p53 expression by decreasing p53 degradation, and shifted p53 localization to the cytoplasm. In vitro methylation assays further demonstrated that PRMT1 methylates p53. Knockdown of p53 increased Slug levels and enhanced EMT, establishing p53 as a regulator of epicardial EMT through controlling Slug expression. Furthermore, RNAseq experiments in MEC1 cells demonstrated that 40% (545/1,351) of TGF-β-induced transcriptional changes were prevented by PRMT1 siRNA. Furthermore, when p53 and PRMT1 were simultaneously knocked down, TGF-β induced transcriptional control of 37% (201/545) of these PRMT1-dependent genes was restored. Conclusions: The PRMT1-p53-Slug pathway is necessary for epicardial EMT in cultured MEC1 cells as well as in the epicardium in vivo . Epicardial PRMT1 is required for the development of compact myocardium and coronary blood vessels.

scholarly journals Optimization of the Production Technology of Oxidized Cyclodextrin Bisulfite

Processes ◽  
2019 ◽  
Vol 7 (7) ◽  
pp. 426
Author(s):  
Yana-Ya Kostyro ◽  
Anastasiya Soldatenko ◽  
Alexey Levchuk
Keyword(s):  
Influenza A ◽  
Cross Flow ◽  
Active Pharmaceutical Ingredient ◽  
Environmental Friendliness ◽  
Whole Process ◽  
In Vivo Experiments ◽  
Cross Flow Filtration ◽  
Influenza A H1n1

The A.E. Favorsky Irkutsk Institute of Chemistry, Siberian Branch of the Russian Academy of Sciences has developed an original active pharmaceutical ingredient based on an oxidized cyclodextrin oligosaccharide, which is a bisulfite derivative. Conducted pharmacological studies proved its antiviral activity in vitro and in vivo experiments against the influenza A (H1N1) virus. The aim of this work was to optimize the technology of obtaining the active pharmaceutical ingredient based on the bisulfite derivative of oxidized cyclodextrin to increase the efficiency and safety of the process. For this, a scaled method of oligosaccharide oxidation was tested on pilot plants in accordance with the requirements of green chemistry. As a result, the reaction time was reduced from three to five days (laboratory conditions) to 1.5 h, and the safety and environmental friendliness of process was ensured. The use of cross-flow filtration and the method of freeze-drying eliminated 96% of ethyl alcohol, reduced the laboriousness and energy consumption of the technological operations for purification and isolation of the final product, and also increased the productivity of the whole process (output increased to 98%). The results are confirmed by data obtained by physicochemical methods.

scholarly journals Heterosubtypic Protection Conferred by the Human Monoclonal Antibody PN-SIA28 against Influenza A Virus Lethal Infections in Mice

2015 ◽  
Vol 59 (5) ◽  
pp. 2647-2653 ◽  
Author(s):  
Miguel Retamal ◽  
Yacine Abed ◽  
Chantal Rhéaume ◽  
Francesca Cappelletti ◽  
Nicola Clementi ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Body Weight ◽  
Influenza Virus ◽  
Influenza A ◽  
Human Monoclonal Antibody ◽  
Influenza A Viruses ◽  
Virus Challenge ◽  
Influenza A H1n1

ABSTRACTPN-SIA28 is a human monoclonal antibody (Hu-MAb) targeting highly conserved epitopes within the stem portion of the influenza virus hemagglutinin (HA) (N. Clementi, et al, PLoS One 6:e28001, 2011,http://dx.doi.org/10.1371/journal.pone.0028001). Previousin vitrostudies demonstrated PN-SIA28 neutralizing activities against phylogenetically divergent influenza A subtypes. In this study, the protective activity of PN-SIA28 was evaluated in mice inoculated with lethal influenza A/WSN/33 (H1N1), A/Quebec/144147/09 (H1N1)pdm09, and A/Victoria/3/75 (H3N2) viruses. At 24 h postinoculation (p.i.), animals received PN-SIA28 intraperitoneally (1 or 10 mg/kg of body weight) or 10 mg/kg of unrelated Hu-MAb (mock). Body weight loss and mortality rate (MR) were recorded for 14 days postinfection (p.i.). Lung viral titers (LVT) were determined at day 5 p.i. In A/WSN/33 (H1N1)-infected groups, all untreated and mock-receiving mice died, whereas MRs of 87.5% and 25% were observed in mice that received PN-SIA28 1 and 10 mg/kg, respectively. In influenza A(H1N1) pdm09-infected groups, an MR of 75% was recorded for untreated and mock-treated groups, whereas the PN-SIA28 1-mg/kg and 10-mg/kg groups had rates of 62.5% and 0%, respectively. In A/Victoria/3/75 (H3N2)-infected animals, untreated and mock-treated animals had MRs of 37.5% and 25%, respectively, and no mortalities were recorded after PN-SIA28 treatments. Accordingly, PN-SIA28 treatments significantly reduced weight losses and resulted in a ≥1-log reduction in LVT compared to the control in all infection groups. This study confirms that antibodies targeting highly conserved epitopes in the influenza HA stem region, like PN-SIA28, not only neutralize influenza A viruses of clinically relevant subtypesin vitrobut also, more importantly, protect from a lethal influenza virus challengein vivo.

scholarly journals Synergistic Antiviral Activity of S-033188/S-033447, a Novel Inhibitor of Influenza Virus Cap-Dependent Endonuclease, in Combination with Neuraminidase Inhibitors In Vitro

2017 ◽  
Vol 4 (suppl_1) ◽  
pp. S371-S371 ◽  
Author(s):  
Mitsutaka Kitano ◽  
Atsuko Yamamoto ◽  
Takeshi Noshi ◽  
Makoto Kawai ◽  
Ryu Yoshida ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Cultured Cells ◽  
H1n1 Virus ◽  
Mdck Cells ◽  
Active Form ◽  
Neuraminidase Inhibitors ◽  
Dependent Endonuclease ◽  
Influenza A H1n1

Abstract Background S-033447, an active form of orally available prodrug S-033188, is a novel small molecule inhibitor of cap-dependent endonuclease that is essential for influenza virus transcription and replication. In this study, we evaluated the inhibitory effect of S-033188 in combination with neuraminidase inhibitors on the replication of influenza A/H1N1 virus in cultured cells. Methods The inhibitory effects of S-033447 in combination with NA inhibitors on the cytopathic effect of A/PR/8/34 strain in Madin–Darby canine kidney cells cultured for 2 days were tested and EC50 were determined. The combination index (CI), which were obtained when S-033188 and NA inhibitor were added at the closest ratio of each EC50 value, were used for the evaluation of these combinational effects (Table 1). CI values were calculated by the Chou and Talalay method, in which combinational effect were determined according to the criteria as follows: synergistic if CI ≤ 0.8, additive if 0.8 < CI < 1.2, and antagonistic if CI ≥ 1.2. CI = (DA/A + B)/DA + (DB/A + B)/DB + (DA/A + B × DB/A + B)/(DA × DB) DA: the EC50 of S-033447 DB: the EC50 of NA inhibitor DA/A + B: the concentration of S-033447 giving 50% inhibition in combination with NA inhibitor at the closest ratio of each EC50 value DB/A + B: the concentration of NA inhibitor giving 50% inhibition in combination with S-033447 at the closest ratio of each EC50 value Results All CI values were lower than 0.8, under the condition that both S-033447 and NA inhibitor (oseltamivir acid, zanamivir hydrate, laninamivir, or peramivir trihydrate) were added at the closest ratio of each EC50 value (Table 1). Conclusion S-033447 in combination with oseltamivir acid, zanamivir hydrate, laninamivir, or peramivir trihydrate synergistically inhibited the replication of influenza A/H1N1 virus in MDCK cells. Disclosures All authors: No reported disclosures.

scholarly journals Role of neuromedin B and its receptor in the innate immune responses against influenza A virus infection in vitro and in vivo

2019 ◽  
Vol 50 (1) ◽  
Author(s):  
Guihong Yang ◽  
Huipeng Huang ◽  
Mengyao Tang ◽  
Zifeng Cai ◽  
Cuiqin Huang ◽  
...  
Keyword(s):  
Virus Infection ◽  
Influenza A Virus ◽  
Influenza A ◽  
A549 Cells ◽  
Virus Challenge ◽  
Neuromedin B ◽  
Influenza A Virus Infection

Abstract The peptide neuromedin B (NMB) and its receptor (NMBR) represent a system (NMB/NMBR) of neuromodulation. Here, it was demonstrated that the expression of NMBR in cells or murine lung tissues was clearly upregulated in response to H1N1/PR8 influenza A virus infection. Furthermore, the in vitro and in vivo activities of NMB/NMBR during PR8 infection were investigated. It was observed that A549 cells lacking endogenous NMBR were more susceptible to virus infection than control cells, as evidenced by the increased virus production in the cells. Interestingly, a significant decrease in IFN-α and increased IL-6 expression were observed in these cells. The role of this system in innate immunity against PR8 infection was probed by treating mice with NMB. The NMB-treated mice were less susceptible to virus challenge, as evidenced by increased survival, increased body weight, and decreased viral NP expression compared with the control animals. Additionally, the results showed that exogenous NMB not only enhanced IFN-α expression but also appeared to inhibit the expression of NP and IL-6 in PR8-infected cells and animals. As expected, opposing effects were observed in the NMBR antagonist-treated cells and mice, which further confirmed the effects of NMB. Together, these data suggest that NMB/NMBR may be an important component of the host defence against influenza A virus infection. Thus, these proteins may serve as promising candidates for the development of novel antiviral drugs.

scholarly journals Differential Viral-Host Immune Interactions Associated with Oseltamivir-Resistant H275Y and Wild-Type H1N1 A(pdm09) Influenza Virus Pathogenicity

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 794
Author(s):  
Beatriz Vidaña ◽  
Pamela Martínez-Orellana ◽  
Jaime Martorell ◽  
Massimiliano Baratelli ◽  
Jorge Martínez ◽  
...  
Keyword(s):  
Influenza A ◽  
Clinical Signs ◽  
Viral Fitness ◽  
Type I ◽  
Lung Pathology ◽  
Wild Type ◽  
Viral Kinetics ◽  
Influenza A H1n1

Oseltamivir is a common therapy against influenza A virus (IAV) infections. The acquisition of oseltamivir resistance (OR) mutations, such as H275Y, hampers viral fitness. However, OR H1N1 viruses have demonstrated the ability to spread throughout different populations. The objective of this work was to compare the fitness of two strains of OR (R6 and R7) containing the H275Y mutation, and a wild-type (F) pandemic influenza A (H1N1) 2009 (pdm09) virus both in vitro and in vivo in mice and to select one OR strain for a comparison with F in ferrets. R6 showed faster replication and pathogenicity than R7 in vitro and in mice. Subsequently, R6 was selected for the fitness comparison with the F strain in ferrets. Ferrets infected with the F virus showed more severe clinical signs, histopathological lung lesions, and viral quantification when compared to OR R6-infected animals. More importantly, differential viral kinetics correlated with differential pro-inflammatory host immune responses in the lungs of infected ferrets, where OR-infected animals developed a protective higher expression of type I IFN and Retinoid acid Inducible Gene I (RIG-I) genes early after infection, resulting in the development of milder disease. These results suggest the presence of early specific viral-host immune interactions relevant in the development of influenza-associated lung pathology.

Variable association of complement activation by rituximab and paclitaxel in cancer patients in vivo and in their screening serum in vitro with clinical manifestations of hypersensitivity: a pilot study

2015 ◽  
Vol 7 (4) ◽  
Author(s):  
Gergely Tibor Kozma ◽  
Tamás Mészáros ◽  
Zsóka Weiszhár ◽  
Tamás Schneider ◽  
András Rosta ◽  
...  
Keyword(s):  
Pilot Study ◽  
Cancer Patients ◽  
Anticancer Drugs ◽  
Complement Activation ◽  
Clinical Manifestations ◽  
Hypersensitivity Reactions

AbstractTo explore the role of complement (C) activation in the hypersensitivity reactions (HSRs) to some anticancer drugs, as well as the use of the C activation biomarkers (C

The Role of Microscopy in Understanding Atherosclerotic Lysosomal Lipid Metabolism

2003 ◽  
Vol 9 (1) ◽  
pp. 54-67 ◽  
Author(s):  
W. Gray Jerome ◽  
Patricia G. Yancey
Keyword(s):  
Cultured Cells ◽  
Foam Cell ◽  
Critical Role ◽  
Cell Formation ◽  
Foam Cell Formation ◽  
Cholesteryl Esters ◽  
Normal Metabolism

Microscopy has played a critical role in first identifying and then defining the role of lysosomes in formation of atherosclerotic foam cells. We review the evidence implicating lysosomal lipid accumulation as a factor in the pathogenesis of atherosclerosis with reference to the role of microscopy. In addition, we explore mechanisms by which lysosomal lipid engorgement occurs. Low density lipoproteins which have become modified are the major source of lipid for foam cell formation. These altered lipoproteins are taken into the cell via receptor-mediated endocytosis and delivered to lysosomes. Under normal conditions, lipids from these lipoproteins are metabolized and do not accumulate in lysosomes. In the atherosclerotic foam cell, this normal metabolism is inhibited so that cholesterol and cholesteryl esters accumulate in lysosomes. Studies of cultured cells incubated with modified lipoproteins suggests this abnormal metabolism occurs in two steps. Initially, hydrolysis of lipoprotein cholesteryl esters occurs normally, but the resultant free cholesterol cannot exit the lysosome. Further lysosomal cholesterol accumulation inhibits hydrolysis, producing a mixture of cholesterol and cholesteryl esters within swollen lysosomes. Various lipoprotein modifications can produce this lysosomal engorgement in vitro and it remains to be seen which modifications are most important in vivo.

scholarly journals Impaired hyperphosphorylation of rotavirus NSP5 in cells depleted of casein kinase 1α is associated with the formation of viroplasms with altered morphology and a moderate decrease in virus replication

2007 ◽  
Vol 88 (10) ◽  
pp. 2800-2810 ◽  
Author(s):  
Michela Campagna ◽  
Mauricio Budini ◽  
Francesca Arnoldi ◽  
Ulrich Desselberger ◽  
Jorge E. Allende ◽  
...  
Keyword(s):  
Cytoplasmic Inclusion ◽  
Rna Replication ◽  
Viral Rna ◽  
Structural Protein ◽  
Cytoplasmic Inclusion Bodies ◽  
Replicative Cycle ◽  
In Cells

The rotavirus (RV) non-structural protein 5, NSP5, is encoded by the smallest of the 11 genomic segments and localizes in ‘viroplasms’, cytoplasmic inclusion bodies in which viral RNA replication and packaging take place. NSP5 is essential for the replicative cycle of the virus because, in its absence, viroplasms are not formed and viral RNA replication and transcription do not occur. NSP5 is produced early in infection and undergoes a complex hyperphosphorylation process, leading to the formation of proteins differing in electrophoretic mobility. The role of hyperphosphorylation of NSP5 in the replicative cycle of rotavirus is unknown. Previous in vitro studies have suggested that the cellular kinase CK1α is responsible for the NSP5 hyperphosphorylation process. Here it is shown, by means of specific RNA interference, that in vivo, CK1α is the enzyme that initiates phosphorylation of NSP5. Lack of NSP5 hyperphosphorylation affected neither its interaction with the virus VP1 and NSP2 proteins normally found in viroplasms, nor the production of viral proteins. In contrast, the morphology of viroplasms was altered markedly in cells in which CK1α was depleted and a moderate decrease in the production of double-stranded RNA and infectious virus was observed. These data show that CK1α is the kinase that phosphorylates NSP5 in virus-infected cells and contribute to further understanding of the role of NSP5 in RV infection.

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