In-vitro validation of self designed siRNA targeting non-structural 1 gene of Influenza A virus

2015 ◽  
Vol 4 (6) ◽  
pp. 315-322
Author(s):  
Bhawana Jain ◽  
Amita Jain ◽  
Om Prakash ◽  
Ajay K. Singh ◽  
Tanushree Dangi ◽  
...  
Keyword(s):  
Cell Line ◽  
Influenza A Virus ◽  
Mdck Cell ◽  
Influenza A ◽  
Screening Method ◽  
Viral Gene ◽  
Mdck Cell Line ◽  
Ns1 Gene ◽  
Conserved Region

  The genomic variability makes Influenza A virus (IAV) difficult to be con-trolled by existing vaccines or anti-influenza drugs. Viral gene targeting siRNA induces the RNAi mechanism in the host and silents the gene by cleaving mRNA. Objective was to develop siRNA targeting non-structural 1 gene and to validate its efficiency in vitro. siRNA was designed rationally, targeting the most conserved region (delineated with the help of multiple sequence align-ment) of NS1 gene of IAV strains. To choose the most efficient siRNA, three levels screening method was developed. Ultimately one siRNA duplex was selected on the basis of its unique position in conserved region. siRNA effica-cy was confirmed in vitro on commonly used Madin Darby Canine Kidney (MDCK) cell line for IAV propagation using two clinical isolates i.e. Influenza A/H3N2 [A/India/LKO864/2011(H3N2)] and Influenza A/pdmH1N1 [A/India/LKO2151/2012(H1N1)]. Of total 173 strains worldwide and 30 strains from India, 32 bp long (position 561 - 592) conserved region was identified. The longest ORF of NS1 gene was targeted by the selected siRNA, which showed 65.5% inhibition in replication of Influenza A/pdmH1N1 and 67.2% inhibition in replication of Influenza A/H3N2 at 48 hpi on MDCK cell line. This study shows that siRNA targeting NS1 may be quite effective in controlling IAV rep-lication so can be used as anti-IAV therapeutic agent.

Optimization of trypsins for influenza A/H1N1 virus replication in MDCK SI-6 cells, a novel MDCK cell line

2018 ◽  
Vol 252 ◽  
pp. 94-99
Author(s):  
Viska I. Iskandar ◽  
Yutaka Sasaki ◽  
Naoto Yoshino ◽  
Raden Z.R. Abubakar ◽  
Shigehiro Sato ◽  
...  
Keyword(s):  
Cell Line ◽  
Virus Replication ◽  
Mdck Cell ◽  
Influenza A ◽  
H1n1 Virus ◽  
Mdck Cell Line ◽  
Influenza A H1n1

scholarly journals Pigmented-MDCK (P-MDCK) Cell Line with Tunable Melanin Expression: An in Vitro Model for the Outer Blood–Retinal Barrier

2012 ◽  
Vol 9 (11) ◽  
pp. 3228-3235 ◽  
Author(s):  
Rajendra S. Kadam ◽  
Robert. I. Scheinman ◽  
Uday B. Kompella
Keyword(s):  
Cell Line ◽  
Mdck Cell ◽  
In Vitro Model ◽  
Blood Retinal Barrier ◽  
Mdck Cell Line ◽  
Vitro Model

C-Met signalling in an HGF/SF-insensitive variant MDCK cell line with constitutive motile/invasive behaviour

1996 ◽  
Vol 109 (9) ◽  
pp. 2371-2381
Author(s):  
C.P. Webb ◽  
K. Lane ◽  
A.P. Dawson ◽  
G.F. Vande Woude ◽  
R.M. Warn
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Tyrosine Phosphorylation ◽  
Mdck Cell ◽  
Mdck Cells ◽  
Cell Surface Expression ◽  
Cell Phenotype ◽  
Mdck Cell Line

The Met protein is a receptor tyrosine kinase for hepatocyte growth factor/scatter factor (HGF/SF), a multifunctional growth factor with mitogenic, motogenic and morphogenic properties. A morphologically altered variant of the MDCK cell line, MDCK-1, spontaneously exhibits a number of features associated with a partial HGF/SF-Met induced phenotype (less adhesive colonies in culture, enhanced invasion and motility, nascent tubule formation), but paradoxically does not respond to HGF/SF treatment. Although the overall cell surface expression and distribution of Met were found to be similar in parental MDCK cells and the MDCK-1 cell line, p145met autophosphorylation (+/ HGF/SF) was significantly reduced in MDCK-1 cells in vitro and in vivo when compared with parental MDCK cells. In contrast, EGF induced cell proliferation and EGF receptor autophosphorylation to similar levels in both cell lines. The basal levels of protein tyrosine phosphorylation were higher in MDCK-1 cells when compared with parental MDCK cells, including that of two prominent proteins with molecular masses of approximately 185 kDa and 220 kDa. Moreover, both p185 and p220 are present and tyrosine phosphorylated in Met immunoprecipitates from MDCK-1 cells (+/-HGF/SF), but not parental MDCK cells. In addition, Met immunocomplexes from MDCK-1 cells exhibited an approximately 3-fold increased tyrosine kinase activity in vitro when compared with MDCK cells, correlating with the higher basal levels of total phosphotyrosine. Treatment of MDCK-1 cells with the tyrosine kinase inhibitor herbimycin A reverted the cell phenotype to a more MDCK-like morphology in culture, with a concomitant reduction in the tyrosine phosphorylation predominantly of p220. Taken together these data suggest that aberrations in Met activity and associated signalling render MDCK-1 cells insensitive to HGF/SF, and may also mediate alterations in MDCK-1 cell behaviour.

scholarly journals Expanding the Efflux In Vitro Assay Toolbox: A CRISPR-Cas9 Edited MDCK Cell Line with Human BCRP and Completely Lacking Canine MDR1

2021 ◽  
Vol 110 (1) ◽  
pp. 388-396 ◽  
Author(s):  
Christine Wegler ◽  
Meryem Gazit ◽  
Karolina Issa ◽  
Sujay Subramaniam ◽  
Per Artursson ◽  
...  
Keyword(s):  
Cell Line ◽  
Mdck Cell ◽  
In Vitro Assay ◽  
Vitro Assay ◽  
Mdck Cell Line

scholarly journals An alternative AUG codon in segment 5 of the 2009 pandemic influenza A virus is a swine-derived virulence motif

2019 ◽  
Author(s):  
Helen M. Wise ◽  
Eleanor Gaunt ◽  
Jihui Ping ◽  
Barbara Holzer ◽  
Seema Jasim ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Untranslated Region ◽  
Influenza A ◽  
H1n1 Virus ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Genetic Features ◽  
Np Gene

AbstractThe 2009 influenza A virus (IAV) pandemic (pdm2009) was caused by a swine H1N1 virus with several atypical genetic features. Here, we investigate the origin and significance of an upstream AUG (uAUG) codon in the 5’-untranslated region of the NP gene. Phylogeny indicated that the uAUG codon arose in the classical swine IAV lineage in the mid 20th Century, and has become fixed in the current triple reassortant, variant pdm2009 swine IAV and human pdm2009 lineages. Functionally, it supports leaky ribosomal initiation in vitro and in vivo to produce two isoforms of NP: canonical, and a longer “eNP”. The uAUG codon had little effect on viral gene expression or replication in vitro. However, in both murine and porcine models of IAV infection, removing the uAUG codon gene attenuated pdm2009 virus pathogenicity. Thus, the NP uAUG codon is a virulence factor for swine IAVs with proven zoonotic ability.

scholarly journals PB2 and Hemagglutinin Mutations Are Major Determinants of Host Range and Virulence in Mouse-Adapted Influenza A Virus

2010 ◽  
Vol 84 (20) ◽  
pp. 10606-10618 ◽  
Author(s):  
Jihui Ping ◽  
Samar K. Dankar ◽  
Nicole E. Forbes ◽  
Liya Keleta ◽  
Yan Zhou ◽  
...  
Keyword(s):  
Host Range ◽  
Influenza A Virus ◽  
Influenza A ◽  
Lethal Dose ◽  
Polymerase Activity ◽  
Mouse Lung ◽  
Viral Gene ◽  
Adaptive Mutations

ABSTRACT Serial mouse lung passage of a human influenza A virus, A/Hong Kong/1/68 (H3N2) (HK-wt), produced a mouse-adapted variant, MA, with nine mutations that was >103.8-fold more virulent. In this study, we demonstrate that MA mutations of the PB2 (D701N) and hemagglutinin (HA) (G218W in HA1 and T156N in HA2) genes were the most adaptive genetic determinants for increased growth and virulence in the mouse model. Recombinant viruses expressing each of the mutated MA genome segments on the HK-wt backbone showed significantly increased disease severity, whereas only the mouse-adapted PB2 gene increased virulence, as determined by the 50% lethal dose ([LD50] >101.4-fold). The converse comparisons of recombinant MA viruses expressing each of the HK-wt genome segments showed the greatest decrease in virulence due to the HA gene (102-fold), with lesser decreases due to the M1, NS1, NA, and PB1 genes (100.3- to 100.8-fold), and undetectable effects on the LD50 for the PB2 and NP genes. The HK PB2 gene did, however, attenuate MA infection, as measured by weight loss and time to death. Replication of adaptive mutations in vivo and in vitro showed both viral gene backbone and host range effects. Minigenome transcription assays showed that PB1 and PB2 mutations increased polymerase activity and that the PB2 D701N mutation was comparable in effect to the mammalian adaptive PB2 E627K mutation. Our results demonstrate that host range and virulence are controlled by multiple genes, with major roles for mutations in PB2 and HA.

scholarly journals Eosinophil Responses at the Airway Epithelial Barrier during the Early Phase of Influenza a Virus Infection in C57BL/6 Mice

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 509 ◽  
Author(s):  
Meenakshi Tiwary ◽  
Robert J. Rooney ◽  
Swantje Liedmann ◽  
Kim S. LeMessurier ◽  
Amali E. Samarasinghe
Keyword(s):  
Epithelial Cells ◽  
Influenza A Virus ◽  
Early Phase ◽  
Influenza A ◽  
Airway Epithelial Cells ◽  
Epithelial Barrier ◽  
Virus Infectivity ◽  
Airway Epithelial ◽  
In Vivo Models

Eosinophils, previously considered terminally differentiated effector cells, have multifaceted functions in tissues. We previously found that allergic mice with eosinophil-rich inflammation were protected from severe influenza and discovered specialized antiviral effector functions for eosinophils including promoting cellular immunity during influenza. In this study, we hypothesized that eosinophil responses during the early phase of influenza contribute to host protection. Using in vitro and in vivo models, we found that eosinophils were rapidly and dynamically regulated upon influenza A virus (IAV) exposure to gain migratory capabilities to traffic to lymphoid organs after pulmonary infection. Eosinophils were capable of neutralizing virus upon contact and combinations of eosinophil granule proteins reduced virus infectivity through hemagglutinin inactivation. Bi-directional crosstalk between IAV-exposed epithelial cells and eosinophils occurred after IAV infection and cross-regulation promoted barrier responses to improve antiviral defenses in airway epithelial cells. Direct interactions between eosinophils and airway epithelial cells after IAV infection prevented virus-induced cytopathology in airway epithelial cells in vitro, and eosinophil recipient IAV-infected mice also maintained normal airway epithelial cell morphology. Our data suggest that eosinophils are important in the early phase of IAV infection providing immediate protection to the epithelial barrier until adaptive immune responses are deployed during influenza.

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