Measurement of γHV68 Infection in Mice

Summary

γ-Herpesviruses (γ-HVs) establish life-long persistency in their host. Infection of mice with γ-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of γHVs. This protocol describes the detection and quantitation of γHV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.

Abstract

γ-Herpesviruses (γ-HVs) are notable for their ability to establish latent infections of lymphoid cells¹. The narrow host range of human γ-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine γ-herpesvirus 68 (γHV68) shares extensive genetic and biological similarities with human γ-HVs and is a natural pathogen of murid rodents². As such, evaluation of γHV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during γ-HVs infection.

Upon intranasal inoculation, γHV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host^3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of γHV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.

Protocol

The following protocol will describe the study of virus titers and viral genome loads in the lytic and latent infection cycle of γHV68 in mice, which can theoretically be used to evaluate infection by other viruses sharing similar lifestyle to that of γHV68.

1. Amplification of γHV68

1. Thaw a frozen vial of γHV68 in 37 °C water bath.
2. Add viral inoculums to NIH3T12 or 3T3 cell culture (~50% confluency) in 10 cm dishes and culture the infected cells at 37 °C in 5% CO₂.
3. Examine the culture by microscopy for cytopathic effect (CPE) and collect the media at a time when greater than 80% of cells show CPE 4....

Discussion

γHV68 has been widely used as a model to understand the pathogenesis of human γ-HVs^2,4,5. In this protocol, we described three routinely used methods, including plaque assay for infectious virus titer, IC assay for viral latent load, and qPCR for viral genome load, to evaluate the acute and latent infection of γHV68 after intranasal inoculation in mice.

The plaque assay has been used extensively to determine the virus titer in infected cells or tissues, but the op.......