This work was supported in part by the Research Program on Hepatitis from Japan Agency for Medical Research and Development (AMED) and by Grants-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

1. Production of Recombinant HBV Encoding the Reporter Protein
<ol>
	<li>Preparation of HepG2 cells
	<ol>
		<li>Prepare cell culture medium (Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 &#181;g/ml streptomycin, and 100 U/ml nonessential amino acids).</li>
		<li>Plate 4 x 106 HepG2 cells in a 10-cm collagen-coated dish in 10 ml of culture medium the day before transfection. Incubate HepG2 cells at 37 &#176;C in a humidified 5% CO2 incubator. 
		NOTE: When approximately 4 x 106 HepG2 cells are plated in a 10 cm collagen-coated dish in 10 ml of culture medium, they will be 70-90% confluent the next day.</li>
	</ol>
	</li>
	<li>Transfection
	<ol>
		<li>Transfect HepG2 cells with 5 &#181;g of pUC1.2HBV delta epsilon15 and 5 &#181;g of pUC1.2HBV/NL15 using a transfection reagent as per manufacturer&#39;s instructions.</li>
		<li>The next day, remove the culture medium and add 10 ml of fresh culture medium.</li>
		<li>One week after transfection, transfer the culture medium containing the recombinant HBV to a 50 ml-tube and proceed to step 1.3.1. Add 10 ml of fresh culture medium to the plate containing the transfected cells. 
		NOTE: Production of the recombinant HBV is maintained for 4 weeks. The culture medium containing recombinant HBV can be stored at 4 &#176;C for 1 month.</li>
	</ol>
	</li>
	<li>Purification of recombinant HBV
	<ol>
		<li>Remove cell debris from the culture medium containing recombinant HBV by centrifugation (2,300 x g for 5 min).</li>
		<li>Pass the supernatant through a 0.45 &#181;m membrane filter.</li>
		<li>Add an equal volume of 26% PEG/1.5 M NaCl (polyethylene glycol 6000: 130 g, NaCl, 49 g, 1 ml of 0.5 M EDTA pH 8.0 and 5 ml of 1 M HEPES pH 7.6 in 500 ml) to the culture medium containing recombinant HBV and mix gently. Incubate overnight at 4 &#176;C.</li>
		<li>Centrifuge at 2,300 x g for 20 min at 4 &#176;C.</li>
		<li>Discard the supernatant and dissolve the pellet in 0.5 ml of TNE (10 mM Tris, 50 mM NaCl, 1 mM EDTA).</li>
		<li>Remove the debris by centrifugation at 2,300 x g for 5 min.</li>
		<li>Load 0.5 ml of TNE containing recombinant HBV onto 0.8 ml of 20% sucrose in TNE.</li>
		<li>Centrifuge at 100,000 &#215; g for 3 hr at 15 &#176;C.</li>
		<li>Discard as much of the supernatant as possible, and save the pellet. Resuspend it in 1 ml of serum free DMEM per 40 ml of starting culture medium.</li>
		<li>Incubate overnight at 4 &#176;C.</li>
		<li>Filter through a 0.45 &#181;m filter. Prepare 0.5 ml aliquots and store at -80 &#176;C. 
		NOTE: If reporter protein contamination of the original virus sample is observed, purify the virus by density gradient ultra-centrifugation of 5-30% sucrose in TNE at 100,000 x g for 2 hr, or CsCl density equilibrated centrifugation from 1.1-1.6 g/ml at 150,000 x g for 50 hr.</li>
	</ol>
	</li>
</ol>
2. Infection of Recombinant HBV
<ol>
	<li>Culture HepG2 cells stably expressing NTCP (HepG2/NTCP)15 that are susceptible to HBV infection in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 &#181;g/ml streptomycin, 250 &#181;g/ml G-418 and 100 U/ml nonessential amino acids at 37 &#176;C in a humidified 5% CO2 incubator.</li>
	<li>One day before infection, plate approximately 5 x 104 HepG2/NTCP cells14 into a 96-well collagen coated plate in 0.1 ml of culture medium.</li>
	<li>Thaw recombinant HBV in a 37 &#176;C water bath until there is a small bit of ice remaining in the vial.</li>
	<li>Prepare the medium for infection by combining the following:10 &#181;l of 40% PEG8000 in 1x phosphate buffered saline (PBS), 2 &#181;l of dimethyl sulfoxide (DMSO), 10 &#181;l of recombinant HBV and 78 &#181;l of fresh culture medium per well of a 96-well plate.</li>
	<li>Add 100 &#181;l of recombinant HBV solution to one well of the 96-well plate.</li>
	<li>One day after infection, wash the infected cells 3 times with 300 &#181;l PBS per well to remove the contaminating reporter protein from the virus fraction.</li>
	<li>Incubate infected cells in 200 &#181;l of culture medium containing 2% DMSO for one week or less.</li>
</ol>
3. Analysis
<ol>
	<li>One week after infection, wash the infected cells 3 times with PBS.</li>
	<li>Add 50 &#181;l of lysis buffer to the infected cells.</li>
	<li>Rock the culture plate for 5 min, and then centrifuge at 2,000 x g for 5 min.</li>
	<li>Add 50 &#181;l of the reporter substrate into the luminometer plate.</li>
	<li>Add 20 &#181;l of cell lysate to a luminometer plate containing the reporter substrate. Mix by vortexing briefly.</li>
	<li>Place the plate in the luminometer and initiate reading15.</li>
</ol>

<ol>
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The authors declare that they have no competing financial interests.

The HBV genome has four primary open reading frames (ORFs) including the core, polymerase, surface and X ORFs (Figure 1). Transcription of these four HBV ORFs is tightly regulated by four promoters: the precore/core promoter containing enhancer II, S1 promoter, S2 promoter and X promoter containing enhancer I18. The 3.5 kb and 3.4 kb mRNAs are translated into PreCore and Core proteins, respectively. The large envelop protein (L) is produced from the largest subgenomic mRNA (2.5 kb...

Chronic infection with hepatitis B virus (HBV) is a major risk factor for chronic liver diseases1. Although current therapeutic strategies are based on nucleotide analogs that inhibit HBV pol function and/or the administration of type I interferon that activates immune responses in infected individuals as well as indirectly suppressing HBV proliferation through interferon-stimulated gene functions2, these treatments cannot eliminate HBV DNA completely3. Moreover, the emergence of HBVs that are resistant to anti-pol agents is of concern4. The administration of combined anti-viral agents directly targeting the different steps of human immunodeficiency virus (HIV) or hepatitis C virus (HCV) life cycle was shown to successfully suppress or eradicate the virus(es). Similar to this idea, the development of anti-HBV agent(s) that act directly on the different stages of the HBV life cycle is important for establishing future HBV therapies.
In general, the establishment of a simple in vitro culture system of the target virus facilitates the development of anti-virus agents. However, there are at least two barriers to the development of in vitro culture systems to screen anti-HBV agents. The first is the lack of a convenient in vitro cell culture system for HBV infection/proliferation. Unlike other viruses, such as HIV and HCV, which are propagated in established cell lines, it is difficult to cultivate HBV in vitro because of experimental limitations including a narrow host range. The use of specific cell culture systems such as the human hepatoma cell line HepaRG, which is susceptible to HBV infection,5,6,7 have been developed to overcome these problems. Moreover, PXB cells, isolated from urokinase-type plasminogen activator transgenic/SCID mice inoculated with primary human hepatocyte (PHH), were shown to be susceptible to HBV infection and replication8. However, HBV replication levels in HepaRG are dependent on the cellular differentiation state after culture, which can cause inconsistent and irreproducible results of HBV infection/replication levels. PXB is commonly used for HBV infection experiments but is limited by its availability. A tetracycline inducible HBV expression cell line, HepAD38, has also been widely used to study HBV replication, but this system only allows evaluation after transcription and not at the entry step of HBV infection9. Recently, the identification of sodium taurocholate cotransporting polypeptide (NTCP) as a functional receptor for HBV has allowed the development of a variable HBV culture system10. Indeed, NTCP expression in non-susceptible hepatocarcinoma cells such as Huh7 and HepG2 enables HBV infection10 and thus, the choice of HBV susceptible cell lines has been expanded, resolving many of the experimental limitations. The second problem is the lack of a simple assay system to evaluate HBV infection and replication. Evaluation of HBV infection is usually conducted by analyzing HBV DNA, RNA and proteins. However, quantification of these virus markers is time consuming, often costly and not always simple. Therefore, the development of a simple assay system, such as using a reporter gene, might overcome problems associated with HBV assay systems.
However, because the genome size that can be packaged into an HBV capsid is limited &#8212; less than 3.7 kb11 &#8212; the size of a reporter gene should be as short as possible. Furthermore, the presence of multiple cis elements scattered throughout the genome, which are essential for viral replication, limits the positions available for insertion of the reporter gene into the genome. Several reports have attempted to insert foreign genes, including HIV-1 Tat, green fluorescent protein, and DsRed, into the HBV genome11,12,13. However, these recombinant HBVs are not useful for screening HBV infection/replication, or for the high-throughput screening of factors affecting HBV infection/replication. This is mainly because of the low productivity of recombinant viruses and the reduced intensity of reporter gene expression caused by inefficient virus production.
To overcome these issues, we constructed a reporter HBV with a high yield of virus production. This virus is highly sensitive for monitoring the early stages of the HBV replication cycle, from entry to transcription. To achieve this, NanoLuc (NL) was chosen as a marker gene because it is a small (171 amino acids) engineered luminescent reporter14. Moreover, NL is approximately 150-fold brighter than firefly or Renilla luciferase, and the luminescent reaction is ATP-independent, suggesting that the false hit rate will be low for high-throughput screening. The production efficiency of the recombinant HBV is approximately 1/5 of the parent HBV, and similar to levels reported for previous HBV recombinant viruses; however, the brightness of NL overcomes virus productivity issues so it can be used for the mass screening of anti-HBV agents.
Screening of anti-HBV agents using primary hepatocytes, HepaRG, HepAD38 and NTCP-transduced hepatocytes might be useful for the screening of anti-HBV agents by conventional method(s). However, the system described here has various advantages such as simple handling, high sensitivity, and low cost for screening. These advantages are suitable for high throughput assays to develop and identify new HBV agents for therapeutic purposes.

<table border="0" cellpadding="0" cellspacing="0" width="781">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">Nano-Glo Luciferase Assay Regent</td>
			<td style="width:201px;">Promega</td>
			<td style="width:160px;">N1110</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin Mixed Solution</td>
			<td>Nacalai tesque</td>
			<td>09367-34&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM Non-Essential Amino Acids Solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>11995065</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Opti-MEM I Reduced Serum Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:332px;">100 mm/collagen-coated dish</td>
			<td>Iwaki</td>
			<td>4020-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipofectamine 3000 Transfection Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>L3000001&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyethylene glycol (PEG) 6000&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>81255</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyethylene glycol (PEG) 8000&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>89510</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Nacalai tesque</td>
			<td>31319-45&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5 mol/l-EDTA Solution</td>
			<td>Nacalai tesque</td>
			<td>06894-14&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris-HCl</td>
			<td>Nacalai tesque</td>
			<td>35434-21</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Millex-HP, 0.45 &#956;m, polyethethersulfone, filter</td>
			<td>Merck Millipore</td>
			<td>SLHP033RS</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagen coated 96-well plate</td>
			<td>Corning</td>
			<td>NO3585</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Passive Lysis 5xBuffer</td>
			<td>Promega</td>
			<td>E1941</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GloMax 96 Microplate Luminometer</td>
			<td>Promega</td>
			<td>E6501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>Nacalai tesque</td>
			<td>30403-55</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Luminometer plate</td>
			<td>Greiner bio-one</td>
			<td>655075</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HepG2-NTCP1-myc-clone22</td>
			<td>-</td>
			<td>-</td>
			<td>Reference 15</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pUC1.2HBV delta epsilon</td>
			<td>-</td>
			<td>-</td>
			<td>Reference 15</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pUC1.2HBV/NL</td>
			<td>-</td>
			<td>-</td>
			<td>Reference 15</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 ml tube</td>
			<td>Violamo</td>
			<td>1-3500-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-Myc antibody</td>
			<td>Sigma-Aldrich</td>
			<td>C3956</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBIG</td>
			<td>Japan Blood Products Organization</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IFN-&#946;</td>
			<td>Mochida Pharmaceutical</td>
			<td>14987224005413</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H3393</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Figure 1 shows a schematic of the HBV genome, transcribed RNAs, virus proteins and their coding region on the genome. The reporter gene and its location in the genome are also indicated. Figure 2 shows the HBV reporter plasmid and helper plasmid containing the reporter gene. The pUC1.2HBV/NL reporter was constructed by deleting nucleotide positions 223-811 from the transcription initiation site of the preCore mRNA of pUC1.2HBV16 an...

Watch this Scientific Journal Video about Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle at JoVE.com

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

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), ...

development-hepatitis-b-virus-reporter-system-to-monitor-early-stages

Research

JoVE Journal

Immunology and Infection

13.1K Views.  National Center for Global Health and Medicine. 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.

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

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

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 ...

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony forming units (CFU-Es). After CFU-Es, cells are morphologically recognizable and generally termed terminal erythroblasts. One of the challenges for the study of terminal erythropoiesis is the lack of experimental approaches to dissect gene functions in a chronological manner. In this protocol, we describe a unique strategy to determine gene functions in the early and late stages of terminal erythropoiesis. In this system, mouse fetal liver TER119 (mature erythroid cell marker) negative erythroblasts were purified and transduced with exogenous expression of cDNAs or small hairpin RNAs (shRNAs) for the genes of interest. The cells were subsequently cultured in medium containing growth factors other than erythropoietin (Epo) to maintain their progenitor stage for 12 hr while allowing the exogenous cDNAs or shRNAs to express. The cells were changed to Epo medium after 12 hr to induce cell differentiation and proliferation while the exogenous genetic materials were already expressed. This protocol facilitates analysis of gene functions in the early stage of terminal erythropoiesis. To study late stage terminal erythropoiesis, cells were immediately cultured in Epo medium after transduction. In this way, the cells were already differentiated to the late stage of terminal erythropoiesis when the transduced genetic materials were expressed. We recommend a general application of this strategy that would help understand detailed gene functions in different stages of terminal erythropoiesis.

We present an in vitro mouse fetal liver erythroblast culture system that dissects the early and late stages of terminal erythropoiesis. This system facilitates functional analysis of specific genes in different developmental stages.

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a three host tick that requires a blood meal from a vertebrate host for each development stage, and the adult females require a blood meal for reproduction. Larval ticks attach to their host for 3 - 5 days for feeding and drop off the host when fully engorged. This dependency on several different hosts and the lengthy attachment time for engorgement complicates tick rearing in the laboratory setting. However, to understand tick biology and tick-pathogen interactions, the production of healthy, laboratory-reared ticks is essential. Here, we demonstrate a simple, cost-effective protocol for immature tick feeding on mice. We modified the existing protocols for decreased stress on mice and increased tick feeding success and survival by using disposable cages without mesh bottoms to avoid contact of ticks with water contaminated with mice urine and feces.

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

The production of healthy laboratory-reared ticks is essential to studies on tick biology, and tick-pathogen interactions. Here we demonstrate a simple protocol for immature tick feeding that is cost-effective and less stressful to mice.

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Assembly and Purification of Prototype Foamy Virus Intasomes

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an integrase (IN) enzyme that catalyzes the covalent joining of viral to host DNA, which is known as strand transfer. Integration may be modeled in vitro with recombinant retroviral IN and DNA oligomers mimicking the ends of the viral genome. In order to more closely recapitulate the integration reaction that occurs in vivo, integration complexes are assembled from recombinant IN and synthetic oligomers by dialysis in a reduced salt concentration buffer. The integration complex, called an intasome, may be purified by size exclusion chromatography. In the case of prototype foamy virus (PFV), the intasome is a tetramer of IN and two DNA oligomers and is readily separated from monomeric IN and free oligomer DNA. The integration efficiency of PFV intasomes may be assayed under a variety of experimental conditions to better understand the dynamics and mechanics of retroviral integration.

Recombinant retroviral integrase and DNA oligomers mimicking viral DNA ends can form an enzymatically active complex known as an intasome. Intasomes may be used for biochemical, structural, and kinetic studies. This protocol details how to assemble and purify prototype foamy virus intasomes.

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...