Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by Real‐Time Reverse Transcription–PCR In Vivo: Detection of Abortive Viral Replication

Marina S. Boukhvalova1, Kevin C. Yim1, Gregory A. Prince1, Jorge C.G. Blanco1

1 Virion Systems, Rockville, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 26.6
DOI:  10.1002/0471143030.cb2606s46
Online Posting Date:  March, 2010
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Abstract

Viral infection is normally detected either by viral culture or by PCR methods. Rarely is a combination of the two techniques used in the same study. Yet, when applied simultaneously, viral culture and PCR may reveal important features of viral biology, such as an abortive replication, as in the case of respiratory syncytial virus (RSV) infection. In this unit, we describe methods for detecting abortive RSV replication in a cotton rat model by using the plaque‐forming unit assay and the real‐time reverse‐transcription PCR (qRT‐PCR) assay. All steps of the process of monitoring viral replication in vivo are described, starting from the design of animal infection protocols. We continue on to the methods for extracting and processing lung samples for viral culture and RNA extraction, and finish with the actual methods of viral titration by the qRT‐PCR and the plaque‐forming unit assays. Curr. Protoc. Cell Biol. 46:26.6.1‐26.6.19. © 2010 by John Wiley & Sons, Inc.

Keywords: abortive replication; RSV; cotton rat

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Plaque‐Forming Unit Assay of RSV Production
  • Basic Protocol 2: Real‐Time Reverse Transcription PCR (qRT‐PCR) Analysis of RSV Infection
  • Support Protocol 1: Infection of Cotton Rats with RSV and Harvesting and Processing of Lungs
  • Support Protocol 2: RSV Infection of A549 Epithelial Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Plaque‐Forming Unit Assay of RSV Production

  Materials
  • HEp‐2 cells (ATCC #ccL23)
  • HEp‐2 growth medium (see recipe)
  • Lung homogenates ( protocol 3)
  • Eagle minimal essential medium (EMEM) with Earle's Balanced Salt Solution (BSS)
  • Methyl cellulose overlay medium (see recipe)
  • Crystal violet fixative/stain (see recipe)
  • 24‐well tissue culture plates
  • Dissecting microscope
  • Additional reagents and equipment for basic cell culture techniques (unit 1.1)

Basic Protocol 2: Real‐Time Reverse Transcription PCR (qRT‐PCR) Analysis of RSV Infection

  Materials
  • RNeasy Midi kit (Qiagen) including:
    • RLT buffer
    • RNeasy Midi columns
    • Reagents for DNase treatment
  • RLT/ME buffer: RLT buffer from the RNeasy kit supplemented with 10 µl/ml 2‐mercaptoethanol
  • Frozen lung tissue for analysis ( protocol 3), including tissue from two animals sacrificed on day 4 after primary RSV infection for construction of standard curve
  • 70% ethanol
  • DEPC‐treated H 2O ( appendix 2A)
  • 0.5 µg/ml oligo(dT) primer
  • 10× PCR buffer (without MgCl 2; Invitrogen)
  • 50 mM MgCl 2 (Invitrogen)
  • 10 mM dNTP mix (Invitrogen)
  • 200 U/µl SR II reverse transcriptase (Invitrogen)
  • iQ SYBR Green Supermix (BioRad)
  • 10 µM forward and reverse PCR primers (Table 26.6.1)
  • 14‐ml polypropylene round‐bottom tubes
  • OMNI TH homogenizer with 7‐mm stainless‐steel generator probe (OMNI International)
  • 15‐ml conical polypropylene tubes
  • Centrifuge with plate carriers
  • 96‐well PCR plates
  • Thermal cycler
  • Bio‐Rad iCycler with iQ5 software
  • iQ 96‐well PCR plates (BioRad)
  • Microseal ‘B’ adhesive seals (BioRad)

Support Protocol 1: Infection of Cotton Rats with RSV and Harvesting and Processing of Lungs

  Materials
  • Cotton rats (S.hispidus, 4 to 8 weeks old; from a colony maintained at Virion Systems, Inc.)
  • RSV A/Long stock solution (>107 pfu/ml: original strain was obtained from ATCC (#VR26); virus was serially plaque purified to reduce defective‐interfering particles)
  • Phosphate‐buffered saline, pH 7.4 ( appendix 2A)
  • Liquid nitrogen
  • Homogenization medium (see recipe)
  • Isoflurane anesthesia chamber (Davis, )
  • 1‐ml syringes
  • 21‐G needles
  • CO 2 chamber (Donovan and Brown, )
  • Dissecting forceps
  • Dissecting scissors
  • Sterile Tenbroeck tissue homogenizers
  • Sterile polystyrene round‐bottom tubes with caps, 12 × 75 mm
  • Centrifuge, refrigerated
  • Sterile 4‐ml glass sample vials with caps
  • Additional reagents and equipment for isoflurane anesthesia in rodents (Davis, ) and euthanasia of rodents by CO 2 asphyxiation (Donovan and Brown, )

Support Protocol 2: RSV Infection of A549 Epithelial Cells

  Materials
  • Human alveolar epithelial type II cell (A549) culture (ATCC #CCL185)
  • A549 growth medium (see recipe)
  • RSV A/Long stock solution (>107 pfu/ml): original strain was obtained from ATCC (#VR26); virus was serially plaque purified to reduce defective‐interfering particles
  • DMEM medium (serum‐free; e.g., Invitrogen)
  • RNeasy Midi kit (Qiagen) including:
    • RLT buffer
    • RNeasy Midi columns
    • Reagents for DNase treatment
  • RLT/ME buffer: RLT buffer from the RNeasy Midi kit (Qiagen) supplemented with 10 µl/ml 2‐mercaptoethanol
  • 70% ethanol
  • PCR primers for viral genes and β‐actin (Table 26.6.1)
  • 75‐ to 175‐cm2 tissue culture flasks
  • 6‐well tissue culture plates
  • Additional reagents and equipment for basic cell culture techniques (unit 1.1), plaque assay ( protocol 1), and preparation/PCR amplification of cDNA ( protocol 2)
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.NOTE: All cell culture incubations should be carried out in a 37°C, 5% CO 2 humidified incubator.
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Figures

  •   FigureFigure 26.6.1 Establishing real‐time PCR assay of RSV replication. (A) Amplification of various RSV genes from RSV antigenome inserted into a vector. Six 10‐fold dilutions of antigenome‐containing vector were used to generate a standard curve. Ct values were plotted against log10 quantity of antigenome used as a template. Correlation coefficients ( R2) of the best fit are shown next to the symbols corresponding to the 7 RSV genes analyzed. Each point on this graph includes symbols for all 7 genes. (B) Expression of RSV genes in infected A549 cells. A549 cells were infected with RSV at MOI 0.1. At time points 6 and 24 hr after infection, total RNA was extracted from infected cells and reverse transcribed using oligo(dT) primer. Expression of mRNA for each RSV gene indicated was measured by qRT‐PCR. The relative level of each amplicon was normalized by β‐actin mRNA level (also measured by qRT‐PCR) in the corresponding sample, and expressed relative to the level of NS1 amplicon at 24 hr. The insert shows a gradient of RSV gene expression at 6 hr on an adjusted y ‐scale to highlight differences between levels of various amplicons. The results represent the mean ±SEM for two different wells per time point.
  •   FigureFigure 26.6.2 RSV replication in vivo: evidence of abortive replication in the reinfection model. Cotton rats were infected with RSV once for primary infection studies or twice (with an interval of 21 days) for secondary infection studies. At the indicated time points after the final challenge, lungs were collected for viral titer determination by plaque assay (A) or for analysis of RSV replication by qRT‐PCR (B, C, D). (A) Pulmonary viral titers in cotton rats with primary RSV infection (“prim”, light gray bars) and secondary RSV infection (“sec”, no virus detected). Viral titers were determined by plaque assay as previously described (Prince et al., ; limit of detection 2 log10 pfu/g). (B) RSV genome and antigenome replication in primary and secondary RSV infection. RNA was extracted from the lungs of infected animals and reversed transcribed using primers complementary to the genomic or antigenomic strand of RSV. Relative genome (and antigenome, insert) amounts were determined by qRT‐PCR, normalized by the β‐actin mRNA level in the corresponding sample, and expressed relative to the level of genome (antigenome, for insert) detected in the lungs of animals with primary RSV infection on day 4. (C) Expression of RSV genes during primary and secondary RSV infection. RNA extracted from animals described above was reverse‐transcribed using oligo (dT) primer. Expression of NS1 mRNA was measured by qRT‐PCR, normalized by β‐actin as described above, and expressed relative to the level of NS1 mRNA detected in the lungs of animals with primary RSV infection on day 4. (D) Gradient of RSV gene expression at 12 hr post‐infection in primary and secondary RSV disease. NS1, M, and L amplicon levels were measured by qRT‐PCR in cDNA prepared using oligo(dT) primer. Expression of each amplicon was normalized by β‐actin, and expressed relative to the level of NS1 mRNA detected in the lungs of animals with primary RSV infection. The results represent the mean ±SEM for four animals per time point for each primary and secondary infection.

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Literature Cited

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