A Rapid Microscopic Method for the Confirmation of Rhinoviruses in Cell Culture

Abstract

Objective: Culture-confirmation of rhinovirus is done using acid lability testing, a laborious and time-consuming method which delays the reporting of patient results by 1–2 days. A fluorescent monoclonal antibody pool (Light Diagnostics Pan-Enterovirus Blend; Millipore Inc., Temecula, Calif., USA) developed to identify various enterovirus isolates in culture was recently reported to also cross-react with rhinoviruses. We evaluated the use of this cross-reacting antibody, used in tandem with non-cross-reacting enterovirus antibodies (D³ IFA; Diagnostic Hybrids Inc., Athens, Ohio, USA) to rapidly identify rhinoviruses in cell culture. Methods: Microscope slides were prepared from cell cultures of 11 rhinovirus clinical isolates and a variety of other respiratory viruses. Slides were stained using both enterovirus antibody pools and examined for fluorescent activity. Results: Positive fluorescence was observed in all the rhinovirus isolates tested using the pan-enterovirus antibody blend, but yielded negative results when stained using the D³ antibodies. Both antibody products produced positive fluorescence for enterovirus isolates and produced negative results when a variety of other respiratory viruses were examined. Conclusion: Staining suspected rhinovirus isolates with each antibody pool affords a rapid means of identifying rhinoviruses and distinguishing them from enteroviruses, without the need for acid lability testing.

Human Rhinoviruses

“…The viruses are distinguished by acid stability testing: EVs are relatively resistant to low pHs compared to HRV, and a 2-to 3-log reduction in titers after exposure to pH 3.0 will indicate that the virus is HRV (181). A more rapid confirmation method was recently reported, utilizing a combination of staining with a pan-enterovirus reagent which cross-reacts with EV-and HRV-positive cultures and non-crossreactive EV antibodies (196). Serotyping with specific antisera can be performed with immunofluorescence-labeled antisera but is increasingly being replaced by molecular genotyping (described below).…”

Rapid Identification of 2009 H1N1 Influenza A Virus Using Fluorescent Antibody Methods

“…Cells were then transferred on glass slides at the bedside by rolling the swab on the slide surface. Positive slides for RSV (5) and influenza A2009 H1N1 (2) and 5 negative slides were stained with the 2009 H1N1 reagent. The low quantity of bedside prepared samples precluded their value in sensitivity and specificity analysis; however, they were used to contrast different direct-specimen preparation methods with fluorescent staining quality.…”

“…Rhinovirus was confirmed using a combination of 2 enterovirus FA reagents (D 3 IFA-enterovirus and Light Diagnostics Panenterovirus blend, Millipore, Temecula, CA), as previously described. 5 The second monolayer was stained with the new 2009 H1N1 reagent. Direct specimens were also stained and confirmed as described.…”

“…Two enterovirus antibody pools were used to identify rhinovirus, as described. 5 Subtype analysis was done using neutralization techniques (adenoviruses and enteroviruses) and reverse transcriptase-polymerase chain reaction testing for seasonal and 2009 H1N1 strains of influenza A (Pro Flu, Gen Probe, San Diego, CA). Uninoculated cultures and negative slides served as control samples.…”