assay to determine the virus infectious dose of drosophila c virus in cultured insect cells

Assay to Determine the Virus Infectious Dose of Drosophila C Virus in Cultured Insect Cells

To determine the average virus tissue culture infective dose, first, seed 1 x 105 S2 star-cells in 100 microliters of complete medium into eight wells per column in 12 rows of a 96-well plate. Then, return the plate to the 25-degree Celsius incubator.
Randomly select five tubes of virus from minus 80 degrees Celsius storage. While the cells are settling, fill 10 sterile 1.5-milliliter microcentrifuge tubes with 450 microliters of sterile complete medium, and add 50 microliters of virus stock to the first 1.5-milliliter tube containing 450 microliters of sterile complete medium, diluting the suspensions tenfold per step to the 12th well.
At the end of the incubation add 50 microliters of each dilution to the appropriate well in each column for that tube of virus, and add 50 microliters of culture medium without virus to one well per column, as the negative control well. Then, place the plate in the 25-degree Celsius incubator for three days, and assess the cytopathic effect of each virus stock under a bright field microscope at a 20 times magnification daily.
Classify a well in which the cells look blurry and the medium is full of fragments as a positive well, and a well in which the cell morphology is normal as a negative well.

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1. Drosophila C virus load measure by CPE assay
<ol>
	<li>Pick 5 random virus-containing tubes to determine the average virus tissue culture infective dose (TCID50) by a cytopathic effect (CPE) assay (1 unit of TCID50= 0.7 plaque-forming unit [PFU]). 
	NOTE: CPE refers to structural changes in host cells caused by viral invasion. The infecting virus causes lysis of the host cell or when the cell dies without lysis due to an inability to reproduce.</li>
	<li>On day 1, collect prepared S2* cells by pipetting. Count the cell number. Centrifuge cells for 300 x g for 4 min and discard the supernatant. Dilute the cells to the density of 1 x 106 cells/mL with a complete medium for S2* cells.</li>
	<li>Seed 1 x 105 S2*cells (100 &#956;L) per well to the flat-bottom 96-well-plate (~30% confluency).</li>
	<li>Perform serial 10-fold dilutions of the DCV stock with complete medium for S2* cells. Add 50 &#181;L dilutions into the well. Add 50 &#181;L of culture medium without virus to the well classified as the negative control well. 
	NOTE: For each dilution rate, 8 wells were used. As the typical DCV yield is around 108-109 PFU/mL, the dilution should at least cover ~10&#8722;5-10&#8722;10.</li>
	<li>On day 4, observe CPE with a bright-field microscope with a 20X or 40X objective. Classify a well in which the cells look blurry and the medium is full of fragments as a &#34;positive well&#34;, and a well in which the cell morphology is normal as a &#34;negative well&#34;.</li>
	<li>Mark CPE positive or negative wells with + or &#8211;, respectively. Calculate the virus TCID50 by the Reed and Muench method.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 ml Microcentrifuge tubes</td>
			<td>Brand</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm cell culture dish</td>
			<td>Sigma</td>
			<td>CLS430167</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Cell Incubator</td>
			<td>Sanyo</td>
			<td>MIR-553</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424R</td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila Incubator</td>
			<td>Percival</td>
			<td>I-41NL</td>
			<td>Rearing Drosophila</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Invitrogen</td>
			<td>12657-029</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Schneider&#39;s Insect Medium</td>
			<td>Sigma</td>
			<td>S9895</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Statistical software</td>
			<td>GraphPad Prism 7</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flat bottom 96-well-plate</td>
			<td>Sigma</td>
			<td>CLS3922</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Cell Incubator</td>
			<td>Sanyo</td>
			<td>MIR-553</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>CKX41</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Yang, S. et al., <a href="https://app.jove.com/v/58845">Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster.</a> J. Vis. Exp. (2019)
In this video, we demonstrate the cytopathic effect assay to determine the tissue culture infective viral dose of Drosophila C virus on Drosophila S2* cells. A range of viral concentrations are used to infect the cultured cells. The proportion of cells exhibiting structural changes is determined, and this data is used to calculate the tissue culture infective dose.

1. Drosophila C virus load measure by CPE assay

	Pick 5 random virus-containing tubes to determine the average virus tissue culture infective dose ...

Drosophila C virus, or DCV, a positive single-stranded RNA virus, commonly infects Drosophila melanogaster.
During infection, DCV uses host cell machinery to replicate the viral genome, eventually producing virus particles. Infected cells exhibit cytopathic effects, CPE, and undergo lysis or die without lysis due to their inability to divide.
To determine the average virus tissue culture infective dose, the DCV concentration at which half the infected cells display CPE, begin with serially diluted DCV suspensions.
Transfer to a multi-well plate containing semi-adherent Drosophila S2* cells derived from late-stage embryos of Drosophila melanogaster, a natural DCV host. Wells containing medium and cells serve as negative controls.
Incubate for the virus to infect the cells. Virus-infected cells display CPE via cell damage and death. Post-incubation, observe the wells under the microscope.
Identify CPE-positive and negative wells across dilutions. Use a suitable method to determine the average virus tissue culture infective dose of DCV.

35 Aufrufe. Assay zur Bestimmung der infektiösen Virusdosis des Drosophila-C-Virus in kultivierten Insektenzellen

Serie: Assay zur Bestimmung der infektiösen Virusdosis des Drosophila-C-Virus in kultivierten Insektenzellen - Experiment

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

Erzeugung, Verstärkung und Titration von rekombinanten Respiratory Syncytial Viren

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

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JoVE Journal

Immunologie und Infektion

Plaquing of Herpes Simplex Viruses

There are numerous published protocols for plaquing viruses, including references within primary literature for methodology. However, plaquing viruses can be difficult to perform, requiring focus on its specifications and refinement. It is an incredibly challenging method for new students to master, mainly because it requires meticulous attention to the most minute details. This demonstration of plaquing herpes simplex viruses should help those who have struggled with visualizing the method, especially its nuances, over the years. While this manuscript is based on the same principles of standard plaquing methodology, it differs in that it contains a detailed description of (1) how best to handle host cells to avoid disruption during the process, (2) a more useful viscous medium than agarose to limit the diffusion of virions, and (3) a simple fixation and staining procedure that produces reliably reproducible results. Furthermore, the accompanying video helps demonstrate the finer distinctions in the process, which are frequently missed when instructing others on conducting plaque assays.

Plaquing is a routine method used to quantify live viruses in a population. Though plaquing is frequently taught in various microbiology curricula with bacteria and bacteriophages, plaquing of mammalian viruses is more complex and time-consuming. This protocol describes the procedures that function reliably for regular work with herpes simplex viruses.

Plaquierung von Herpes-simplex-Viren

There are numerous published protocols for plaquing viruses, including references within primary literature for methodology. However, plaquing viruses can ...

Use of Crystal Violet to Improve Visual Cytopathic Effect-based Reading for Viral Titration using TCID50 Assays

Viral titration is a key assay for virology research. The detection of cytopathic effect (CPE) via TCID50 assays and plaque-forming units (PFU) assays are the two main methods to calculate the titer of a virus stock and are often based on microscopy detection or cell staining for visualization. In the case of TCID50 assay, objective visualization is commonly based on immunocytochemical (ICC) staining of intracellular virus to calculate titers combined with visual CPE detection via microscopy. However, ICC staining is costly and time consuming. In this study, we compared visual CPE observation via microscopy, ICC staining and crystal violet staining to determine the titers of two CPE-forming viruses, Influenza A virus (IAV) of swine origin and Porcine Reproductive and Respiratory Syndrome virus (PRRSV). We show that both crystal violet and ICC staining are more accurate than visual CPE detection, presenting nearly identical levels of precision on both IAV and PRRSV. For this reason, here we present crystal violet staining as a faster and more affordable way to determine viral titrations on a TCID50 assay for CPE-forming viruses titrated in cell lines.

This protocol shows an accurate and objective approach to visualize viral titrations using crystal violet, by comparing it with optical microscopy and immunocytochemical staining.

Verwendung von Crystal Violet zur Verbesserung des visuellen zytopathischen Effekt-basierten Lesens für die virale Titration mit TCID50-Assays

Viral titration is a key assay for virology research. The detection of cytopathic effect (CPE) via TCID50 assays and plaque-forming units (PFU) assays are ...

Assay to Determine the Virus Infectious Dose of Drosophila C Virus in Cultured Insect Cells

Transkript

Assay to Determine the Virus Infectious Dose of Drosophila C Virus in Cultured Insect Cells