Production- and Purification-Relevant Properties of Human and Murine Cytomegalovirus.

There is a large unmet need for a prophylactic vaccine against human cytomegalovirus (HCMV) to combat the ubiquitous infection that is ongoing with this pathogen. A vaccination against HCMV could protect immunocompromised patients and prevent birth defects caused by congenital HCMV infections. Moreover, cytomegalovirus (CMV) has a number of features that make it a very interesting vector platform for gene therapy. In both cases, preparation of a highly purified virus is a prerequisite for safe and effective application. Murine CMV (MCMV) is by far the most studied model for HCMV infections with regard to the principles that govern the immune surveillance of CMVs. Knowledge transfer from MCMV and mice to HCMV and humans could be facilitated by better understanding and characterization of the biological and biophysical properties of both viruses. We carried out a detailed investigation of HCMV and MCMV growth kinetics as well as stability under the influence of clarification and different storage conditions. Further, we investigated the possibilities to concentrate and purify both viruses by ultracentrifugation and ion-exchange chromatography. Defective enveloped particles were not separately analyzed; however, the behavior of exosomes was examined during all experiments. The effectiveness of procedures was monitored using CCID50 assay, Nanoparticle tracking analysis, ELISA for host cell proteins, and quantitative PCR for host cell DNA. MCMV generally proved to be more robust in handling. Despite its greater sensitivity, HCMV was efficiently (100% recovery) purified and concentrated by anion-exchange chromatography using QA monolithic support. The majority of the host genomic DNA as well as most of the host cell proteins were removed by this procedure.

A viral kinase counteracts in vivo restriction of murine cytomegalovirus by SAMHD1.

The deoxynucleotide triphosphate (dNTP) hydrolase SAMHD1 inhibits retroviruses in non-dividing myeloid cells. Although antiviral activity towards DNA viruses has also been demonstrated, the role of SAMHD1 during cytomegalovirus (CMV) infection remains unclear. To determine the impact of SAMHD1 on the replication of CMV, we used murine CMV (MCMV) to infect a previously established SAMHD1 knockout mouse model and found that SAMHD1 inhibits the replication of MCMV in vivo. By comparing the replication of MCMV in vitro in myeloid cells and fibroblasts from SAMHD1-knockout and control mice, we found that the viral kinase M97 counteracts SAMHD1 after infection by phosphorylating the regulatory residue threonine 603. The phosphorylation of SAMHD1 in infected cells correlated with a reduced level of dNTP hydrolase activity and the loss of viral restriction. Together, we demonstrate that SAMHD1 acts as a restriction factor in vivo and we identify the M97-mediated phosphorylation of SAMHD1 as a previously undescribed viral countermeasure.

Murine Cytomegalovirus Spread Depends on the Infected Myeloid Cell Type.

Cytomegaloviruses (CMVs) colonize blood-borne myeloid cells. Murine CMV (MCMV) spreads from the lungs via infected CD11c+ cells, consistent with an important role for dendritic cells (DC). We show here that MCMV entering via the olfactory epithelium, a natural transmission portal, also spreads via infected DC. They reached lymph nodes, entered the blood via high endothelial venules, and then entered the salivary glands, driven by constitutive signaling of the viral M33 G protein-coupled receptor (GPCR). Intraperitoneal infection also delivered MCMV to the salivary glands via DC. However, it also seeded F4/80+ infected macrophages to the blood; they did not enter the salivary glands or require M33 for extravasation. Instead, they seeded infection to a range of other sites, including brown adipose tissue (BAT). Peritoneal cells infected ex vivo then adoptively transferred showed similar cell type-dependent differences in distribution, with abundant F4/80+ cells in BAT and CD11c+ cells in the salivary glands. BAT colonization by CMV-infected cells was insensitive to pertussis toxin inhibition of the GPCR signaling through Gi/o substrate, whereas salivary gland colonization was sensitive. Since salivary gland infection required both M33 and Gi/o-coupled signaling, whereas BAT infection required neither, these migrations were mechanistically distinct. MCMV spread from the lungs or nose depended on DC, controlled by M33. Infecting other monocyte populations resulted in unpredictable new infections.IMPORTANCE Cytomegaloviruses (CMVs) spread through the blood by infecting monocytes, and this can lead to disease. With murine CMV (MCMV) we can track infected myeloid cells and so understand how CMVs spread. Previous experiments have injected MCMV into the peritoneal cavity. MCMV normally enters mice via the olfactory epithelium. We show that olfactory infection spreads via dendritic cells, which MCMV directs to the salivary glands. Peritoneal infection similarly reached the salivary glands via dendritic cells. However, it also infected other monocyte types, and they spread infection to other tissues. Thus, infecting the "wrong" monocytes altered virus spread, with potential to cause disease. These results provide a basis for understanding how the monocyte types infected by human CMV might promote different infection outcomes.

Caspase-8-dependent control of NK- and T cell responses during cytomegalovirus infection.

Caspase-8 (CASP8) impacts antiviral immunity in expected as well as unexpected ways. Mice with combined deficiency in CASP8 and RIPK3 cannot support extrinsic apoptosis or RIPK3-dependent programmed necrosis, enabling studies of CASP8 function without complications of unleashed necroptosis. These extrinsic cell death pathways are naturally targeted by murine cytomegalovirus (MCMV)-encoded cell death suppressors, showing they are key to cell-autonomous host defense. Remarkably, Casp8-/-Ripk3-/-, Ripk1-/-Casp8-/-Ripk3-/- and Casp8-/-Ripk3K51A/K51A mice mount robust antiviral T cell responses to control MCMV infection. Studies in Casp8-/-Ripk3-/- mice show that CASP8 restrains expansion of MCMV-specific natural killer (NK) and CD8 T cells without compromising contraction or immune memory. Infected Casp8-/-Ripk3-/- or Casp8-/-Ripk3K51A/K51A mice have higher levels of virus-specific NK cells and CD8 T cells compared to matched RIPK3-deficient littermates or WT mice. CASP8, likely acting downstream of Fas death receptor, dampens proliferation of CD8 T cells during expansion. Importantly, contraction proceeds unimpaired in the absence of extrinsic death pathways owing to intact Bim-dependent (intrinsic) apoptosis. CD8 T cell memory develops in Casp8-/-Ripk3-/- mice, but memory inflation characteristic of MCMV infection is not sustained in the absence of CASP8 function. Despite this, Casp8-/-Ripk3-/- mice are immune to secondary challenge. Interferon (IFN)γ is recognized as a key cytokine for adaptive immune control of MCMV. Ifngr-/-Casp8-/-Ripk3-/- mice exhibit increased lifelong persistence in salivary glands as well as lungs compared to Ifngr-/- and Casp8-/-Ripk3-/- mice. Thus, mice deficient in CASP8 and RIPK3 are more dependent on IFNγ mechanisms for sustained T cell immune control of MCMV. Overall, appropriate NK- and T cell immunity to MCMV is dependent on host CASP8 function independent of RIPK3-regulated pathways.

Potential Application of TALENs against Murine Cytomegalovirus Latent Infections.

Cytomegalovirus (CMV) infections are still a global health problem, because the latent viruses persist in humans and cause recurring disease. Currently, there are no therapies for CMV latent infections and the therapies for active infections are limited by side effects and other problems. It is impossible to eradicate latent viruses in animals. HCMV (human CMV) is specific to human diseases; however, it is difficult to study HCMV due to its host specificity and long life cycle. Fortunately, MCMV (murine CMV) provides an excellent animal model. Here, three specific pairs of transcription activator-like effector nuclease (TALEN) plasmids (MCMV1-2, 3-4, and 5-6) were constructed to target the MCMV M80/80.5 sequence in order to test their efficacy in blocking MCMV lytic replication in NIH3T3 cell culture. The preliminary data showed that TALEN plasmids demonstrate specific targeting and cleavage in the MCMV M80/80.5 sequence and effectively inhibit MCMV growth in cell culture when the plasmid transfection is prior to the viral infection. The most specific pairs of TALEN plasmids (MCMV3-4) were further used to confirm the negative regulation of latent MCMV replication and gene expression in Balb/c mice. The injection of specific TALEN plasmids caused significant inhibition in the copy number level of immediately early gene (ie-1) DNA in five organs of mice, when compared with the controls. The result demonstrated that TALENs potentially provide an effective strategy to remove latent MCMV in animals.

Caspase-8 restricts natural killer cell accumulation during MCMV Infection.

Natural killer (NK) cells provide important host defense against herpesvirus infections and influence subsequent T cell control of replication and maintenance of latency. NK cells exhibit phases of expansion, contraction and memory formation in response to the natural mouse pathogen murine cytomegalovirus (MCMV). Innate and adaptive immune responses are tightly regulated in mammals to avoid excess tissue damage while preventing acute and chronic viral disease and assuring resistance to reinfection. Caspase (CASP)8 is an autoactivating aspartate-specific cysteine protease that initiates extrinsic apoptosis and prevents receptor interacting protein (RIP) kinase (RIPK)1-RIPK3-driven necroptosis. CASP8 also promotes death-independent signal transduction. All of these activities make contributions to inflammation. Here, we demonstrate that CASP8 restricts NK cell expansion during MCMV infection but does not influence NK memory. Casp8-/-Ripk3-/- mice mount higher NK response levels than Casp8+/-Ripk3-/- littermate controls or WT C57BL/6 J mice, indicating that RIPK3 deficiency alone does not contribute to NK response patterns. MCMV m157-responsive Ly49H+ NK cells support increased expansion of both Ly49H- NK cells and CD8 T cells in Casp8-/-Ripk3-/- mice. Surprisingly, hyperaccumulation of NK cells depends on the pronecrotic kinase RIPK1. Ripk1-/-Casp8-/-Ripk3-/- mice fail to show the enhanced expansion of lymphocytes observed in Casp8-/-Ripk3-/- mice even though development and homeostasis are preserved in uninfected Ripk1-/-Casp8-/-Ripk3-/- mice. Thus, CASP8 naturally regulates the magnitude of NK cell responses in response to infection where strong activation signals depend on another key regulator of death signaling, RIPK1. In addition, the strong NK cell response promotes survival of effector CD8 T cells during their expansion. Thus, hyperaccumulation of NK cells and crosstalk with T cells becomes amplified in the absence of extrinsic cell death machinery.

Immunometabolic phenotype of BV-2 microglia cells upon murine cytomegalovirus infection.

Microglia are resident brain macrophages with key roles in development and brain homeostasis. Cytomegalovirus (CMV) readily infects microglia cells, even as a possible primary target of infection in development. Effects of CMV infection on a cellular level in microglia are still unclear; therefore, the aim of this research was to assess the immunometabolic changes of BV-2 microglia cells following the murine cytomegalovirus (MCMV) infection. In light of that aim, we established an in vitro model of ramified BV-2 microglia (BV-2∅FCS, inducible nitric oxide synthase (iNOSlow), arginase-1 (Arg-1high), mannose receptor CD206high, and hypoxia-inducible factor 1α (HIF-1αlow)) to better replicate the in vivo conditions by removing FCS from the cultivation media, while the cells cultivated in 10% FCS DMEM displayed an ameboid morphology (BV-2FCS high, iNOShigh, Arg-1low, CD206low, and HIF-1αhigh). Experiments were performed using both ramified and ameboid microglia, and both of them were permissive to productive viral infection. Our results indicate that MCMV significantly alters the immunometabolic phenotypic properties of BV-2 microglia cells through the manipulation of iNOS and Arg-1 expression patterns, along with an induction of a glycolytic shift in the infected cell cultures.

Function of the cargo sorting dileucine motif in a cytomegalovirus immune evasion protein.

As an immune evasion mechanism, cytomegaloviruses (CMVs) have evolved proteins that interfere with cell surface trafficking of MHC class-I (MHC-I) molecules to tone down recognition by antiviral CD8 T cells. This interference can affect the trafficking of recently peptide-loaded MHC-I from the endoplasmic reticulum to the cell surface, thus modulating the presentation of viral peptides, as well as the recycling of pre-existing cell surface MHC-I, resulting in reduction of the level of overall MHC-I cell surface expression. Murine cytomegalovirus (mCMV) was paradigmatic in that it led to the discovery of this immune evasion strategy of CMVs. Members of its m02-m16 gene family code for type-I transmembrane glycoproteins, proven or predicted, most of which carry cargo sorting motifs in their cytoplasmic, C-terminal tail. For the m06 gene product m06 (gp48), the cargo has been identified as being MHC-I, which is linked by m06 to cellular adapter proteins AP-1A and AP-3A through the dileucine motif EPLARLL. Both APs are involved in trans-Golgi network (TGN) cargo sorting and, based on transfection studies, their engagement by the dileucine motif was proposed to be absolutely required to prevent MHC-I exposure at the cell surface. Here, we have tested this prediction in an infection system with the herein newly described recombinant virus mCMV-m06AA, in which the dileucine motif is destroyed by replacing EPLARLL with EPLARAA. This mutation has a phenotype in that the transition of m06-MHC-I complexes from early endosomes (EE) to late endosomes (LE)/lysosomes for degradation is blocked. Consistent with the binding of the MHC-I α-chain to the luminal domain of m06, the m06-mediated disposal of MHC-I did not require the ß2m chain of mature MHC-I. Unexpectedly, however, disconnecting MHC-I cargo from AP-1A/3A by the motif mutation in m06 had no notable rescuing impact on overall cell surface MHC-I, though it resulted in some improvement of the presentation of viral antigenic peptides by recently peptide-loaded MHC-I. Thus, the current view on the mechanism by which m06 mediates immune evasion needs to be revised. While the cargo sorting motif is critically involved in the disposal of m06-bound MHC-I in the endosomal/lysosomal pathway at the stage of EE to LE transition, this motif-mediated disposal is not the critical step by which m06 causes immune evasion. We rather propose that engagement of AP-1A/3A by the cargo sorting motif in m06 routes the m06-MHC-I complexes into the endosomal pathway and thereby detracts them from the constitutive cell surface transport.

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.

CD4 T cells are required for maintenance of CD8 TRM cells and virus control in the brain of MCMV-infected newborn mice.

Cytomegalovirus (CMV) infection is a significant public health problem. Congenital CMV infection is a leading infectious cause of long-term neurodevelopmental sequelae, including mental retardation and sensorineural hearing loss. Immune protection against mouse cytomegalovirus (MCMV) is primarily mediated by NK cells and CD8+ T cells, while CD4+ T cells are not needed for control of MCMV in majority of organs in immunocompetent adult mice. Here, we set out to determine the role of CD4+ T cells upon MCMV infection of newborn mice. We provide evidence that CD4+ T cells are essential for clearance of MCMV infection in brain of neonatal mice and for prevention of recurrence of latent MCMV. In addition, we provide evidence that CD4+ T cells are required for induction and maintenance of tissue-resident memory CD8+ T cells in the brain of mice perinatally infected with MCMV.

Persistent viral replication and the development of T-cell responses after intranasal infection by MCMV.

Natural transmission of cytomegalovirus (CMV) has been difficult to observe. However, recent work using the mouse model of murine (M)CMV demonstrated that MCMV initially infects the nasal mucosa after transmission from mothers to pups. We found that intranasal (i.n.) inoculation of C57BL/6J mice resulted in reliable recovery of replicating virus from the nasal mucosa as assessed by plaque assay. After i.n. inoculation, CD8+ T-cell priming occurred in the mandibular, deep-cervical, and mediastinal lymph nodes within 3 days of infection. Although i.n. infection induced "memory inflation" of T cells specific for the M38316-323 epitope, there were no detectable CD8+ T-cell responses against the late-appearing IE3416-423 epitope, which contrasts with intraperitoneal (i.p.) infection. MCMV-specific T cells migrated into the nasal mucosa where they developed a tissue-resident memory (TRM) phenotype and this could occur independently of local virus infection or antigen. Strikingly however, virus replication was poorly controlled in the nasal mucosa and MCMV was detectable by plaque assay for at least 4 months after primary infection, making the nasal mucosa a second site for MCMV persistence. Unlike in the salivary glands, the persistence of MCMV in the nasal mucosa was not modulated by IL-10. Taken together, our data characterize the development of local and systemic T-cell responses after intranasal infection by MCMV and define the nasal mucosa, a natural site of viral entry, as a novel site of viral persistence.

Interleukin-17D and Nrf2 mediate initial innate immune cell recruitment and restrict MCMV infection.

Innate immune cells quickly infiltrate the site of pathogen entry and not only stave off infection but also initiate antigen presentation and promote adaptive immunity. The recruitment of innate leukocytes has been well studied in the context of extracellular bacterial and fungal infection but less during viral infections. We have recently shown that the understudied cytokine Interleukin (IL)-17D can mediate neutrophil, natural killer (NK) cell and monocyte infiltration in sterile inflammation and cancer. Herein, we show that early immune cell accumulation at the peritoneal site of infection by mouse cytomegalovirus (MCMV) is mediated by IL-17D. Mice deficient in IL-17D or the transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), an inducer of IL-17D, featured an early decreased number of innate immune cells at the point of viral entry and were more susceptible to MCMV infection. Interestingly, we were able to artificially induce innate leukocyte infiltration by applying the Nrf2 activator tert-butylhydroquinone (tBHQ), which rendered mice less susceptible to MCMV infection. Our results implicate the Nrf2/IL-17D axis as a sensor of viral infection and suggest therapeutic benefit in boosting this pathway to promote innate antiviral responses.

Human cytomegalovirus US28 allows dendritic cell exit from lymph nodes.

Human cytomegalovirus (HCMV) colonizes blood-borne dendritic cells (DCs). They express US28, a viral G protein-coupled receptor (GPCR). In vitro functions have been described for US28, but how it contributes to host colonization has been unclear. The murine CMV (MCMV) M33 GPCR promotes DC recirculation. We show that US28 shares this function. Thus, DC recirculation is also available to HCMV via US28, and inhibiting US28 G protein-dependent signalling has the potential to reduce systemic infection. We show that M33 also promotes systemic infection through infected DC extravasation.

Inflammation and outer blood-retina barrier (BRB) compromise following choroidal murine cytomegalovirus (MCMV) infections.

Purpose: The purpose of this study was to determine whether the blood-retina barrier is compromised by choroidal murine cytomegalovirus (MCMV) infection, using electron microscopy. Methods: BALB/c mice were immunosuppressed with methylprednisolone and monoclonal antibodies to CD4 and CD8. At several time points post-MCMV intraperitoneal inoculation, the eyes were removed and analyzed with western blotting and immunoelectron microscopy for the presence of MCMV early antigen (EA) and the host protein RIP3. Posterior eyecups from RIP3-/- and RIP3+/+ mice were cultured and inoculated with MCMV. At days 4, 7, and 11 post-infection, cultures were collected and analyzed with plaque assay, immunohistochemical staining, and real-time PCR (RT-PCR). Results: MCMV EA was observed in the nuclei of vascular endothelial cells and pericytes in the choriocapillaris. Disruption of Bruch's membrane was observed, especially at sites adjacent to activated platelets, and a few RPE cells containing some enlarged vesicles were found directly beneath disrupted Bruch's membrane. Some virus particles were also observed in the enlarged vesicles of RPE cells. Levels of the RIP3 protein, which was observed mainly in the RPE cells and the basement membrane of the choriocapillaris, were greatly increased following MCMV infection, while depletion of RIP3 resulted in greatly decreased inflammasome formation, as well as expression of downstream inflammation factors. Conclusions: The results suggest that systemic MCMV spreads to the choroid and replicates in vascular endothelia and pericytes of the choriocapillaris during immunosuppression. Choroidal MCMV infection is associated with in situ inflammation and subsequent disruption of Bruch's membrane and the outer blood-retina barrier.

Nrf2 Induces IL-17D to Mediate Tumor and Virus Surveillance.

Cells undergoing xenobiotic or oxidative stress activate the transcription factor nuclear factor erythroid-derived 2-like 2 (Nrf2), which initiates an intrinsic "stress surveillance" pathway. We recently found that the cytokine IL-17D effects a form of extrinsic stress surveillance by inducing antitumor immunity, but how IL-17D is regulated remains unknown. Here, we show that Nrf2 induced IL-17D in cancer cell lines. Moreover, both Nrf2 and IL-17D were induced in primary tumors as well as during viral infection in vivo. Expression of IL-17D in tumors and virally infected cells is essential for optimal protection of the host as il17d(-/-) mice experienced a higher incidence of tumors and exacerbated viral infections compared to wild-type (WT) animals. Moreover, activating Nrf2 to induce IL-17D in established tumors led to natural killer cell-dependent tumor regression. These data demonstrate that Nrf2 can initiate both intrinsic and extrinsic stress surveillance pathways and highlight the use of Nrf2 agonists as immune therapies for cancer and infection.

Difference in replication of low-passage MCMV HaNa1 in BALB/c, C57BL/6 and NOD mice and role of different branches of immunity in susceptibility.

Currently, murine cytomegalovirus (MCMV) infections have been studied extensively in inbred mice via intraperitoneal route with highly passaged strains. However, the question how a low-passage MCMV replicates in inbred mice via a natural route remained unanswered. Here, different inbred mice (BALB/c, C57BL/6 and NOD) were inoculated oronasally with a low-passage MCMV strain, HaNa1. Viral replication was evaluated by virus titration and quantitative real-time PCR, and antibody response was assessed by immunoperoxidase cell monolayer assay (IPMA). In BALB/c mice, virus persisted in nasal mucosa (from 3 dpi) and submandibular glands (from 7 dpi) until the end of experiment (49 dpi). In C57BL/6 mice, infectious virus was only detected in nasal mucosa from 3 dpi until 21 dpi; viral genome was still detectable in nasal mucosa until 49 dpi. Although infectious virus was not detected in submandibular glands of C57BL/6 mice, viral genome was detected from 7 dpi until 49 dpi. NOD mice appeared to be even more resistant with absence of any productive infection; viral genome was detected at low levels in nasal mucosa. We demonstrated that there was a strong correlation between on the one hand degree of productive replication and on the other hand the time of first appearance and titer of MCMV-specific IgG antibody. The deficiency of functional T and B cells and interleukin-2 (IL-2) common-γ chain (γc) did not increase the susceptibility to MCMV by the use of NOD.SCID and NSG mice. In addition, using monocytic cells from different inbred mice we found patterns of resistance similar to those seen in vivo, as assessed by viral antigen expression. Taken together, these results demonstrated that upon oronasal inoculation low-passage MCMV HaNa1 replication clearly differs between different inbred mice (BALB/c>C57BL/6>NOD); resistance in vivo to MCMV is partly due to less susceptibility of host target cells and is independent of T, B cells and γc signaling cytokine-dependent NK cell activities.

Sequence variability among murine herpesvirus isolates shows possible effect of long-term in vitro passaging on their genome.

No abstract Keywords: murine herpesvirus 68; virus isolates; sequence analysis; restriction fragment length polymorphism.

Murine cytomegalovirus strains co-replicate at multiple tissue sites and establish co-persistence in salivary glands in the absence of Ly49H-mediated competition.

Infection with multiple genetically distinct strains of pathogen is common and can lead to positive (complementation) or negative (competitive) within-host interactions. These interactions can alter aspects of the disease process and help shape pathogen evolution. Infection of the host with multiple strains of cytomegalovirus (CMV) occurs frequently in humans and mice. Profound, NK-cell-mediated (apparent) competition has been identified in C57BL/6 mice, and prevented the replication and shedding of certain co-infecting CMV strains. However, the frequency of such strong competition has not been established. Other within-host interactions such as complementation or alternative forms of competition remain possible. Moreover, high rates of recombination in both human CMV and murine CMV (MCMV) suggest prolonged periods of viral co-replication, rather than strong competitive suppression. An established model was employed to investigate the different possible outcomes of multi-strain infection in other mouse strains. In this study, co-replication of up to four strains of MCMV in the spleen, liver and salivary glands was observed in both MCMV-susceptible and MCMV-resistant mice. In the absence of apparent competition, no other forms of competition were unmasked. In addition, no evidence of complementation between viral strains was observed. Importantly, co-replication of MCMV strains was apparent for up to 90 days in the salivary glands. These data indicated that competition was not the default outcome of multi-strain CMV infection. Prolonged, essentially neutral, co-replication may be the norm, allowing for multi-strain transmission and prolonged opportunities for recombination.

Nodular inflammatory foci are sites of T cell priming and control of murine cytomegalovirus infection in the neonatal lung.

Neonates, including mice and humans, are highly susceptible to cytomegalovirus (CMV) infection. However, many aspects of neonatal CMV infections such as viral cell tropism, spatio-temporal distribution of the pathogen as well as genesis of antiviral immunity are unknown. With the use of reporter mutants of the murine cytomegalovirus (MCMV) we identified the lung as a primary target of mucosal infection in neonatal mice. Comparative analysis of neonatal and adult mice revealed a delayed control of virus replication in the neonatal lung mucosa explaining the pronounced systemic infection and disease in neonates. This phenomenon was supplemented by a delayed expansion of CD8(+) T cell clones recognizing the viral protein M45 in neonates. We detected viral infection at the single-cell level and observed myeloid cells forming "nodular inflammatory foci" (NIF) in the neonatal lung. Co-localization of infected cells within NIFs was associated with their disruption and clearance of the infection. By 2-photon microscopy, we characterized how neonatal antigen-presenting cells (APC) interacted with T cells and induced mature adaptive immune responses within such NIFs. We thus define NIFs of the neonatal lung as niches for prolonged MCMV replication and T cell priming but also as sites of infection control.

Ablation of the regulatory IE1 protein of murine cytomegalovirus alters in vivo pro-inflammatory TNF-alpha production during acute infection.

Little is known about the role of viral genes in modulating host cytokine responses. Here we report a new functional role of the viral encoded IE1 protein of the murine cytomegalovirus in sculpting the inflammatory response in an acute infection. In time course experiments of infected primary macrophages (MΦs) measuring cytokine production levels, genetic ablation of the immediate-early 1 (ie1) gene results in a significant increase in TNFα production. Intracellular staining for cytokine production and viral early gene expression shows that TNFα production is highly associated with the productively infected MΦ population of cells. The ie1- dependent phenotype of enhanced MΦ TNFα production occurs at both protein and RNA levels. Noticeably, we show in a series of in vivo infection experiments that in multiple organs the presence of ie1 potently inhibits the pro-inflammatory cytokine response. From these experiments, levels of TNFα, and to a lesser extent IFNß, but not the anti-inflammatory cytokine IL10, are moderated in the presence of ie1. The ie1- mediated inhibition of TNFα production has a similar quantitative phenotype profile in infection of susceptible (BALB/c) and resistant (C57BL/6) mouse strains as well as in a severe immuno-ablative model of infection. In vitro experiments with infected macrophages reveal that deletion of ie1 results in increased sensitivity of viral replication to TNFα inhibition. However, in vivo infection studies show that genetic ablation of TNFα or TNFRp55 receptor is not sufficient to rescue the restricted replication phenotype of the ie1 mutant virus. These results provide, for the first time, evidence for a role of IE1 as a regulator of the pro-inflammatory response and demonstrate a specific pathogen gene capable of moderating the host production of TNFα in vivo.