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1Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles
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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.
Pirooz, S. D., Lee, J., Zhao, Z., Ni, D., Oh, S., Liang, C. Measurement of γHV68 Infection in Mice. J. Vis. Exp. (57), e3472, doi:10.3791/3472 (2011).
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 host3,4. 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.
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
2. Intranasal Infection of Mice with γHV68
3. Plaque Assay to Determine the Virus Titer in Acutely Infected Lungs
4. Infectious Center Assay to Determine the Virus Load in Latently Infected Splenocytes
5. Quantification of the Viral Genome
6. Representative Results:
Figure 1 depicts the overall scheme of the experiments for the measurement of γHV68 infection in mice in vivo. Representative results of virus titers in the lungs during acute infection of γHV68, as determined by plaque assay, were shown in Figure 2A. A mutant strain of γHV68 containing non-functional viral Bcl-2 (vBcl-2) replicated at levels comparable to wild-type γHV68 in the lungs after 7 days intranasal infection of BALB/c mice (6 - 7 mice per group). No statistically significant differences in the lung titers of the virus were detected between two groups. This data indicates that vBcl-2 is not a crucial factor for the acute infection of γHV68 in mice. However, by 28 days postinfection, the titer of vBcl-2 mutant virus in the spleens dropped 6- to 10- fold compared to the WT, as measured by infectious center assay (Figure 2B), suggesting that the vBcl-2 mutant γHV68 virus is defective in the maintenance of splenic latency after infection. In agreement with the reduced infectious center titers, the viral genome load of the vBcl-2 mutant virus was severely reduced compared to that of WT viruses at day 28 in repeated experiments, as shown in Figure 2C. The close correlation between viral genome loads and the frequency of latent-HV68vBcl-2 mutant virus reactivation ex vivo at latent stage of infection highlights a latency defect of this mutant virus infection.
Figure 1. Schematic diagram for the measurement of γHV68 infection in mice by means of plaque formation, infectious center, and qPCR assays.
Figure 2. Lytic and latent infection of the WT and mutant γHV68 viruses in vivo. Acute replication (A) of the WT and mutant vBcl-2 γHV68 viruses in the lungs of the BALB/c mice at 7 dpi (day post infection) was determined by plaque assay. (B and C) Splenic infectious centers (B) and viral genome load (C) in the spleens of infected mice were measured at 28 dpi by infectious center assay and qPCR, respectively. Red lines, the averages of indicated values. n.s., not significant.
γHV68 has been widely used as a model to understand the pathogenesis of human γ-HVs2,4,5. 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 optimal conditions vary for the virus being used. This is largely because the capacity of different viruses to produce plaques (countable lesions) on a cell monolayer is different. γHV68 normally produces obvious plaques on the monolayer of monkey Vero cells or cultured mouse cells such as NIH3T12 or 3T3, 6 - 7 days after infection. Notably, the cell sensitivity to viral infection declines as the monolayer ages or becomes overcrowded Thus, we usually use less than 50% confluency of Vero cells for plaque assay, which, in our experience, demonstrates the best results for γHV68. In addition, an evenly distributed and freshly-made monolayer is highly recommended for the accuracy of plaque counting. While making the serial dilution of the virus samples, caution must be taken to avoid bubbles and pipet tips should be changed between each titration. For high-titered virus samples, 10-fold serial dilutions are recommended; otherwise, 2- to 5-fold serial dilutions are used.
Unlike acute infection in which infectious virus can be directly assayed by the plaque-forming ability, latently-infected tissues/samples of γ-HVs do not usually contain detectable preformed infectious virus, instead, viral DNA is maintained as a circular episome with few genes expressed6,7. In this case, the viral latency load can be determined by the frequency of ex vivo reactivation of the virus from in vitro explants of latently-infected cells onto a permissive indicator cell culture (e.g. Vero cells)8. To this end, single cell suspension of infected tissues is prepared, counted, and plated onto monolayers of susceptible cells, which are then overlaid with plaque assay medium. In this protocol, we have described how to prepare single cell suspensions of spleen for IC assay to measure the amount of latent virus that has the ability to reactivate per spleen or per population of splenocytes4. Since only a small population of splenocytes is latently infected, we usually prepare serial low-fold dilutions of splenocytes suspension for infectious titer testing. Extreme caution must be taken during the entire procedure to avoid harsh pipetting or vortexing cells. It is important to note that explanted B cells from spleen generally show poor viability9, which might affect the efficiency of ex vivo reactivation. Any gene that improves the survival of latently infected cells could indirectly facilitate the efficiency of the ex vivo reactivation of the virus.
Compared with plaque-forming and IC assays, qPCR provides an effective means to accurately quantify lymphocytic γ-HVs in infected mice10. It is particularly useful for a virus with poor plaque-forming ability due to its minimal cytotoxicity to Vero or other indicator cells, and can be applied for both acutely and chronically infected samples. A standard curve is necessary and important for accurately quantifying viral genomes. We developed γHV68 standard curves based on the ORF56 gene amplification, utilizing a bacmid clone containing the γHV68 genome8. qPCR reaction is extremely sensitive being able to detect even a few copies of viral DNA per qPCR reaction, far beyond the limits of plaque assay. Precautions however must be put in place to avoid cross-contamination between samples while including uninfected samples as necessary negative controls for each reaction. The specificity of qPCR reaction can be a serious issue particularly for infected tissue samples, since enormous amount of cellular DNA may affect the signal of virus-specific amplification. This however can be detected by melting curve analysis for nonspecific amplified products. In naturally infected splenocyte populations, the frequency of γHV68-bearing cells can be very low. In this scenario, as a complement to the qPCR analysis using quasispecies DNA, limiting-dilution PCR (LD-PCR) provides an alternative advantage to evaluate the frequency of viral genome-bearing cells11,12. Briefly, γ-HVs-infected lymphocytes are serially diluted. DNA extracted from these cells is to be used for a sensitive nested PCR assay, which can detect the presence of single-copy viral DNA in individual samples. A combined analysis of limiting dilution and qPCR will reveal both the frequency of latently infected cells and the viral latent DNA load.
Notably, IC assay cannot be used as a stand-alone method to measure viral latent load, due to the fact that it does not distinguish between reductions in viral latent loads versus a failure of the latent virus itself to reactivate. As a further measure of viral latency, qPCR is used to quantitatively evaluate the viral genome copies in infected tissues. A correlation between reduced viral genome loads and infectious center titer would suggest a latency defect of the virus. In contrast, a disparity between high viral genome loads and low latent viral titer would argue that although the virus is capable of maintaining a latent viral DNA pool, it is unable to efficiently reactivate from latency.
In conclusion, the methods described in this protocol also have general applicability to herpesviruses other than γHV68, for which the targeted cell types and viral genomic sequences are available and allow the unequivocal evaluation of distinct life cycles in vivo. It can also be used to address the biological role of specific virulence factors in the context of mouse infection. For instance, by comparing the replication of different viral Bcl-2 mutant γHV68 with that of wild-type γHV68, our recent work8 allows us to determine which function of vBcl-2 (e.g. apoptosis, autophagy, or both) contributes to the in vivo behavior of this virus in acute and chronic infection in mice. Thus, they also represent important tools to dissect the virus-host interactions during infection.
No conflicts of interest declared.
The authors would like to acknowledge the technical advice and support from Ren Sun (University of California, Los Angeles) and Seungmin Hwang (Washington University). This work was funded by the Baxter Foundation, National Institutes of Health grants (R01 CA140964 and R21 AI083841 to C. Liang).
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