Introduction: Hepatitis C virus (HCV) is a positive stranded RNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma. Aims & Objectives: The study was conducted to establish the transfection of Huh 7.5 derived cell lines with In-vitro transcript of HCV pF6/JFH-1 for production of infectious virus particles in naïve Huh 7.5 cells, its detection by RT-PCR. Materials and Method: Huh 7.5 cells, a highly permissive cell lines for HCV replication, were grown in Dulbecco’s Modified Eagle’s Medium and pFL-J6/JFH plasmid was linearized with XbaI and subjected to in-vitro transcription using MEGAscript Kit (Ambion, USA) Huh-7.5 cells were transfected with 2.5 μg transcript using Lipofectamine 2000 transfection reagent (Invitrogen, USA) . The culture supernatant was collected after 24, 48 and 72 hr after incubation in fresh media and viral RNAs were isolated from it using Trizol LS reagent (Ambion, USA) and quantified by real-time quantitative RT-PCR. Total RNA was extracted from cells using Trizol reagent (Ambion, USA) and then RNA was subjected to cDNA synthesis using RevertAid reverse transcription (Thermo Fisher Scientific, USA). The PCR products were resolved by electrophoresis in 1.5% (w/v) agarose gels and images were captured by a Chemidoc XRS system (Bio-Rad, USA). Results: We observed Huh7.5 cells were cultured in DMEM. Plasmid FL-J6/JFH1 was linearized with the restriction enzyme XbaI and HCV RNA was obtained by In-vitro transcription and was transfected to grown Huh 7.5 cells shown by band on agarose gel and total RNA isolated after 24 hours of post infection followed by RT-PCR gave distinct band on gel whereas 48 and 72 hr did not. Infection of Huh 7.5 cells with cell culture supernatant from cells transfected with HCV in vitro transcript gave a distinct band. This will help in understanding entire viral life cycle and its non-structural gene products like NS4B and NS5A that enhance the replicative capacity of replicons in Huh 7.5 cell lines for development of drug and vaccines.
Dr. Ben Hause - Metagenomic Sequencing for Virus Discovery and CharacterizationJohn Blue
Metagenomic Sequencing for Virus Discovery and Characterization - Dr. Ben Hause, Kansas State University, from the 2015 North American PRRS Symposium, December 4 - 5, 2015, Chicago, IL, USA.
More presentations at http://www.swinecast.com/2015-north-american-prrs-symposium
Dr. Ben Hause - Metagenomic Sequencing for Virus Discovery and CharacterizationJohn Blue
Metagenomic Sequencing for Virus Discovery and Characterization - Dr. Ben Hause, Kansas State University, from the 2015 North American PRRS Symposium, December 4 - 5, 2015, Chicago, IL, USA.
More presentations at http://www.swinecast.com/2015-north-american-prrs-symposium
In Vitro Analog of the Primitive Streak (ANIMATED)Nikolay Turovets
PLEASE DOWNLOAD TO SEE ANIMATION.
In vitro analog of the primitive streak: efficient derivation of highly enriched populations of hepatocytes from various types of human pluripotent stem cells.
May, 2011
Crisper cas-9 is a powerful gene editing technology and helps to solve many problems in shorter time and more precisely. The two main molecules is an enzyme CAS-9 and a piece of RNA known as GUIDE RNA, that introduce a change into the DNA.
Activation of surrogate death receptor signalling triggers peroxynitrite depe...Saurabh Shekhar
includes information about cisplatin resistance cancer cells and their execution through peroxynitrite triggered apoptosis due to death signaling receptors basedon the findings of research article published in cell death and diseases.
Explaining Biocide Tolerance of Gram Negative Bacteria Kate Barlow
Working on multiple organisms and constantly changing gene-targets requires use of an easily optimisable and cheap qPCR method, which is why we use SyBr Green qPCR. We have investigated chlorhexidine resistance in Klebsiella pneumoniae and are about to publish on biocide resistance in Acinetobacter baumannii and Pseudomonas aeruginosa. I will explain our approach and our optimisation and robustness strategies, as well as an overview of the hypotheses we developed and confirmed using our qPCR approach.
Lucy Bock, Senior Scientist/Project Team Leader, Technology Development Group, Public Health England, UK
El ARN del SARS-CoV-2 de transcripción inversa puede integrarse en el genoma de células humanas cultivadas y puede ser expresado en tejidos derivados del paciente
Proceedings of National Academy of SciencesPNAS is a partner of CHORUS, COPE, CrossRef, ORCID, and Research4Life.
In Vitro Analog of the Primitive Streak (ANIMATED)Nikolay Turovets
PLEASE DOWNLOAD TO SEE ANIMATION.
In vitro analog of the primitive streak: efficient derivation of highly enriched populations of hepatocytes from various types of human pluripotent stem cells.
May, 2011
Crisper cas-9 is a powerful gene editing technology and helps to solve many problems in shorter time and more precisely. The two main molecules is an enzyme CAS-9 and a piece of RNA known as GUIDE RNA, that introduce a change into the DNA.
Activation of surrogate death receptor signalling triggers peroxynitrite depe...Saurabh Shekhar
includes information about cisplatin resistance cancer cells and their execution through peroxynitrite triggered apoptosis due to death signaling receptors basedon the findings of research article published in cell death and diseases.
Explaining Biocide Tolerance of Gram Negative Bacteria Kate Barlow
Working on multiple organisms and constantly changing gene-targets requires use of an easily optimisable and cheap qPCR method, which is why we use SyBr Green qPCR. We have investigated chlorhexidine resistance in Klebsiella pneumoniae and are about to publish on biocide resistance in Acinetobacter baumannii and Pseudomonas aeruginosa. I will explain our approach and our optimisation and robustness strategies, as well as an overview of the hypotheses we developed and confirmed using our qPCR approach.
Lucy Bock, Senior Scientist/Project Team Leader, Technology Development Group, Public Health England, UK
El ARN del SARS-CoV-2 de transcripción inversa puede integrarse en el genoma de células humanas cultivadas y puede ser expresado en tejidos derivados del paciente
Proceedings of National Academy of SciencesPNAS is a partner of CHORUS, COPE, CrossRef, ORCID, and Research4Life.
Interleukin16 and Interleukin 28B Genes Polymorphism in HBV Infected Saudi pa...iosrjce
The course of hepatitis B virus (HBV) infection is variable depending on many factors. In this study,
we investigated single nucleotide polymorphisms of interleukin-28B and interleukin-16 as possible host factors
which may determine the occurrence of hepatocellular carcinoma (HCC) in Saudi patients. Chronic hepatitis B
(CHB) patients (75), HCC patients (42), and healthy controls (70) were analyzed for polymorphisms of the IL16
and IL28B genes using PCR and restriction fragment length polymorphism (RFLP). Results showed that HCC
and chronic HBV patients had higher prevalence of rs11556218TG genotype than controls. The rs11556218GG
genotype was higher among HCC (14.4%) compared to chronic HBV (2.7%) patients. The IL-16 genotype
rs4072111CT was higher among HCC (47.6%) and chronic HBV (46.7%) patients than controls (28.6%). The
rs4072111TT genotype was higher among HCC patients compared to the other two groups. The T allele
frequency was higher among HCC patients than controls. The CT and TT of the IL-28B rs12979860 genotype
were significantly less frequent in chronic HBV and HCC patients. The IL-28B rs8099917 TG genotype was
more frequent among HCC (19%) compared to chronic HBV (8%) patients. However, no significant difference
was detected in the allele distribution.
ABSTRACT- Multiple Drug resistance (MDR) tuberculosis timely diagnose is of utmost clinical relevance and needs to be diagnose at initial stages for the proper treatment. The current study was done to detect the several genes for MDR tuberculosis (TB) in clinical isolates by molecular tools. 60 clinical isolates were collected and subjected for AFB smear preparation, Nested PCR (IS6110) for mycobacterium tuberculosis complex detection and MDR TB PCR targeting rpoB, kat G, mab A promoter. 12 came positive for AFB smears, out of which 08 were pulmonary and 04 were extra pulmonary. Nested PCR targeting IS6110 gene was amplified at 123 base pairs with 340 base pairs as IC (internal control) was seen in 25 cases which include 19 pulmonary and 6 extra pulmonary. The Positive TB PCR specimens were subjected for MDRTB PCR Only 06 cases yielded, an amplicon of 315 bp confirming the rpoB gene resistance for resistance for rifampcin drug. In any of the 06 positives none of the other resistance gene other than rpoB was amplified. Targeting multiple genes at once, additional information will be gained from a single test run that otherwise would require several times the reagents and more time to perform. Current study signifies the usage of quick, cost effective, DNA sequences based method for MDR TB detection where disease will be diagnosed earlier and hence treatment would be started at an early stage.
Keywords: Multiple drug resistance, amplicon, Polymerase chain reaction, Nested PCR, Rifampicin.
The emerging CRISPR/Cas9 gene editing technology greatly accelerates the R&D process in life sciences. Here, we briefly introduce CRISPR/Cas9 and its delivery strategies.
I reviewed several manuscripts, books, grants and project proposals. This is one of the paper I reviewed recently published in Plant Biotechnology Journal
Chronic Hepatitis C Virus Infection and Carcinoma Cervix – Report of a Case ...Apollo Hospitals
We report a case of a 54 year old woman with carcinoma cervix and chronic hepatitic C infection. Hepatitis C Virus was isolated from the malignant cervical tissue which caused Chronic Hepatitis and may have had a direct role in the development and pathogenesis of cervical cancer.
Deep Behavioral Phenotyping in Systems Neuroscience for Functional Atlasing a...Ana Luísa Pinho
Functional Magnetic Resonance Imaging (fMRI) provides means to characterize brain activations in response to behavior. However, cognitive neuroscience has been limited to group-level effects referring to the performance of specific tasks. To obtain the functional profile of elementary cognitive mechanisms, the combination of brain responses to many tasks is required. Yet, to date, both structural atlases and parcellation-based activations do not fully account for cognitive function and still present several limitations. Further, they do not adapt overall to individual characteristics. In this talk, I will give an account of deep-behavioral phenotyping strategies, namely data-driven methods in large task-fMRI datasets, to optimize functional brain-data collection and improve inference of effects-of-interest related to mental processes. Key to this approach is the employment of fast multi-functional paradigms rich on features that can be well parametrized and, consequently, facilitate the creation of psycho-physiological constructs to be modelled with imaging data. Particular emphasis will be given to music stimuli when studying high-order cognitive mechanisms, due to their ecological nature and quality to enable complex behavior compounded by discrete entities. I will also discuss how deep-behavioral phenotyping and individualized models applied to neuroimaging data can better account for the subject-specific organization of domain-general cognitive systems in the human brain. Finally, the accumulation of functional brain signatures brings the possibility to clarify relationships among tasks and create a univocal link between brain systems and mental functions through: (1) the development of ontologies proposing an organization of cognitive processes; and (2) brain-network taxonomies describing functional specialization. To this end, tools to improve commensurability in cognitive science are necessary, such as public repositories, ontology-based platforms and automated meta-analysis tools. I will thus discuss some brain-atlasing resources currently under development, and their applicability in cognitive as well as clinical neuroscience.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
In vitro transcription and transfection of HCV genomic replicon
1. International Journal of Scientific and Research Publications, Volume 9, Issue 3, March 2019 419
ISSN 2250-3153
http://dx.doi.org/10.29322/IJSRP.9.03.2019.p8764 www.ijsrp.org
In-Vitro Transcription and Transfection of HCV
Genomic Replicons
Binod Gupta
Functional Genomic Labs, Centre for Advance Study, Department of Genetics, University College of science and technology, University of
Calcutta, 35, Ballygunge Circular Road, Kolkata-700019, India
Email:bgtauras@gmail.com
DOI: 10.29322/IJSRP.9.03.2019.p8764
http://dx.doi.org/10.29322/IJSRP.9.03.2019.p8764
ABSTRACT:
Introduction: Hepatitis C virus (HCV) is a positive stranded RNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma.
Aims & Objectives: The study was conducted to establish the transfection of Huh 7.5 derived cell lines with In-vitro transcript of HCV
pF6/JFH-1 for production of infectious virus particles in naïve Huh 7.5 cells, its detection by RT-PCR. Materials and Method: Huh 7.5 cells, a
highly permissive cell lines for HCV replication, were grown in Dulbecco’s Modified Eagle’s Medium and pFL-J6/JFH plasmid was linearized
with XbaI and subjected to in-vitro transcription using MEGAscript Kit (Ambion, USA) Huh-7.5 cells were transfected with 2.5 µg transcript
using Lipofectamine 2000 transfection reagent (Invitrogen, USA) . The culture supernatant was collected after 24, 48 and 72 hr after incubation
in fresh media and viral RNAs were isolated from it using Trizol LS reagent (Ambion, USA) and quantified by real-time quantitative RT-PCR.
Total RNA was extracted from cells using Trizol reagent (Ambion, USA) and then RNA was subjected to cDNA synthesis using RevertAid
reverse transcription (Thermo Fisher Scientific, USA). The PCR products were resolved by electrophoresis in 1.5% (w/v) agarose gels and
images were captured by a Chemidoc XRS system (Bio-Rad, USA). Results: We observed Huh7.5 cells were cultured in DMEM. Plasmid FL-
J6/JFH1 was linearized with the restriction enzyme XbaI and HCV RNA was obtained by In-vitro transcription and was transfected to grown
Huh 7.5 cells shown by band on agarose gel and total RNA isolated after 24 hours of post infection followed by RT-PCR gave distinct band on
gel whereas 48 and 72 hr did not. Infection of Huh 7.5 cells with cell culture supernatant from cells transfected with HCV in vitro transcript gave
a distinct band. This will help in understanding entire viral life cycle and its non-structural gene products like NS4B and NS5A that enhance the
replicative capacity of replicons in Huh 7.5 cell lines for development of drug and vaccines.
KEYWORDS: HCV; HCV NS5B; Huh 7.5; DMEM; Transfection
INTRODUCTION
Hepatitis was considered to be a disease originated from liver complications like anorexia, indigestion, poor assimilation, poor
L.F.T (liver function test), yellowing of skin, dark urine, jaundice, liver cirrhosis, hepatocellular carcinoma. Further researches
concluded that hepatitis is caused by several factors like excessive use of alcohol, use of multiple drugs and some side effects of
other diseases (Keril et al., 2002). But recent researches lead to discovery of Hepatitis-c virus by Dr. Harvery J. Alter, Chief of the
Infectious Disease Section in the Department of Transfusion Medicine at the National Institutes of Health, USA and Dr. Michael
Houghton in 1989 by immunoscreening an expression library with serum from a patient with post-transfusion non-A, non-B
hepatitis. However the virus was not visualized properly and it was unanswerable, the low viral titers in serum and liver tissue
made it impossible for biochemical characterization of native viral products and, most importantly, it was not possible to culture
HCV efficiently in vitro (Takanobu et al., 2007). Despite these obstacles, great progress has been made in the study of HCV over
the past 18 years using heterologous expression systems, functional cDNA clones, a replicon system, pseudo particles (engineered
retroviral particles bearing functional HCV envelope proteins) that enable the study of viral entry under reproducible and
conveniently measurable conditions and, most recently, complete cell-culture systems (Moradpour et al., 2007).
Hepatitis c virus is classified in the Hepacivirus genus within the Flaviviridae family, which is positive strand enveloped RNA
virus (Moradpour et al., 2007). HCV genome consists of RNA molecule of approx. 9.6 kb containing a large Open Reading Frame
flanked by structured 5' & 3' Non Translated Regions (NTRS). The 5’ NTR includes an internal ribosome entry site (IRES)
(Levrero et al., 2006). Viral proteins are translated as a polyprotein precursor from this Internal Ribosome Entry Site (IRES)
located in the 5’ NTR. The polyprotein undergoes a complex series of co-and post-translational cleavage events which is
catalyzed by both host and viral proteinases to yield the individual HCV proteins (Tetsuro et al., 2007).
The structural protein includes the core proteins and the envelope glycoproteins E1 and E2. The non-structural proteins include
the P7 polypeptide, the NS2-3 auto protease and the NS3 serine protease, an RNA helicase located in the C-terminal region of
NS3, the NS4A polypeptide, the NS4B and NS5A proteins, and the NS5B which is an RNA dependent RNA polymerase (Tetsuro
et al., 2007). Hepatitis-c virus infection is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC)
2. International Journal of Scientific and Research Publications, Volume 9, Issue 3, March 2019 420
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http://dx.doi.org/10.29322/IJSRP.9.03.2019.p8764 www.ijsrp.org
worldwide. A protective vaccine is not yet available and therapeutic options are still unlimited. Current therapies include
pegylated interferon-α combined with ribavirin but this treatment is not so effective and only responsive to some patients infected
with particular genotype (Moradpour et al,. 2007). All the genotypes do not respond to this drug.. The hepatitis C virus is usually
detectable by PCR in the blood within one to three weeks after infection, and antibodies to the virus are generally detectable
within 3 to 15 weeks or, chronic refers to infection with the hepatitis C virus persisting for more than six months. Clinically, it is
often asymptomatic (without symptoms) and it is mostly discovered accidentally (e.g. usual checkup). The natural course of
chronic hepatitis C varies considerably from one person to another. It is transmitted through the blood, items contaminated with
infected blood and Sexual transmission is rare (Shimotohno et al., 2000). It is very difficult to study its proper life cycle due to
absence of robust cell culture and small animal model. But now recent development of HCV replicon systems has made progress
for the study of HCV translation and RNA replication in human hepatoma-derived Huh-7 cells derived for HCV genotype 2a
replicon (JFH-1) In- vitro by Wakita and colleagues (Lohmann et al., 1999) . Moreover, these replicons do not replicate efficiently
without adaptive mutation so Huh-7 cells are derived to Huh-7.5 cells and Huh 7.5.1 cells for better replication study. This system
provides a powerful tool for producing infectious HCV viral particles in Huh 7.5 cell lines by In-Vitro transcription of genomic
pFL-JG/JFH by transfection detecting the presence of RNA by RT-PCR and maintaining the replicon in culture cell line, Time
course replication of Hepatitis-c virus for analysis of host-virus interactions that should facilitate the discovery of antiviral drugs
and vaccines for this important human pathogen (Moradpour et al., 2007).
MATERIALS AND METHODS
The hepatic Huh 7.5 cells (a kind of gift from Prof. Charles M. Rice, The Rockefeller University, New York, NY, USA (Blight et
al., 2002) were maintained in complete DMEM (Dulbecco's Modified Eagle's medium, Invitrogen, USA) supplemented with 10%
FBS (Fetal Bovine Serum, Invitrogen), 100µg/ml Penicillin, 100µg/ml streptomycin (HiMedia, India), Glutamax 1% (Invitrogen),
Hygromycin (50µl/ml), Puromycin (1µg/ml) at 37o
C in 5% CO2 and grown for 5-6 days depending upon how much seeding is
needed with frequent changing and checking the media and cells. 10µg of pFL-J6/JFH1 plasmid was linearized with XbaI and
purified by purification as per manufacturer’s protocol.
1µg of Linearized and Purified pFL-J6/JFH genotype 2a construct(a kind of gift from Prof.Charles M. Rice) was subjected to in
vitro transcription for the synthesis of HCV genomic replicon using T-7 Mega script kit (Ambion, USA) as per manufacturer’s
protocol.
In-vitro transcribed genomic HCV RNA was delivered to overnight grown naive Huh 7.5 cells in a 60 mm tissue culture dish
using Lipofectamin 2000 transfecting reagent (Invitrogen), as per manufacturer’s protocol (Mandal et al., 2016)
Total cellular RNA was isolated by TRIZOL (Invitrogen) using manufacturer’s protocol. Then after Reverse Transcriptase
Polymerase Chain Reaction (RT-PCR) for β-actin, NS5B was done by using previously designed and purified oligonucleotides
suitable for priming for β-actin (300nM), NS5B (1µM) of HCV genome. For β- actin, the forward primer sequence were HCV 5’
UTR: forward, 5’- GCGGGAAATCGTGCGTGACATT-3’; reverse, 5’- GATGGAGTTGAAGGTAGTTTCGTG-3’. For NS5B ,
forward , 5’-ACATCAAGTCCGTGTGGAAGG-3’; reverse, 5’-AGCCCGGGCGAGTGGAGTGG-3’ , Sybr green real- time
PCR master mix reagent (Invitrogen) was used for the Assays in a Mastercycler ep RealPlex2 (Eppendorf , Germany) as described
earlier (Mandal et al., 2016)
Synthesis of first strand cDNA for β-actin, NS5B: RT (Reverse Transcription) reaction was done for First strand cDNA synthesis
for β-actin, NS5B by taking total RNA sample (after isolation from transfected Huh 7.5 cells) along with 2 µl of specific primer.
Initially RNA and primer were heated at 65o
C for 5 minutes along with 25mM dNTP (200µM) and then quickly chilled in ice for
10 minutes, and then spinned down. β-actin (300nM), NS5B (1µM) was taken. cDNA synthesis was done on Realplex 2 Thermal
cycler (Eppendorf). Double stranded cDNA was amplified by PCR using 1 µl of first strand cDNA (1.2µg). PCR for β-actin, 0.6µl
of Mgcl2 and for NS5B, 0.75µl of Mgcl2 was added. PCR was done by following thermal profile: Initial denaturation at 94o
C for 5
minutes followed by 30 cycles of denaturation, annealing and extension at 94o
C for 30 seconds, 58o
C for 30 seconds and 72o
C for
30 seconds respectively, followed by a final extension at 72o
C for 10 minutes. The reaction volume for each reaction was 20µl.
Each reaction mix contained ------
Reagents for 1X 4.5X Stock final conc.
Buffer 2 µl 9 µl 10X 1X
Forward primer 1 µl 4.5 µl 12.5µM 1µM
Reverse primer 1 µl 4.5 µl 12.5µM 1µM
25mM dNTP 1 µl 4.5 µl 5mM 200µM
Mgcl2 0.6 µl 2.7 µl 50mM 1.5mM
Enzyme (Taq polymerase) 0.5 µl 2.25 µl 5U/ µl 2.5 units/µl
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H2O (Double distilled water) 12.9 µl 58.05 µl
cDNA 1µl
DNA was electrophoresed on 1.5% Agarose gel, run for 60 minutes at 70-80 volts on 50X TAE buffer system, stained with
ethidium bromide and visualized under UV-light in a UV-trans illuminator analysed on a Gel Doc (Bio-Rad) using standard
protocol. No destaining of the gel was required.
Infection of Naïve Huh 7.5 cells with viral supernatant:
And also to study the HCV infection in Huh 7.5 cell line, Viral supernatant from the previous experiment were used to infect
naïve Huh 7.5 cell line and left for 24 hours. Then the media was changed and fresh media (supplemented with G-418) was added
and incubated.
After 48 hours RNA was isolated by TRIZOL method using standard protocol and then RT-PCR was done. Then the sample was
loaded on the 2% Agarose gel for electrophoresis for the confirmation of the infection.
Time course study of hepatitis-c virus:
Time course study means for how much time the virus remains infective. For this, Viral supernatant was taken and infected the
naïve Huh 7.5 cells and left for 24 hours for infection. Then the media was changed and the Replicon maintaining media was
added and left for 48 hours for post infection. After 48 hours RNA was isolated by TRIZOL method using standard protocol and
then RT-PCR was done and the products were analyzed by Agarose gel electrophoresis using standard methods.
RESULTS:
I. Culture of Huh 7.5 cells:
Cells appeared as spindle shaped. First day had less confluency but third day got more confluent culture.
II. In-vitro transcription of pFL-J6/JFH
Linearization of plasmid pFL-J6/JFH1 with the restriction enzyme XbaI I were optimized at 37o
C for 4 hours and was In- Vitro
transcribed by using Linearized plasmid. The full length HCV genomic replicon was obtained which was confirmed by presence
of distinct band on agarose gel. . For control : pTRI-Xef transcript (1.96 kb) was used which gave successful band on agarose gel.
Fig1: Linearization of pFL-J6/JFH1 Vector with Xba I.
. Here, plasmid was found to be fully linearized at above optimum condition. Lane 1 loaded with uncut pFL-J6/JFH1 gave three
forms of plasmid and lane 2 loaded with XbaI I treated plasmid gave single form of plasmid. This Linearized plasmid was used
for In vitro transcription.
Lane 1: Uncut pFL-J6/JFH1,
Lane 2: pFL-J6/JFH1 linearized with XbaI
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Fig2: In vitro transcription using linearized pFL-J6/JFH1 to generate the full length HCV genome.
Lane 2 was loaded with pTRI-Xef transcript (1.96kb) as a control and lane 3 with the pFL-J6/JFH1 transcript (9.6kb) full length
which was in-vitro transcribed using linearized one. .
Lane2: pTRI-Xef transcript (1.96 kb),
Lane 3: pFL-J6/JFH1 transcript (9.6 kb)
The presence of distinct band shows the successful in vitro transcription.
III. HCV replicon Transfection:
Total RNA was isolated after 48 hours post transfection followed by RT-PCR for NS5B region gave a distinct band on agarose
gel confirmed successful transfection.
Fig8: Transfection of 24hrs grown Huh7.5 cells with 2.5µg HCV in vitro transcript using Lipofactamin 2000 reagent
(Invitrogen).
Different lanes in the gel were loaded with different samples as given below:-
Lane1: NS5B (PCR control)
Lane2:NS5B (Transfected Huh7.5 cells),
Lane3:NS5B (Naive Huh7.5 cells)
Thus from the picture above it is clear that PCR for NS5B occurred properly showing the presence of HCV RNA. Thus,
experiment giving successful transfection.
IV. Infection of HCV in Huh 7.5 cells:
The cell culture supernatant was obtained from tissue culture dish containing cells transfected with HCV In- Vitro transcript gave
a successful band on agarose gel.
1 2 3
1 2 3 4 5 6 7 8
1 2 3 4
5 6 7 8
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Fig9: Infection of Huh7.5 cells with cell culture supernatant from cells transfected with HCV in vitro transcript
Lane 1: PCR control β actin was loaded and distinct band was obtained.
Lane2: RT control β actin, was loaded and distinct band was obtained.
Lane3: β- actin (naïve Huh 7.5), it also showed the distinct band confirming its presence in naïve cells.
Lane4: was loaded with β actin obtained from 24 hour grown infected Huh 7.5 cells. A distinct band was obtained confirming
PCR has occurred correctly.
Lane5: was loaded with NS5B (PCR control)
Lane 6: was loaded with NS5B (24 hour Infected Huh 7.5 cells). This lane shows the clear distinct band confirming the successful
infection.
Lane 7- was loaded with the NS5B obtained by 48 hour of post infection. This lane does not show any band pattern suggesting no
infection established after 48 hours.
Lane 8-was loaded with the NS5B obtained by 72 hour of post infection. Here it also does not have any band suggesting no
establishment of infection.
DISCUSSION:
FL-J6/JFH1 is a full-length, chimeric, genotype 2a HCV genome containing the core-NS2 coding region from the J6 HCV isolate
and the NS3-NS5B coding region of HCV strain JFH-1. This genome includes nucleotides (nt) 1-300 of the JFH-1 strain, nt 301-
3430 of the J6 strain, and nt 3431-9678 of the JFH-1 strain. These genomes were created by using standard molecular biology
techniques and were maintained as cloned cDNAs within the plasmids pFL-J6/JFH (Hugle et al., 2001)
The pFL-J6/JFH plasmids were now linearized at the 3’end of the HCV cDNA by XbaI digestion. The linearized DNA was then
purified and used as a template for in-vitro transcription. Infectious RNAs were synthesized with T7 RNA Polymerase via
standard In- vitro transcription reactions. Then the RNA was purified using standard protocol and finally quantified.
From the data provided above, distinct DNA band of NS5B shows that pFL-J6/JFH plasmid construct is able to replicate
efficiently and produce infectious virus particles in Huh 7.5 cultured cells whereas β-actin is used as PCR control because it is
housekeeping gene of mammalian cells that is always expressed and when loaded on gel after PCR always showed a distinct band
confirming that PCR has occurred correctly. This cell lines are thus capable of contributing to efficient viral production and
infection in this system. Also to see that the viral supernatant is infective or not, the naïve Huh 7.5 cells were infected with the
viral supernatant and by following the protocol, RT- PCR and gel electrophoresis it was found that it can generate the infection
and produce infectious viral particles in naïve Huh 7.5 cell lines.
CONCLUSION:
Thus it was possible to produce the infectious HCV viral particles in Huh 7.5 cell lines by In-vitro transcription of genomic pFL-
JG/JFH by transfection and it could be also serially passaged to naïve cells. The ability of this cell culture technique created an
opportunity to address the aspects of virus life cycle, its infective nature, the time course of infection that somehow created the
knowledge of new investigative opportunities for the discovery of antiviral drugs and vaccines. But the main challenging question
is that ‘For how much time these cells remain infective?’ For this, the time course replication study was done by first taking the
viral supernatant and infecting the naïve Huh7.5 and following incubation detecting the presence of RNA by RT-PCR and again
taking the viral supernatant for the next experiment and again infecting the naïve Huh 7.5 and so on. By this it was possible to get
the infectivity for just 24 hour as shown by the data above. But in general it is difficult to get the infectivity for so long time that
could be preserved, it automatically disappears. Due to this it has not been possible to maintain the replicon in culture cell line and
future genetic studies on HCV proteins and RNA element; it still remains in the dark. Hence due to this, the dangerous HCV viral
lifecycle still remains unclear and development of drug and vaccines also remains to be discovered.
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