RSV A2 was kindly provided by Dr. R. de Swart (Erasmus MC, Rotterdam, The Netherlands). MV and GF are supported by the Virgo consortium, funded by the Dutch government project number FES0908, and by the Netherlands Genomics Initiative (NGI) project number 050-060-452. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Note:
Experiments with HRSV must be performed in a biological safety cabinet in a biosafety level 2 laboratory. Collect all viral waste and material that has been in contact with the virus for proper disposal due to biological hazards.
Special precaution must be taken when using potentially infected human blood.
It is crucial to use endotoxin-free reagents throughout all protocols.
1. Culturing of HRSV A2
<ol>
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<ol>
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In this study, we demonstrate that infection of PBMC with HRSV is a fast and reliable model system in which inflammatory pathways can be studied. In order to stimulate PBMCs an HRSV stock has to be prepared and quantified.
Multiple cell lines are susceptible to HRSV infection. Cell lines most used for HRSV culturing are HEp-2, HeLa,&#160;and Vero cells. However, we would not recommend the usage of Vero cells for culturing of HRSV because it has been shown that culturing of HRSV in Vero cells r...

Human Respiratory Syncytial Virus (HRSV) is the most common cause of lower respiratory tract infections in children. Each year, over 33 million children under the age of five are infected with HRSV, leading to over three million hospitalizations and almost 200,000 deaths1. A growing body of evidence suggests that HRSV also poses a significant threat to the elderly and adults with underlying chronic illnesses2.
The majority of HRSV infections in young children presents with mild symptoms, comparable to the common cold, and do not require clinical intervention. However, a small proportion of patients require hospitalization and mechanical ventilation due to severe bronchiolitis.
Part of the pathogenesis of HRSV disease is the host&#39;s overexuberant and inadequate immune response to infection3,4. This is illustrated by several observations. The period of maximal illness is often preceded by the peak of viral infection and coincides better with cellular infiltration of infected tissues and the release of inflammatory cytokines3. Another line of evidence comes from the formalin inactivated HRSV (FI-RSV) trials in the 1960s. Instead of inducing protection in young infants, the vaccine resulted in an exaggerated immune response resulting in enhanced respiratory disease and higher morbidity and mortality.
Several models have been developed to study the pathogenesis of HRSV infections. Continuous cell lines, like HEp-2 and A549, have been used extensively to study HRSV infection in vitro. Epithelial cells are the primary targets of HRSV infection5, therefore a lot of focus has been on these cell types. However, the in vivo situation is much more complex and not limited to one cell type. In order to examine these complex interactions, several animal infection models have been developed to study HRSV pathogenesis. Thus far, two different strategies have been employed, focusing either on heterologous (nonhuman) models as well as cognate host-pneumovirus models. Examples of heterologous models for HRSV include chimpanzees, sheep, cotton rats and mice. RSV infection in its natural host has been studied in cattle and in mice, using bovine RSV and pneumovirus, respectively.
Whilst each of these models has provided important insights into disease pathogenesis, HRSV is highly adapted to humans. Therefore, as an addition to the cell lines and animal models currently used, we propose to use human primary PBMCs as a model for stimulation with HRSV. Human PBMCs can be obtained from a range of individuals to address specific research questions, including young children6, immunocompromised individuals as well as healthy adults7 or elderly individuals8. PBMCs can be used to study innate aspects of infection as well as the adaptive response to the virus. Activated inflammatory pathways important for the pathogenesis of HRSV disease can be studied at the mRNA and protein level. Further, viral infection of (subsets of) immune cells can be determined by flow cytometry. We have used this model previously to identify the synergistic effects of HRSV infection together with the bacterial ligand muramyl dipeptide (MDP) on the induction of proinflammatory cytokines7. We propose to use HRSV stimulation of human primary PBMCs as a robust, easy and fast in vitro model to study inflammatory pathways involved in the pathogenesis of HRSV disease.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Lonza</td>
			<td>BE17-516F</td>
			<td>PBS without Ca2+ Mg2+ or phenol red</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Invitrogen</td>
			<td>14025-100</td>
			<td>HBSS, Calcium, Magnesium, no Phenol Red</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>31966-047</td>
			<td>DMEM, High Glucose, GlutaMAX, Pyruvate</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Invitrogen</td>
			<td>72400054</td>
			<td>RPMI 1640 Medium, GlutaMAX, HEPES</td>
		</tr>
		<tr>
			<td>Reduced Serum Medium (Opti-MEM)</td>
			<td>Invitrogen</td>
			<td>51985-026</td>
			<td>Opti-MEM I Reduced Serum Medium, GlutaMAX</td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Greiner Bio-one</td>
			<td></td>
			<td>FBS (Fetal Bovine Serum)</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich</td>
			<td>A7030</td>
			<td>Albumin from bovine serum</td>
		</tr>
		<tr>
			<td>P/S</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>Penicillin-Streptomycin, liquid</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Invitrogen</td>
			<td>25300-054</td>
			<td>0.05% Trypsin-EDTA (1x), Phenol Red</td>
		</tr>
		<tr>
			<td>HeLa cells</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>A549 cells</td>
			<td>ATCC</td>
			<td>CCL-185</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S7903</td>
			<td>Sucrose BioXtra, &#8805;99.5%</td>
		</tr>
		<tr>
			<td>IgG2 anti-mouse-FITC</td>
			<td>BD Pharmingen</td>
			<td>555057</td>
			<td>Ms IgG2b K FITC isotype control</td>
		</tr>
		<tr>
			<td>Anti-NP-RSV-FITC</td>
			<td>Abcam</td>
			<td>ab25849</td>
			<td>Anti-Respiratory Syncytial Virus antibody [671] (FITC)</td>
		</tr>
		<tr>
			<td>Density gradient medium (Lymphoprep)</td>
			<td>Axis-Shield</td>
			<td>1114545</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA tubes</td>
			<td>BD Biosciences</td>
			<td>367525</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Merck Millipore</td>
			<td>1000141000</td>
			<td>Acetone for analysis</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Material </td>
		</tr>
		<tr>
			<td>Centrifuge Tubes</td>
			<td>Beckman Coulter</td>
			<td>326823</td>
			<td>Thinwall, Polyallomer, 38.5 ml, 25 mm x 89 mm</td>
		</tr>
		<tr>
			<td>SureSpin 630 Rotor with 36 ml buckets</td>
			<td>Sorvall</td>
			<td>79368</td>
			<td>Swinging bucket titanium rotor</td>
		</tr>
		<tr>
			<td>Sorvall WX80 Ultracentrifuge</td>
			<td>Sorvall</td>
			<td>46900</td>
			<td></td>
		</tr>
		<tr>
			<td>CB 150l CO2 Incubator</td>
			<td>Binder</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro model to study immune responses of human peripheral blood mononuclear cells to human respiratory syncytial virus infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

The HRSV A2 cultivation and purification method described here will give a yield between 2 x&#160;107 and 2 x&#160;108 FITC-50 quantified infectious viral particles/ml. Results of microscopic analysis, where FITC-stained infected cells were counted (results not shown), were compared to results of quantification by FACS (Figure 1). Both methods resulted in similar titers, although the FACS method had a much lower standard deviation between different experiments and was less laborious...

Watch this Scientific Journal Video about An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection at JoVE.com

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

an-vitro-model-to-study-immune-responses-human-peripheral-blood

Research

JoVE Journal

Immunology and Infection

7.8K vistas.  Radboud university medical center. Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.