Cytomegalovirus durably primes neutrophil oxidative burst.

Cytomegalovirus (CMV) is a ubiquitous herpes virus that infects most humans, thereafter persisting lifelong in tissues of the host. It is a known pathogen in immunosuppressed patients, but its impact on immunocompetent hosts remains less understood. Recent data have shown that CMV leaves a significant and long-lasting imprint in host immunity that may confer some protection against subsequent bacterial infection. Such innate immune activation may come at a cost, however, with potential to cause immunopathology. Neutrophils are central to many models of immunopathology, and while acute CMV infection is known to influence neutrophil biology, the impact of chronic CMV infection on neutrophil function remains unreported. Using our murine model of CMV infection and latency, we show that chronic CMV causes persistent enhancement of neutrophil oxidative burst well after resolution of acute infection. Moreover, this in vivo priming of marrow neutrophils is associated with enhanced formyl peptide receptor expression, and ultimately constitutive c-Jun N-terminal kinase phosphorylation and enhanced CD14 expression in/on circulating neutrophils. Finally, we show that neutrophil priming is dependent on viral load, suggesting that naturally infected human hosts will show variability in CMV-related neutrophil priming. Altogether, these findings represent a previously unrecognized and potentially important impact of chronic CMV infection on neutrophil responsiveness in immunocompetent hosts.

nanoString evaluation of murine Cytomegalovirus transcription in vivo and in vitro.

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.

Stochastic Episodes of Latent Cytomegalovirus Transcription Drive CD8 T-Cell "Memory Inflation" and Avoid Immune Evasion.

Acute infection with murine cytomegalovirus (mCMV) is controlled by CD8+ T cells and develops into a state of latent infection, referred to as latency, which is defined by lifelong maintenance of viral genomes but absence of infectious virus in latently infected cell types. Latency is associated with an increase in numbers of viral epitope-specific CD8+ T cells over time, a phenomenon known as "memory inflation" (MI). The "inflationary" subset of CD8+ T cells has been phenotyped as KLRG1+CD62L- effector-memory T cells (iTEM). It is agreed upon that proliferation of iTEM requires repeated episodes of antigen presentation, which implies that antigen-encoding viral genes must be transcribed during latency. Evidence for this has been provided previously for the genes encoding the MI-driving antigenic peptides IE1-YPHFMPTNL and m164-AGPPRYSRI of mCMV in the H-2d haplotype. There exist two competing hypotheses for explaining MI-driving viral transcription. The "reactivation hypothesis" proposes frequent events of productive virus reactivation from latency. Reactivation involves a coordinated gene expression cascade from immediate-early (IE) to early (E) and late phase (L) transcripts, eventually leading to assembly and release of infectious virus. In contrast, the "stochastic transcription hypothesis" proposes that viral genes become transiently de-silenced in latent viral genomes in a stochastic fashion, not following the canonical IE-E-L temporal cascade of reactivation. The reactivation hypothesis, however, is incompatible with the finding that productive virus reactivation is exceedingly rare in immunocompetent mice and observed only under conditions of compromised immunity. In addition, the reactivation hypothesis fails to explain why immune evasion genes, which are regularly expressed during reactivation in the same cells in which epitope-encoding genes are expressed, do not prevent antigen presentation and thus MI. Here we show that IE, E, and L genes are transcribed during latency, though stochastically, not following the IE-E-L temporal cascade. Importantly, transcripts that encode MI-driving antigenic peptides rarely coincide with those that encode immune evasion proteins. As immune evasion can operate only in cis, that is, in a cell that simultaneously expresses antigenic peptides, the stochastic transcription hypothesis explains why immune evasion is not operative in latently infected cells and, therefore, does not interfere with MI.

Transcripts expressed in cytomegalovirus latency coding for an antigenic IE/E phase peptide that drives "memory inflation".

Roizman's definition of herpesviral latency, which applies also to cytomegaloviruses (CMVs), demands maintenance of reactivation-competent viral genomes after clearance of productive infection. It is more recent understanding that failure to complete the productive viral cycle for virus assembly and release does not imply viral gene silencing at all genetic loci and all the time. It rather appears that CMV latency is transcriptionally "noisy" in that silenced viral genes get desilenced from time to time in a stochastic manner, leading to "transcripts expressed in latency" (TELs). If a TEL happens to code for a protein that contains a CD8 T cell epitope, protein processing can lead to the presentation of the antigenic peptide and restimulation of cognate CD8 T cells during latency. This mechanism is discussed as a potential driver of epitope-selective accumulation of CD8 T cells over time, a phenomenon linked to CMV latency and known as "memory inflation" (MI). So far, expression of an epitope-encoding TEL was shown only for the major immediate-early (MIE) gene m123/ie1 of murine cytomegalovirus (mCMV), which codes for the prototypic MI-driving antigenic peptide YPHFMPTNL that is presented by the MHC class-I molecule Ld. The only known second MI-driving antigenic peptide of mCMV in the murine MHC haplotype H-2d is AGPPRYSRI presented by the MHC-I molecule Dd. This peptide is very special in that it is encoded by the early (E) phase gene m164 and by an overlapping immediate-early (IE) transcript governed by a promoter upstream of m164. If MI is driven by presentation of TEL-derived antigenic peptides, as the hypothesis says, one should find corresponding TELs. We show here that E-phase and IE-phase transcripts that code for the MI-driving antigenic peptide AGPPRYSRI are independently and stochastically expressed in latently infected lungs.

Cytomegalovirus promotes murine glioblastoma growth via pericyte recruitment and angiogenesis.

Cytomegalovirus (CMV) has been implicated in glioblastoma (GBM); however, a mechanistic connection in vivo has not been established. The purpose of this study is to characterize the effects of murine CMV (MCMV) on GBM growth in murine models. Syngeneic GBM models were established in mice perinatally infected with MCMV. We found that tumor growth was markedly enhanced in MCMV+ mice, with a significant reduction in overall survival compared with that of controls (P < 0.001). We observed increased angiogenesis and tumor blood flow in MCMV+ mice. MCMV reactivation was observed in intratumoral perivascular pericytes and tumor cells in mouse and human GBM specimens, and pericyte coverage of tumor vasculature was strikingly augmented in MCMV+ mice. We identified PDGF-D as a CMV-induced factor essential for pericyte recruitment, angiogenesis, and tumor growth. The antiviral drug cidofovir improved survival in MCMV+ mice, inhibiting MCMV reactivation, PDGF-D expression, pericyte recruitment, and tumor angiogenesis. These data show that MCMV potentiates GBM growth in vivo by increased pericyte recruitment and angiogenesis due to alterations in the secretome of CMV-infected cells. Our model provides evidence for a role of CMV in GBM growth and supports the application of antiviral approaches for GBM therapy.

Cytomegalovirus immunoglobulin G titers do not predict reactivation risk in immunocompetent hosts.

Cytomegalovirus (CMV) reactivation occurs in roughly one-third of immunocompetent patients during critical illness, and is associated with worse outcomes. These outcomes have prompted consideration of early antiviral prophylaxis, but two-third of patients would receive unnecessary treatment. Tissue viral load has been associated with risk of reactivation in murine models, and recent work has suggested a relationship between immune responses to CMV and underlying viral load. We, therefore, sought to confirm the hypothesis that serum CMV-specific immunoglobulin G (IgG) correlates with tissue viral load, and might be used to predict the risk of reactivation during critical illness. We confirm that there is a good correlation between tissue viral load and serum CMV-specific IgG after laboratory infection of inbred mice. Further, we show that naturally infected outbred hosts have variable tissue viral DNA loads that do not correlate well with serum IgG. Perhaps as a consequence, CMV-specific IgG was not predictive of reactivation events in immunocompetent humans. When reactivation did occur, those with the lowest IgG levels had longer durations of reactivation, but IgG quartiles were not associated with differing peak DNAemia. Together our data suggest that CMV-specific IgG titers diverge from tissue viral loads in outbred immunocompetent hosts, and their importance for the control of reactivation events remains unclear.

Determination of suitable reference genes for RT-qPCR analysis of murine Cytomegalovirus in vivo and in vitro.

Reverse transcription quantitative PCR (RT-qPCR) is the most commonly used method to evaluate gene expression. Reliable qPCR results are highly dependent on accurate normalization using suitable reference genes. We investigated expression of commonly used reference genes during murine Cytomegalovirus (mCMV) infection and latency to determine those genes least perturbed by infection. Following mCMV infection in BALB/c mice, lung, salivary gland, liver, spleen and kidney were evaluated. Liver sinusoidal endothelial cells and NIH-3T3 cells were also evaluated. RT-qPCR was performed during acute and latent mCMV infection for 11 commonly used reference genes with comparisons made to uninfected samples. Normfinder, BestKeeper, GeNorm and the comparative delta CT method produced comparable analyses that were combined in RefFinder to generate an overall ranking. Ppia, B2m and Gapdh are the most stable reference genes for in vitro infection studies. For in vivo studies the most suitable reference genes were highly tissue and cell type dependent. Comparing infected and uninfected groups revealed viral influence on transcription of some genes. We provide reference gene guidelines for investigations of gene expression for mCMV Smith strain infection of Balb/cJ mice or NIH-3T3 cells. These results also suggest careful consideration of reference genes for different host tissues evaluated.

Cytomegalovirus Reinfections Stimulate CD8 T-Memory Inflation.

Cytomegalovirus (CMV) has been shown to induce large populations of CD8 T-effector memory cells that unlike central memory persist in large quantities following infection, a phenomenon commonly termed "memory inflation". Although murine models to date have shown very large and persistent CMV-specific T-cell expansions following infection, there is considerable variability in CMV-specific T-memory responses in humans. Historically such memory inflation in humans has been assumed a consequence of reactivation events during the life of the host. Because basic information about CMV infection/re-infection and reactivation in immune competent humans is not available, we used a murine model to test how primary infection, reinfection, and reactivation stimuli influence memory inflation. We show that low titer infections induce "partial" memory inflation of both mCMV specific CD8 T-cells and antibody. We show further that reinfection with different strains can boost partial memory inflation. Finally, we show preliminary results suggesting that a single strong reactivation stimulus does not stimulate memory inflation. Altogether, our results suggest that while high titer primary infections can induce memory inflation, reinfections during the life of a host may be more important than previously appreciated.

Broncholaveolar lavage to detect cytomegalovirus infection, latency, and reactivation in immune competent hosts.

Roughly 1/3rd of immune competent patients will reactivate latent cytomegalovirus (CMV) during critical illness. There are no standard methods to detect reactivation, and some investigators have postulated that presence of DNA in BAL fluid is indicative of viral replication. To test this hypothesis, we used a murine model that allows inclusion of matched healthy controls which is not possible in human studies. BALB/c mice infected with Smith-murine CMV or PBS (mock) had BAL evaluated 7, 14, or 21 days after acute infections, during latency, or during bacterial sepsis. Plaque assay, PCR, and rtPCR were performed on BALs and concomitantly obtained lung tissue. BAL cellular compositions, including tetramer evaluation of CMV-specific T cells were evaluated by flow cytometry. CMV DNA were detected in BAL at all time-points during acute infection, becoming undetectable in all mice during latency, then were detected again during bacterial sepsis, peaking 3 weeks after onset. mCMV specific T-cells were most numerous in BAL after acute viral infections, decreasing to low levels during latency, then fluctuating during bacterial sepsis. Specifically, mCMV-specific T-cells contracted at sepsis onset, expanding 2-4 weeks post-sepsis, presumably in response to increased viral loads at that time point. Altogether, our results support the use of BAL PCR for the diagnosis of CMV replication in immune competent hosts. Additionally, we demonstrate dynamic changes in CMV-specific T cells that occur in BAL during CMV infection and during sepsis induced viral reactivation. J. Med. Virol. 88:1408-1416, 2016. © 2016 Wiley Periodicals, Inc.

Sepsis and cytomegalovirus: foes or conspirators?

Cytomegalovirus (CMV) reactivation in non-immune-suppressed critically ill patients is an area of increasing interest. CMV has long been appreciated as a pathogen in immunocompromised hosts. CMV reactivates in approximately one-third of latently infected non-immune-suppressed hosts during critical illness; however, its role as a pathogen in these patients remains unclear. CMV reactivation has been linked to bacterial sepsis and likely results from inflammation, transient immune compromise, and viral epigenetic changes. While CMV may improve immune response to some bacterial infections, other data suggest that CMV induces exaggerated responses to severe infections that may be harmful to latently infected hosts. These results also suggest that previous infection history may explain significant differences seen between human septic responses and murine models of sepsis. While critically ill human hosts clearly have worse outcomes associated with CMV reactivation, determining causality remains an area of investigation, with randomized control trials currently being performed. Here we review the current literature and highlight areas for future investigation.

Viral latency drives 'memory inflation': a unifying hypothesis linking two hallmarks of cytomegalovirus infection.

Low public awareness of cytomegalovirus (CMV) results from the only mild and transient symptoms that it causes in the healthy immunocompetent host, so that primary infection usually goes unnoticed. The virus is not cleared, however, but stays for the lifetime of the host in a non-infectious, replicatively dormant state known as 'viral latency'. Medical interest in CMV results from the fact that latent virus can reactivate to cytopathogenic, tissue-destructive infection causing life-threatening end-organ disease in immunocompromised recipients of solid organ transplantation (SOT) or hematopoietic cell transplantation (HCT). It is becoming increasingly clear that CMV latency is not a static state in which the viral genome is silenced at all its genetic loci making the latent virus immunologically invisible, but rather is a dynamic state characterized by stochastic episodes of transient viral gene desilencing. This gene expression can lead to the presentation of antigenic peptides encoded by 'antigenicity-determining transcripts expressed in latency (ADTELs)' sensed by tissue-patrolling effector-memory CD8 T cells for immune surveillance of latency [In Reddehase et al., Murine model of cytomegalovirus latency and reactivation, Current Topics in Microbiology and Immunology, vol 325. Springer, Berlin, pp 315-331, 2008]. A hallmark of the CD8 T cell response to CMV is the observation that with increasing time during latency, CD8 T cells specific for certain viral epitopes increase in numbers, a phenomenon that has gained much attention in recent years and is known under the catchphrase 'memory inflation.' Here, we provide a unifying hypothesis linking stochastic viral gene desilencing during latency to 'memory inflation.'