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

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.

Antibody response to the central unglycosylated region of the respiratory syncytial virus attachment protein in mice

“…[15,16] Clarified extracts of infected cells were titered for RSV and stored at −80°C until use within 2 months. [17,18]…”

Limited Type I Interferons and Plasmacytoid Dendritic Cells during Neonatal Respiratory Syncytial Virus Infection Permit Immunopathogenesis upon Reinfection

“…At 4 days after the initial RSV infection, lungs were isolated, weighed, and frozen until analysis by either plaque assay (24) or real-time reverse transcription-PCR (RT-PCR) methods (25). For plaque assays, supernatants from centrifuged lung homogenates were added onto Vero cells and overlaid with 0.3% agarose (MP Biochemicals, Santa Ana, CA).…”

“…Real-time RT-PCR was then performed using a SuperScript III Platinum one-step quantitative RT-PCR (qRT-PCR) kit from Life Technologies (Carlsbad, CA). The primers (25) used for the reaction were NS1 forward primer (5=-CACAACAATGCCAGTGCTACAA-3=) and NS1 reverse primer (5=-TTAGACCATTAGGTTGAGAGCAATGT-3=).…”

Respiratory syncytial virus infection of primary human mast cells induces the selective production of type I interferons, CXCL10, and CCL4

“…Plaque assays were performed as previously described. 42,43 Fluorescence-activated cell sorting staining and analysis For CD117/c-Kit staining, CBMCs were incubated with allophycocyaninconjugated anti-human CD117 mAb or mouse IgG 1 (eBioscience, San Diego, Calif) at a concentration of 1 mg/mL. For intracellular RSV antigen staining, cells were fixed with 1% paraformaldehyde, permeabilized in 0.2% saponin (Sigma-Aldrich), and incubated with biotin-conjugated goat anti-RSV serum (Meridian Life Sciences, Memphis, Tenn) at 5 mg/mL.…”

“…Commercial primer pairs (Qiagen) for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1), CCL4, CCL5, CXCL10, IFNA1, IFNB, interferon-stimulated gene 56 (ISG56), and myxovirus resistance gene 1 (MX1 [MxA]) were used to assess the relative expression of genes. RSV-specific primers for NS1, N, and F (Sigma) were as described by Boukhvalova et al 43 The QuantiFast SYBR Green PCR Kit (Qiagen) was used to perform duplicate quantitative PCR (qPCR) reactions in the MX-3000P QPCR System (Stratagene, La Jolla, Calif). Data were analyzed with MxPro Software (Stratagene), and the fold induction of gene expression was calculated by using the Pfaffl method, 44 or when appropriate, they were normalized to HPRT1 and GAPDH and expressed as 2 to the power of the difference between the threshold cycles for amplification of the reference gene and target gene.…”