Application of Modified Shell Vial Culture Procedure for Arbovirus Detection

Tadeusz J Kochel,Edna R Caceda,Joel Montgomery

doi:10.1371/journal.pone.0001034

Tadeusz J Kochel, Edna R Caceda + Show 1 more

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https://doi.org/10.1371/journal.pone.0001034

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Journal: PLoS ONE	Publication Date: Oct 17, 2007
Citations: 18	License type: CC0 1.0

Affiliation: Naval Medical Research Unit Six

Abstract

The isolation of arboviruses from patient's low titer sera can be difficult. Here we compared the detection efficiency of Dengue (DEN), Yellow Fever (YF), Saint Louis Encephalitis (SLE), West Nile (WN), Ilheus (ILH), Group C (GC), Oropouche (ORO), Mayaro (MAY) and Venezuela Encephalitis Equine (VEE) viruses using a Modified Shell Vial Culture (MSVC) protocol to a Standard Cell Culture (SCC) protocol. First the MSVC and SCC protocols were compared using five dilutions for each of the following stock viruses: DEN-1, DEN-2, DEN-3, DEN-4, YF, SLE, WN, ILH, GC, ORO, MAY and VEE. Next, patients' original sera from which viruses (DEN-1, DEN-2, DEN-3, YF, GC, ORO, MAY and VEE) had been previously isolated were compare by the two methods using five sera dilutions. In addition, seven sera that were positive for DEN-3 by RT-PCR and negative by SCC were processed by MSVC. The MSVC protocol was consistently 1-2 logs higher virus dilution more sensitive for virus detection than the SCC protocol for all stock Flaviviruses tested (DEN-1, DEN-2, DEN-3, DEN-4, YF, SLE, WN and ILH). MSVC was equal to or one log more sensitive for virus detection than SCC for the stock Bunyaviruses (GC and ORO). For the stock Alphavirus MAY, MSVC was equally or one log more sensitive for virus detection than SCC, while for VEE SCC was equally or one log more sensitive for virus detection than MSVC. MSVC was consistently one to two sera dilutions more sensitive than SCC for the detection of Flaviviruses from patients' sera. Both methods were approximately equally sensitive for the detection of Bunyaviruses from patients' sera and equal or one dilution less sensitive for the detection of Alphaviruses from patients' sera. Additionally, MSVC detected DEN virus in five of seven DEN-3 RT-PCR positive, SCC negative patients' sera.

Highlights

The isolation of arboviruses from patient’s low titer sera can be difficult.Shell vial culture centrifugation methods for virus isolation have been shown to be more sensitive than standard isolation methods for members of the Paramyxoviradae [1,2,3,4], Herpesviridae [5,6], Orthomyxoviridae [7] and Flaviviridae [8] families
The cell pellets were reconstituted with PBS and spotted onto slides and tested by indirect immunofluorescent assay (IFA) using DEN, Yellow Fever (YF), Saint Louis Encephalitis (SLE), West Nile (WN), ILH, Group C (GC), ORO, MAY and Venezuela Encephalitis Equine (VEE) polyclonal antibodies
The cell pellets were reconstituted with PBS and spotted onto slides and tested by IFA using DEN, YF, SLE, WN, ILH, GC, ORO, MAY and VEE polyclonal antibodies

Summary

Introduction

The isolation of arboviruses from patient’s low titer sera can be difficult.Shell vial culture centrifugation methods for virus isolation have been shown to be more sensitive than standard isolation methods for members of the Paramyxoviradae [1,2,3,4], Herpesviridae [5,6], Orthomyxoviridae [7] and Flaviviridae [8] families. We have compared the MSVC and standard cell culture (SCC) methods of virus detection for the Flaviviruses: DEN-1, DEN-2, DEN-3, DEN-4, YF, SLE, ILH and WN; the Bunyaviruses: GC, ORO and the Alphaviruses: MAY and VEE. Sera were collected from febrile subjects and processed for virus isolation following the SCC method (1:5 inoculum, 10 day incubation, C636 and/or Vero culture) and stored at 280uC until use.

Results

Conclusion