Immune surveillance of cytomegalovirus in tissues.

Cytomegalovirus (CMV), a representative member of the Betaherpesvirinae subfamily of herpesviruses, is common in the human population, but immunocompetent individuals are generally asymptomatic when infected with this virus. However, in immunocompromised individuals and immunologically immature fetuses and newborns, CMV can cause a wide range of often long-lasting morbidities and even death. CMV is not only widespread throughout the population but it is also widespread in its hosts, infecting and establishing latency in nearly all tissues and organs. Thus, understanding the pathogenesis of and immune responses to this virus is a prerequisite for developing effective prevention and treatment strategies. Multiple arms of the immune system are engaged to contain the infection, and general concepts of immune control of CMV are now reasonably well understood. Nonetheless, in recent years, tissue-specific immune responses have emerged as an essential factor for resolving CMV infection. As tissues differ in biology and function, so do immune responses to CMV and pathological processes during infection. This review discusses state-of-the-art knowledge of the immune response to CMV infection in tissues, with particular emphasis on several well-studied and most commonly affected organs.

Reovirus infection of tumor cells reduces the expression of NKG2D ligands, leading to impaired NK-cell cytotoxicity and functionality.

In recent years, reoviruses have been of major interest in immunotherapy because of their oncolytic properties. Preclinical and clinical trials, in which reovirus was used for the treatment of melanoma and glioblastoma, have paved the way for future clinical use of reovirus. However, little is known about how reovirus infection affects the tumor microenvironment and immune response towards infected tumor cells. Studies have shown that reovirus can directly stimulate natural killer (NK) cells, but how reovirus affects cellular ligands on tumor cells, which are ultimately key to tumor recognition and elimination by NK cells, has not been investigated. We tested how reovirus infection affects the binding of the NK Group-2 member D (NKG2D) receptor, which is a dominant mediator of NK cell anti-tumor activity. Using models of human-derived melanoma and glioblastoma tumors, we demonstrated that NKG2D ligands are downregulated in tumor cells post-reovirus-infection due to the impaired translation of these ligands in reovirus-infected cells. Moreover, we showed that downregulation of NKG2D ligands significantly impaired the binding of NKG2D to infected tumor cells. We further demonstrated that reduced recognition of NKG2D ligands significantly alters NK cell anti-tumor cytotoxicity in human primary NK cells and in the NK cell line NK-92. Thus, this study provides novel insights into reovirus-host interactions and could lead to the development of novel reovirus-based therapeutics that enhance the anti-tumor immune response.

Two murine cytomegalovirus microRNAs target the major viral immediate early 3 gene.

Human cytomegalovirus is responsible for morbidity and mortality in immune compromised patients and is the leading viral cause of congenital infection. Virus-encoded microRNAs (miRNAs) represent interesting targets for novel antiviral agents. While many cellular targets that augment productive infection have been identified in recent years, regulation of viral genes such as the major viral immediate early protein 72 (IE72) by hcmv-miR-UL112-1 may contribute to both the establishment and the maintenance of latent infection. We employed photoactivated ribonucleotide-enhanced individual nucleotide resolution crosslinking (PAR-iCLIP) to identify murine cytomegalovirus (MCMV) miRNA targets during lytic infection. While the PAR-iCLIP data were of insufficient quality to obtain a comprehensive list of cellular and viral miRNA targets, the most prominent PAR-iCLIP peak in the MCMV genome mapped to the 3' untranslated region of the major viral immediate early 3 (ie3) transcript. We show that this results from two closely positioned binding sites for the abundant MCMV miRNAs miR-M23-2-3p and miR-m01-2-3p. Their pre-expression significantly impaired viral plaque formation. However, mutation of the respective binding sites did not alter viral fitness during acute or subacute infection in vivo. Furthermore, no differences in the induction of virus-specific CD8+ T cells were observed. Future studies will probably need to go beyond studying immunocompetent laboratory mice housed in pathogen-free conditions to reveal the functional relevance of viral miRNA-mediated regulation of key viral immediate early genes.

Characterization of M116.1p, a Murine Cytomegalovirus Protein Required for Efficient Infection of Mononuclear Phagocytes.

Broad tissue tropism of cytomegaloviruses (CMVs) is facilitated by different glycoprotein entry complexes, which are conserved between human CMV (HCMV) and murine CMV (MCMV). Among the wide array of cell types susceptible to the infection, mononuclear phagocytes (MNPs) play a unique role in the pathogenesis of the infection as they contribute both to the virus spread and immune control. CMVs have dedicated numerous genes for the efficient infection and evasion of macrophages and dendritic cells. In this study, we have characterized the properties and function of M116, a previously poorly described but highly transcribed MCMV gene region that encodes M116.1p, a novel protein necessary for the efficient infection of MNPs and viral spread in vivo. Our study further revealed that M116.1p shares similarities with its positional homologs in HCMV and RCMV, UL116 and R116, respectively, such as late kinetics of expression, N-glycosylation, localization to the virion assembly compartment, and interaction with gH-a member of the CMVs fusion complex. This study, therefore, expands our knowledge about virally encoded glycoproteins that play important roles in viral infectivity and tropism. IMPORTANCE Human cytomegalovirus (HCMV) is a species-specific herpesvirus that causes severe disease in immunocompromised individuals and immunologically immature neonates. Murine cytomegalovirus (MCMV) is biologically similar to HCMV, and it serves as a widely used model for studying the infection, pathogenesis, and immune responses to HCMV. In our previous work, we have identified the M116 ORF as one of the most extensively transcribed regions of the MCMV genome without an assigned function. This study shows that the M116 locus codes for a novel protein, M116.1p, which shares similarities with UL116 and R116 in HCMV and RCMV, respectively, and is required for the efficient infection of mononuclear phagocytes and virus spread in vivo. Furthermore, this study establishes the α-M116 monoclonal antibody and MCMV mutants lacking M116, generated in this work, as valuable tools for studying the role of macrophages and dendritic cells in limiting CMV infection following different MCMV administration routes.

Fast, Reliable, and Simple Point-of-Care-like Adaptation of RT-qPCR for the Detection of SARS-CoV-2 for Use in Hospital Emergency Departments.

During COVID-19 pandemics, the availability of testing has often been a limiting factor during patient admissions into the hospital. To circumvent this problem, we adapted an existing diagnostic assay, Seegene Allplex SARS-CoV-2, into a point-of-care-style direct qPCR (POC dqPCR) assay and implemented it in the Emergency Department of Clinical Hospital Center Rijeka, Croatia. In a 4-month analysis, we tested over 10,000 patients and demonstrated that POC-dqPCR is robust and reliable and can be successfully implemented in emergency departments and similar near-patient settings and can be performed by medical personnel with little prior experience in qPCR.

Cytomegalovirus restricts ICOSL expression on antigen-presenting cells disabling T cell co-stimulation and contributing to immune evasion.

Viral infections are controlled, and very often cleared, by activated T lymphocytes. The inducible co-stimulator (ICOS) mediates its functions by binding to its ligand ICOSL, enhancing T-cell activation and optimal germinal center (GC) formation. Here, we show that ICOSL is heavily downmodulated during infection of antigen-presenting cells by different herpesviruses. We found that, in murine cytomegalovirus (MCMV), the immunoevasin m138/fcr-1 physically interacts with ICOSL, impeding its maturation and promoting its lysosomal degradation. This viral protein counteracts T-cell responses, in an ICOS-dependent manner, and limits virus control during the acute MCMV infection. Additionally, we report that blockade of ICOSL in MCMV-infected mice critically regulates the production of MCMV-specific antibodies due to a reduction of T follicular helper and GC B cells. Altogether, these findings reveal a novel mechanism evolved by MCMV to counteract adaptive immune surveillance, and demonstrates a role of the ICOS:ICOSL axis in the host defense against herpesviruses.

Cytomegalovirus protein m154 perturbs the adaptor protein-1 compartment mediating broad-spectrum immune evasion.

Cytomegaloviruses (CMVs) are ubiquitous pathogens known to employ numerous immunoevasive strategies that significantly impair the ability of the immune system to eliminate the infected cells. Here, we report that the single mouse CMV (MCMV) protein, m154, downregulates multiple surface molecules involved in the activation and costimulation of the immune cells. We demonstrate that m154 uses its cytoplasmic tail motif, DD, to interfere with the adaptor protein-1 (AP-1) complex, implicated in intracellular protein sorting and packaging. As a consequence of the perturbed AP-1 sorting, m154 promotes lysosomal degradation of several proteins involved in T cell costimulation, thus impairing virus-specific CD8+ T cell response and virus control in vivo. Additionally, we show that HCMV infection similarly interferes with the AP-1 complex. Altogether, we identify the robust mechanism employed by single viral immunomodulatory protein targeting a broad spectrum of cell surface molecules involved in the antiviral immune response.

The complex of MCMV proteins and MHC class I evades NK cell control and drives the evolution of virus-specific activating Ly49 receptors.

CMVs efficiently target MHC I molecules to avoid recognition by cytotoxic T cells. However, the lack of MHC I on the cell surface renders the infected cell susceptible to NK cell killing upon missing self recognition. To counter this, mouse CMV (MCMV) rescues some MHC I molecules to engage inhibitory Ly49 receptors. Here we identify a new viral protein, MATp1, that is essential for MHC I surface rescue. Rescued altered-self MHC I molecules show increased affinity to inhibitory Ly49 receptors, resulting in inhibition of NK cells despite substantially reduced MHC I surface levels. This enables the virus to evade recognition by licensed NK cells. During evolution, this novel viral immune evasion mechanism could have prompted the development of activating NK cell receptors that are specific for MATp1-modified altered-self MHC I molecules. Our study solves a long-standing conundrum of how MCMV avoids recognition by NK cells, unravels a fundamental new viral immune evasion mechanism, and demonstrates how this forced the evolution of virus-specific activating MHC I-restricted Ly49 receptors.

Mouse cytomegalovirus encoded immunoevasins and evolution of Ly49 receptors - Sidekicks or enemies?

Cytomegaloviruses (CMVs) have dedicated a large portion of their genome towards immune evasion targeting many aspects of the host immune system, particularly NK cells. However, the host managed to cope with the infection by developing multiple mechanisms to recognize viral threat and counterattack it, thus illustrating never-ending evolutionary interplay between CMV and its host. In this review, we will focus on several mechanisms of NK cell evasion by mouse CMV (MCMV), the role of host inhibitory and activating Ly49 receptors involved in the virus control and acquisition of adaptive features by NK cells as a consequence of MCMV infection.

IL-33/ST2 pathway drives regulatory T cell dependent suppression of liver damage upon cytomegalovirus infection.

Regulatory T (Treg) cells dampen an exaggerated immune response to viral infections in order to avoid immunopathology. Cytomegaloviruses (CMVs) are herpesviruses usually causing asymptomatic infection in immunocompetent hosts and induce strong cellular immunity which provides protection against CMV disease. It remains unclear how these persistent viruses manage to avoid induction of immunopathology not only during the acute infection but also during life-long persistence and virus reactivation. This may be due to numerous viral immunoevasion strategies used to specifically modulate immune responses but also induction of Treg cells by CMV infection. Here we demonstrate that liver Treg cells are strongly induced in mice infected with murine CMV (MCMV). The depletion of Treg cells results in severe hepatitis and liver damage without alterations in the virus load. Moreover, liver Treg cells show a high expression of ST2, a cellular receptor for tissue alarmin IL-33, which is strongly upregulated in the liver of infected mice. We demonstrated that IL-33 signaling is crucial for Treg cell accumulation after MCMV infection and ST2-deficient mice show a more pronounced liver pathology and higher mortality compared to infected control mice. These results illustrate the importance of IL-33 in the suppressive function of liver Treg cells during CMV infection.

Comparative in vitro study of cholinium-based ionic liquids and deep eutectic solvents toward fish cell line.

With the advent of ionic liquids, much was expected concerning their applicability as an alternative to organic solvents in the chemical technology and biotechnology fields. However, the most studied and commonly used ionic liquids based on imidazolium and pyridinium were found not to be as environmentally friendly as it was first expected. Therefore, a new generation of alternative solvents named natural ionic liquids and deep eutectic solvents, composed of natural and/or renewable compounds, have come into focus in recent years. Since the number of newly synthesized chemicals increases yearly, simple and reliable methods for their ecotoxicological assessment are necessary. Permanent fish cell lines can serve as a test system for the evaluation of a chemical's cytotoxicity. This paper presents research results on the cytotoxic effects on Channel Catfish Ovary (CCO) cell line induced by fifteen cholinium-based ionic liquids and deep eutectic solvents. Based on the decrease in cell viability, the most obvious toxic effect on CCO cells was caused by ionic liquid choline oxalate, while other solvents tested exhibited low cytotoxicity. Therefore, we can conclude that cholinium-based ionic liquids and deep eutectic solvents are comparatively less toxic to CCO cells than conventional ionic liquids.