We are grateful to Suzanne Emerson for the hepatitis E virus p6 clone. HEV-specific rabbit hyperimmune serum was kindly provided by Rainer Ulrich, Friedrich Loeffler Institute, Germany. Moreover, we thank all members of the Department of Molecular and Medical Virology at the Ruhr University Bochum for their support and discussion. Figures 1-7 were generated with BioRender.com.

The authors have nothing to disclose.

Starting with the plasmid preparation, DNA yields should exceed 150 ng/&#181;L to be able to perform&#160;multiple linearization from the same plasmid stock, which minimizes the risk of bacteria-induced mutagenesis of crucial genome sequences. Furthermore, it is important to check the restriction digest for the complete plasmid linearization by gel electrophoresis (Figure 8b). A lack of linearized plasmid DNA would induce rolling circle amplification causing the in vitro transcription to be ...

Hepatitis E is a fairly&#160;underestimated disease with increasing prevalence worldwide. About 20 million infections result in more than 70,000 deaths per year1. The underlying agent, the Hepatitis E virus (HEV), was reassigned recently and is now classified within the family Hepeviridae including the genera Orthohepevirus and Piscihepevirus. HEV of various origins are classed within the species Orthohepevirus A-D including isolates from humans, swine, rabbits, rats, birds and other mammals2. At present, eight different genotypes (GT) of the positive-orientated, single-stranded RNA virus have been identified2. Although they differ in their sequence identity, routes of transmission and geographical distribution, their genomic structure is highly conserved. More specific, the 7.2 kbp HEV genome is divided into 3 major open reading frames (ORF1-3). While ORF1 encodes all enzymes needed for a successful replication within the host cell, ORF2 encodes the capsid protein, and the ORF3 protein operates as a functional ion channel required for assembly and release of infectious particles3. Once released into the basal or apical lumen HEV exists in both, quasienveloped and non-enveloped/naked species depending on whether the virus originates from blood or feces, respectively4,5.
While GT1 and GT2 are mainly found in developing countries solely infecting humans6 via the fecal-oral route, GT3, GT4 and GT7 predominantly occur in developed countries1,7 with a variety of species serving as reservoirs, e.g., swine8, rat9, chicken10,11, deer12, mongoose13, bat14, rabbit15,16, wild boar17 and many more7,18,19, providing evidence of zoonosis7,20,21,22. In addition to inadequate sanitary conditions23 and contaminated food products12,24,25,26, transmission via blood transfusion and organ transplantations is also possible27,28. HEV is a common cause of liver cirrhosis and liver failure29 especially in patients with pre-existing liver disease, immunocompromised individuals (genotype 3, 4 and 7) and pregnant women (genotype 1). Of note, there are also extrahepatic manifestations such as hematopoietic disease30,31,32, neurological disorders33 and renal injury34.
To date, the off-label drug ribavirin (RBV) is the treatment of choice for many infected patients35,36. However, cases of treatment failure and poor clinical long-term outcomes have been reported. Treatment failure has been linked to viral mutagenesis and increased viral heterogeneity in chronically infected patients37,38,39. On the contrary, a recent European retrospective multicenter study was not able to correlate polymerase mutations to RBV treatment failure40. In clinical observations and in vitro experiments, interferon41,42,43, sofosbuvir44,45, zinc salts46 and silvestrol47,48 have also shown antiviral effects. Nonetheless, a specific HEV treatment remains to be found, hampered by the lack of knowledge about the HEV life cycle and its pathogenesis. Therefore, a robust cell culture system for virological studies and the development of new antiviral drugs is urgently needed49.
Unfortunately, like other hepatitis viruses, HEV is difficult to propagate in conventional cell lines and usually progresses very slowly leading to low viral loads. Nevertheless, some groups were able to boost viral loads by the generation of cell line subclones50 or the adjustment of media supplements51. Recently the generation of cDNA clones52 and the adaption of primary patient isolates by passaging53,54 further improved HEV propagation in cell culture55. In this protocol, we used the genome of a cell culture adapted Kernow-C1 strain (referred to as p6_WT)54&#160;and a mutant strain harboring a replication-enhancing mutation (referred to as p6_G1634R)37. Kernow-C1 is the most frequently used strain in HEV cell culture and is capable to produce high viral loads. By assessing viral RNA copy numbers, HEV replication can be monitored in vitro. Nevertheless, these techniques do not allow assessment of the number of infectious particles being produced. Therefore, we have established an immunofluorescence staining to determine Focus Forming Units (FFU/mL).
The here described method56 can be used to produce full-length infectious viral particles that are capable to infect a variety of cell types from diverse origins including primary cells and mammalian cell lines. This is a fundamental prerequisite to decipher important aspects of HEV infection and tropism. There is no need for inoculation with usually limited patient isolates. The production of infectious HEV particles from plasmids poses an infinite source, which makes this protocol comparably efficient. In addition, this system can be used for reverse genetics enabling the study of in vivo identified genome alteration and their impact on HEV replication and fitness. This technique overcomes many limitations and, can path the way for drug development, mutagenesis studies and the evaluation of virus-host interactions such as restriction or entry factors.

<table><tbody><tr><td>0.45 &amp;micro;m mesh</td><td>Sarstedt</td><td>83.1826</td><td>Harvest extracellular Virus</td></tr><tr><td>4 % Histofix</td><td>CarlRoth</td><td>P087.4</td><td>Immunofluorescence</td></tr><tr><td>Acetic acid</td><td>CarlRoth</td><td>6755.1</td><td>Collagen working solution</td></tr><tr><td>Amicon Ultra-15</td><td>Merck Millipore</td><td>UFC910024</td><td>Virus harvesting</td></tr><tr><td>Ampicillin</td><td>Sigma-Aldrich</td><td>A1593</td><td>Selection of transformed bacteria</td></tr><tr><td>ATP</td><td>Roche</td><td>11140965001</td><td>&lt;em&gt;in vitro&lt;/em&gt; transcription and electroporation</td></tr><tr><td>BioRender</td><td>BioRender</td><td></td><td>Figure Generation</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Roth</td><td>5239.2</td><td>Cytomix</td></tr><tr><td>Collagen R solution 0.4 % sterile</td><td>Serva</td><td>47256.01</td><td>Collagen working solution</td></tr><tr><td>CTP</td><td>Roche</td><td>11140922001</td><td>&lt;em&gt;in vitro&lt;/em&gt; transcription</td></tr><tr><td>Cuvette</td><td>Biorad</td><td>165-2088</td><td>Electroporation</td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>D21490</td><td>Immunofluorescence</td></tr><tr><td>DMEM</td><td>gibco</td><td>41965-039</td><td>Cell culture</td></tr><tr><td>DNAse</td><td>Promega</td><td>M6101</td><td>&lt;em&gt;in vitro&lt;/em&gt; transcription</td></tr><tr><td>DTT</td><td>Promega</td><td>included in P2077</td><td>&lt;em&gt;in vitro&lt;/em&gt; transcription</td></tr><tr><td>EGTA</td><td>Roth</td><td>3054.3</td><td>Cytomix</td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt; JM109</td><td>Promega</td><td>L2005</td><td>Transformation</td></tr><tr><td>Fetal bovine serum</td><td>gibco</td><td>10270106</td><td>Cell culture</td></tr><tr><td>Fluoromount</td><td>SouthernBiotech</td><td>0100-01</td><td>Immunofluorescence</td></tr><tr><td>GenePulser Xcell Electroporation System</td><td>BioRad</td><td>1652660</td><td>Electroporation</td></tr><tr><td>Gentamycin</td><td>gibco</td><td>15710049</td><td>Cell culture</td></tr><tr><td>GTP</td><td>Roche</td><td>11140957001</td><td>&lt;em&gt;in vitro&lt;/em&gt; transcription</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Braun</td><td>184238001</td><td>Immunofluorescence</td></tr><tr><td>Hepes</td><td>Invitrogen</td><td>15630-03</td><td>Cytomix</td></tr><tr><td>Horse serum</td><td>gibco</td><td>16050122</td><td>Immunofluorescence</td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Roth</td><td>P749.1</td><td>Cytomix</td></tr><tr><td>KCL</td><td>Roth</td><td>6781.3</td><td>Cytomix</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Roth</td><td>3904.2</td><td>Cytomix</td></tr><tr><td>L-Glutamin</td><td>gibco</td><td>25030081</td><td>Cell culture</td></tr><tr><td>L-Glutathione reduced</td><td>Sigma-Aldrich</td><td>G4251-5G</td><td>Cytomix</td></tr><tr><td>MEM</td><td>gibco</td><td>31095-029</td><td>Cell culture</td></tr><tr><td>MEM NEAA (100&amp;times;)</td><td>gibco</td><td>11140-035</td><td>Cell culture</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Roth</td><td>2189.2</td><td>Cytomix</td></tr><tr><td>Microvolume UV-Vis spectrophotometer NanoDrop One</td><td>Thermo Fisher</td><td>ND-ONE-W</td><td>DNA/RNA concentration</td></tr><tr><td>MluI enzyme</td><td>NEB</td><td>R0198L</td><td>Linearization</td></tr><tr><td>NEB buffer</td><td>NEB</td><td>included in R0198L</td><td>Linearization</td></tr><tr><td>NucleoSpin Plasmid kit</td><td>Macherey &amp;amp; Nagel</td><td>740588.250</td><td>Plasmid preparation</td></tr><tr><td>NucleoSpin RNA Clean-up Kit</td><td>Macherey &amp;amp; Nagel</td><td>740948.250</td><td>RNA purification</td></tr><tr><td>PBS</td><td>gibco</td><td>70011051</td><td>Cell culture</td></tr><tr><td>Pen/Strep</td><td>Thermo Fisher</td><td>15140122</td><td>Cell culture</td></tr><tr><td>Plasmid encoding full-length HEV genome (p6_G1634R)</td><td>Todt et.al</td><td></td><td>Virus production</td></tr><tr><td>Plasmid encoding full-length HEV genome (p6_WT)</td><td>Shukla et al.</td><td>GenBank accession no. JQ679013</td><td>Virus production</td></tr><tr><td>Primary antibody 1E6</td><td>LS-Bio</td><td>C67675</td><td>Immunofluorescence</td></tr><tr><td>Primary antibody 8282</td><td></td><td></td><td>Rainer Ulrich, Friedrich Loeffler Institute, Germany</td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27106</td><td>DNA extraction</td></tr><tr><td>Ribo m&lt;sup&gt;7&lt;/sup&gt;G Cap Analog</td><td>Promega</td><td>P1711</td><td>&lt;em&gt;in vitro&lt;/em&gt; transcription</td></tr><tr><td>RNase away</td><td>CarlRoth</td><td>A998.3</td><td>RNA purification</td></tr><tr><td>RNasin (RNase inhibitor)</td><td>Promega</td><td>N2515</td><td>&lt;em&gt;in vitro&lt;/em&gt; transcription</td></tr><tr><td>Secondary antibody donkey anti-mouse 488</td><td>Thermo Fisher</td><td>A-21202</td><td>Immunofluorescence</td></tr><tr><td>Secondary antibody goat anti-rabbit 488</td><td>Thermo Fisher</td><td>A-11008</td><td>Immunofluorescence</td></tr><tr><td>Sodium Pyruvat</td><td>gibco</td><td>11360070</td><td>Cell culture</td></tr><tr><td>T&lt;sub&gt;7&lt;/sub&gt; RNA polymerase</td><td>Promega</td><td>P2077</td><td>&lt;em&gt;in vitro&lt;/em&gt; transcription</td></tr><tr><td>Transcription Buffer</td><td>Promega</td><td>included in P2077</td><td>&lt;em&gt;in vitro&lt;/em&gt; transcription</td></tr><tr><td>Triton X-100</td><td>CarlRoth</td><td>3051.3</td><td>Immunofluorescence</td></tr><tr><td>Trypsin-EDTA (0.5 %)</td><td>gibco</td><td>15400054</td><td>Cell culture</td></tr><tr><td>ultra-low IgG</td><td>gibco</td><td>1921005PJ</td><td>Cell culture</td></tr><tr><td>UTP</td><td>Roche</td><td>11140949001</td><td>&lt;em&gt;in vitro&lt;/em&gt; transcription</td></tr></tbody></table>

a cell culture model for producing high titer hepatitis e virus stocks

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is predominantly transmitted by blood transfusions, inadequate sanitary conditions and contaminated food products. To date the off-label drug ribavirin (RBV) is the treatment of choice for many patients. Nonetheless, a specific HEV treatment remains to be identified. So far, the knowledge about the HEV life cycle and pathogenesis has been severely hampered because of the lack of an efficient HEV cell culture system. A robust cell culture system is essential for the study of the viral life cycle which also includes the viral pathogenesis. With the method described here one can produce viral titers of up to 3 x 106 focus forming unit/mL (FFU/mL) of non-enveloped HEV and up to 5 x 104 FFU/mL of enveloped HEV. Using these particles, it is possible to infect a variety of cells of diverse origins including primary cells and human, as well as animal cell lines. The production of infectious HEV particles from plasmids poses an infinite source, which makes this protocol exceedingly efficient.

In this protocol, we describe the production of high titer infectious HEVcc. The first step is to isolate plasmid DNA (pBluescript_SK_HEVp654 and pBluescript_SK_HEVp6-G1634R37, Figure 8a), which then is linearized by restriction digestion and purified for in vitro transcription (Figure 1). A successful linearization can be verified by comparing the non-digested plasmid-DNA to the digested plasmid-DNA using gel elec...

Watch this Scientific Journal Video about A Cell Culture Model for Producing High Titer Hepatitis E Virus Stocks at JoVE.com

A Cell Culture Model for Producing High Titer Hepatitis E Virus Stocks

Described here is an effective method on how to produce high viral titer stocks of hepatitis E virus (HEV) to efficiently infect hepatoma cells. With the presented method, both non-enveloped, as well as enveloped viral particles can be harvested and used for inoculating various cell lines.

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is ...

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Research

JoVE Journal

Immunology and Infection

9.6K Views.  Ruhr University Bochum. Described here is an effective method on how to produce high viral titer stocks of hepatitis E virus (HEV) to efficiently infect hepatoma cells. With the presented method, both non-enveloped, as well as enveloped viral particles can be harvested and used for inoculating various cell lines.

Video: A Cell Culture Model for Producing High Titer Hepatitis E Virus Stocks

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the gene of interest in hematopoietic cells and can be used to study the gene's role in normal and/or malignant hematopoiesis. This protocol provides a detailed description on how to perform transduction-transplantation using calreticulin (CALR) mutations recently identified in myeloproliferative neoplasm (MPN) as an example. In this protocol whole bone marrow cells from 5-flurouracil (5-FU) treated donor mice are transduced with a retrovirus encoding mutant CALR and transplanted into lethally irradiated syngeneic hosts. Donor cells expressing mutant CALR are marked with green fluorescent protein (GFP). Transplanted mice develop an MPN phenotype including elevated platelets in the peripheral blood, expansion of megakaryocytes in the bone marrow, and bone marrow fibrosis. We provide a step-by-step account of how to generate retrovirus, calculate viral titer, transduce whole bone marrow cells, and transplant into irradiated recipient mice.

This manuscript provides a description of the methodology used to establish transduction-transplantation mouse models. A detailed account is given of technical errors to avoid when performing bone marrow transplants. A clear understanding should be gained of the importance of high viral titer, transfection/transduction, and irradiation.

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the ...

Cancer Research

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load but not result in complete remission. An efficient hepatocyte model for HBV infection would offer a true-to-life viral life cycle that would be crucial for the screening of therapeutic agents. Most available anti-HBV agents target lifecycle stages post viral entry but not before viral entry. This protocol details the generation of a competent hepatocyte model capable of screening for therapeutic agents targeting pre-viral entry and post viral entry lifecycle stages. This includes the targeting of sodium taurocholate cotransporting polypeptide (NTCP) binding, cccDNA formation, transcription, and viral assembly based on imHC or HepaRG as host cells. Here, the HBV entry inhibition assay used curcumin to inhibit HBV binding and transporting functions via NTCP. The inhibitors were evaluated for binding affinity (KD) with NTCP using isothermal titration calorimetry (ITC)-a universal tool for HBV drug screening based on thermodynamic parameters.

We present a protocol to screen anti-hepatitis B virus (HBV) compounds targeting pre- and post-viral entry lifecycle stages, using isothermal titration calorimetry to measure binding affinity (KD) with host sodium taurocholate cotransporting polypeptide. Antiviral efficacy was determined through the suppression of viral lifecycle markers (cccDNA formation, transcription, and viral assembly).

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load ...

Biochemistry

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV models are limited to hepatoma cell lines (such as HepG2 and Huh7) overexpressing a functional HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Here, we used 293T-NE-3NRs (293T&#160;overexpressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and HepG2-NE (HepG2 overexpressing NTCP) as model cell lines.&#160;HBV infection in these cell lines was performed either by using concentrated HBV virus particles from HepG2.2.15 or co-culturing HepG2.2.15 with the target cell lines. HBcAg immunofluorescence for HBcAg was performed to confirm HBV infection. The two methods presented here will help us study HBV infection in non-hepatic cell lines.&#160;&#160;

This manuscript describes a detailed protocol for Hepatitis B virus (HBV) infection in novel engineered 293T cells (293T-NE-3NRs, expressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and traditional hepatic cells (HepG2-NE, expressing human NTCP).

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV ...

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells.

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...

Biology

Simplified and Efficient AAV Production Using HEK293 Suspension Culture

Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100-fold higher titer (minimally 1 x 1011 genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor- and material-intensive process. This constraint prompted the development of a protocol for suspension-based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 1012 genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 1012 GC in total, and TransIt yielded 2.4 x 1013 GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.

Author Spotlight: Advancing Gene Therapy with High-Yield AAV Vectors Through HEK293 Suspension Cell Cultivation

Here, a suspension HEK293 cell-based AAV production protocol is presented, resulting in reduced time and labor needed for vector production using components that are available for research purposes from commercial vendors.

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, ...