nanoString evaluation of murine Cytomegalovirus transcription in vivo and in vitro

Marion Griessl; Michael Gutknecht; Vanda Juranić-Lisnić; Charles H Cook

doi:10.1016/j.jviromet.2021.114436

nanoString evaluation of murine Cytomegalovirus transcription in vivo and in vitro

J Virol Methods. 2022 Mar:301:114436. doi: 10.1016/j.jviromet.2021.114436. Epub 2021 Dec 17.

Authors

Marion Griessl¹, Michael Gutknecht¹, Vanda Juranić-Lisnić², Charles H Cook³

Affiliations

¹ Department of Surgery, Division of Acute Care Surgery, Trauma and Surgical Critical Care, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States.
² Department of Surgery, Division of Acute Care Surgery, Trauma and Surgical Critical Care, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States; PhD Center for Proteomics Head of graduate programme Biomedicine University of Rijeka, Faculty of Medicine B. Branchetta 20 51000, Rijeka, Croatia.
³ Department of Surgery, Division of Acute Care Surgery, Trauma and Surgical Critical Care, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States. Electronic address: chcook@bidmc.harvard.edu.

PMID: 34929204
DOI: 10.1016/j.jviromet.2021.114436

Abstract

Background: Next Generation Sequencing allows for deep analysis of transcriptional activity in cells and tissues, however it is still a cost intensive method that demands well versed data handling. Reverse transcription quantitative PCR (RT-qPCR) is the most commonly used method to measure gene expression levels, however the information gathered is quite small in comparison to NGS. A newer method called nanoString allows for highly multiplexed gene expression analysis by detecting mRNAs without the use of enzymes for reverse transcription or amplification even for single cells or low input material. The method can be done in 1.5 days and data are quickly analyzed by the accompanied user friendly software. Our aim was to investigate this new method and compare it to the existing alternatives, while investigating murine Cytomegalovirus (mCMV) infection and latency.

Methods: mCMV infected murine embryonic fibroblasts (MEF), lung and salivary glands from BALB/c mice were evaluated at different stages of infection. A set of 30 custom designed nanoString probes were tested, 20 probes specific for mCMV genes, 6 probes for host genes known to be influenced by viral infection and 4 reference gene specific probes. nanoString counts were compared to published RNA-Seq RPKM.

Results: We found that nanoString can be used for analysis of cytomegalovirus gene expression during acute infection in vitro and in vivo, both for virus specific and host genes. Although some transcripts show different expression rates in comparison to NGS data, the most abundant transcripts are comparable. When tissues are infected, there are significantly fewer transcripts than in MEFs, and consistent with previous work there are significant differences in relevant abundance between MEF and tissues. We were unable to detect our viral transcripts of interest in latently infected tissue.

Conclusions: For viruses with annotated transcriptomes, nanoString allows simultaneous quantitation of multiple virus and host genes. One huge advantage of the platform is rapid turnaround and simplicity of analysis. It should prove to be very useful to explore host virus interactions during acute infection, but it is unclear if it has adequate sensitivity for analysis during latency in immunocompetent mice.

Keywords: Murine cytomegalovirus; Viral transcription; nanoString.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Animals
Cytomegalovirus / genetics
Cytomegalovirus Infections*
Mice
Mice, Inbred BALB C
Muromegalovirus* / genetics
Transcriptome