Inactivation of infectious virus and serological detection of virus antigen in Rift Valley fever virus-exposed mosquitoes fixed with paraformaldehyde.

Abstract

Formaldehyde is routinely used to fix tissues in preparation for pathology studies, however concerns remain that treatment of tissues with cellular fixatives may not entirely inactivate infectious virus particles. This concern is of particular regulatory importance for research involving viruses that are classified as select agents such as Rift Valley fever virus (RVFV). Therefore, the specific aims of this study were to (1) assay RVFV-exposed Aedes aegypti mosquitoes fixed in 4% paraformaldehyde for the presence of infectious RVFV particles at various time points following infection and (2) demonstrate the utility of immunofluorescence assay (IFA) for the detection of RVFV antigen in various tissues of paraformaldehyde-fixed mosquitoes. Mosquitoes were administered an infectious blood meal containing one of two strains of RVFV, harvested at various time points following infection, intrathoracically inoculated with 4% paraformaldehyde, and fixed overnight at 4°C. The infection status of a subset of mosquitoes was verified by IFA on leg tissues prior to fixation, and infectivity of RVFV in fixed mosquito carcasses was determined by Vero cell plaque assay. Paraformaldehyde-fixed mosquitoes harvested 14 days post infection were also paraffin-embedded and sectioned for detection of RVFV antigen to particular tissues by IFA. None of the RVFV-exposed mosquitoes tested by Vero cell plaque assay contained infectious RVFV after fixation. Furthermore, incubation of mosquito sections with trypsin prior to antibody staining is recommended for optimal visualization of RVFV antigen in infected mosquito tissues by IFA.

• Authors: Rebekah Kading, Mary Crabtree, Barry Miller
• Journal: Journal of virological methods (J Virol Methods) Vol. 189 Issue 1 Pg. 184-8 (Apr 2013) ISSN: 1879-0984 [Electronic] Netherlands
• PMID: 23391826 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Published by Elsevier B.V.

Chemical References

• Antigens, Viral
• Polymers
• Formaldehyde
• paraform

Topics

• Aedes (immunology, virology)
• Animals
• Antigens, Viral (analysis, immunology)
• Cell Line
• Chlorocebus aethiops
• Fluorescent Antibody Technique
• Formaldehyde
• Insect Vectors (virology)
• Polymers
• Rift Valley Fever (blood, transmission, virology)
• Rift Valley fever virus (immunology, isolation & purification)
• Tissue Fixation
• Vero Cells
• Virus Inactivation