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.

Fluorescent Crimean-Congo hemorrhagic fever virus illuminates tissue tropism patterns and identifies early mononuclear phagocytic cell targets in Ifnar-/- mice.

Crimean-Congo hemorrhagic fever virus (CCHFV, order Bunyavirales, family Nairoviridae, genus Orthonairovirus) is the tick-borne etiological agent of Crimean-Congo hemorrhagic fever (CCHF) in humans. Animals are generally susceptible to CCHFV infection but refractory to disease. Small animal models are limited to interferon-deficient mice, that develop acute fatal disease following infection. Here, using a ZsGreen1- (ZsG) expressing reporter virus (CCHFV/ZsG), we examine tissue tropism and dissemination of virus in interferon-α/ß receptor knock-out (Ifnar-/-) mice. We demonstrate that CCHFV/ZsG retains in vivo pathogenicity comparable to wild-type virus. Interestingly, despite high levels of viral RNA in all organs assessed, 2 distribution patterns of infection were observed by both fluorescence and immunohistochemistry (IHC), corresponding to the permissiveness of organ tissues. To further investigate viral dissemination and to temporally define cellular targets of CCHFV in vivo, mice were serially euthanized at different stages of disease. Flow cytometry was used to characterize CCHFV-associated alterations in hematopoietic cell populations and to classify infected cells in the blood, lymph node, spleen, and liver. ZsG signal indicated that mononuclear phagocytic cells in the lymphatic tissues were early targets of infection; in late-stage infection, overall, the highest levels of signal were detected in the liver, and ZsG was found in both antigen-presenting and lymphocyte cell populations.

Structure-activity relationship of uridine-based nucleoside phosphoramidate prodrugs for inhibition of dengue virus RNA-dependent RNA polymerase.

To identify a potent and selective nucleoside inhibitor of dengue virus RNA-dependent RNA polymerase, a series of 2'- and/or 4'-ribose sugar modified uridine nucleoside phosphoramidate prodrugs and their corresponding triphosphates were synthesized and evaluated. Replacement of 2'-OH with 2'-F led to be a poor substrate for both dengue virus and human mitochondrial RNA polymerases. Instead of 2'-fluorination, the introduction of fluorine at the ribose 4'-position was found not to affect the inhibition of the dengue virus polymerase with a reduction in uptake by mitochondrial RNA polymerase. 2'-C-ethynyl-4'-F-uridine phosphoramidate prodrug displayed potent anti-dengue virus activity in the primary human peripheral blood mononuclear cell-based assay with no significant cytotoxicity in human hepatocellular liver carcinoma cell lines and no mitochondrial toxicity in the cell-based assay using human prostate cancer cell lines.

Avian reovirus replication in mononuclear phagocytes in chicken footpad and spleen after footpad inoculation.

Circulating monocytes and tissue macrophages were suggested to be susceptible to avian reovirus (ARV) infection. To determine if ARV infects and replicates in mononuclear phagocytes (KUL01-positive cells), we infected 3-day-old specific-pathogen-free chickens with ARV strain 2408 by inoculation of the left footpad. The left footpads and spleens were collected for analysis at 1.5 and 2.5 d after inoculation. Replication of ARV in the footpad and spleen was demonstrated by detection of the viral protein σNS using immunohistochemical testing and viral S1 RNA expression by real-time quantitative polymerase chain reaction (qPCR). Furthermore, immunofluorescent double-staining assay of cytocentrifuged cells and cryosections of the footpad and spleen for the viral protein σNS and the surface marker recognized by monoclonal antibody (MAb) KUL01 indicated that KUL01-positive cells costained with MAb H1E1, which recognizes ARV protein σNS. In addition, more ARV S1 RNA was measured by qPCR in the KUL01-positive cell samples prepared from the footpad or spleen 1.5 d after inoculation compared with non-KUL01-positive cell samples. The amounts of ARV S1 RNA in the spleen were significantly lower (P < 0.05) than the amounts in the footpad 1.5 d after inoculation. The results suggest that ARV infects mononuclear phagocytes and then replicates within these cells before migrating to the spleen, where it infects and replicates in KUL01-positive cells.

Il a été suggéré que les monocytes circulants et les macrophages tissulaires étaient sensibles à une infection par le reovirus aviaire (ARV). Afin de déterminer si l'ARV infecte et se réplique dans les phagocytes mononucléaires (cellules KUL01-positives), nous avons infecté des poussins exempts d'agents pathogènes spécifiques âgés de 3 j avec la souche 2408 d'ARV par inoculation dans le coussinet plantaire gauche. Les coussinets plantaires et les rates furent prélevés pour analyse aux jours 1,5 et 2,5 suivant l'inoculation. La réplication d'ARV dans le coussinet plantaire et la rate fut démontrée par détection de la protéine virale σNS par épreuve immunohistochimique et l'expression d'ARN S1 viral par réaction d'amplification en chaîne par la polymérase en temps réel (qPCR). De plus, l'épreuve d'immunofluorescence par double coloration de cellules cytocentrifugées et de coupes congelées du coussinet plantaire et de la rate pour la protéine virale σNS et le marqueur de surface reconnu par l'anticorps monoclonal (AcMo) KUL01 indiquait que les cellules positives pour KUL01se co-coloraient avec l'AcMo H1E1, qui reconnait la protéine σNS de l'ARV. Également, plus d'ARN S1 d'ARV était mesuré par qPCR dans les échantillons de cellules KUL01 positives préparés à partir de coussinets plantaires ou de rates 1,5 j après l'inoculation comparativement à des échantillons de cellules KUL01 négatives. Les quantités d'ARN S1 d'ARV dans la rate étaient significativement plus basses (P < 0,05) que les quantités dans les coussinets plantaires 1,5 j après l'inoculation. Les résultats suggèrent que l'ARV infecte les phagocytes mononucléaires et par la suite se répliquent dans ces cellules avant de migrer à la rate, où il infecte et se réplique dans les cellules KUL01-positives.(Traduit par Docteur Serge Messier).

Role of macrophages and monocytes in hepatitis C virus infections.

A number of studies conducted over many years have shown that hepatitis C virus (HCV) can infect a variety of cell types. In vivo infection of monocytes, macrophages, and dendritic cells by HCV has been frequently shown by a number of researchers. These studies have demonstrated replication of HCV by detecting the presence of both negative genomic strands and a variety of non-structural HCV proteins in infected cells. In addition, analyses of genome sequences have also shown that different cell types can harbor different HCV variants. Investigators have also done preliminary studies of which cellular genes are affected by HCV infection, but there have not yet been a sufficient number of these studies to understand the effects of infection on these cells. Analyses of in vitro HCV replication have shown that monocytes, macrophages and dendritic cells can be infected by HCV from patient sera or plasma. These studies suggest that entry and cellular locations may vary between different cell types. Some studies suggest that macrophages may preferentially allow HCV genotype 1 to replicate, but macrophages do not appear to select particular hypervariable regions. Overall, these studies agree with a model where monocytes and macrophages act as an amplification system, in which these cells are infected and show few cytopathic effects, but continuously produce HCV. This allows them to produce virus over an extended time and allows its spread to other cell types.

SIV infection of rhesus macaques differentially impacts mononuclear phagocyte responses to virus-derived TLR agonists.

BACKGROUND: During progressive simian immunodeficiency virus (SIV) infection, the ability of innate mononuclear phagocytes to function when responding to the invading pathogen has yet to be determined. METHODS: We generated single-stranded RNA (ssRNA) oligonucleotides from the infecting strain of virus and utilized them to stimulate mononuclear phagocytes from blood and lymph nodes of naïve and SIVmac251-infected rhesus macaques. RESULTS: Soon after infection and continuing through to chronic disease, plasmacytoid dendritic cells (pDC), monocytes, and macrophages from SIV-infected macaques were less able to produce pro-inflammatory cytokines after exposure to virus-derived toll-like receptor (TLR) agonists. In contrast, myeloid dendritic cells (mDC) became hyper-responsive during acute and stable chronic infection. CONCLUSIONS: Plasmacytoid dendritic cells, monocytes, and macrophages may not instigate continued immune activation by recognizing the single-stranded RNA from SIV as they are left dysfunctional after infection. Conversely, mDC functionality may be beneficial as their hyper-responsiveness is related to slowed disease progression.

Cytometry, immunology, and HIV infection: three decades of strong interactions.

Flow cytometry (FCM) has been extensively used to investigate immunological changes that occur from infection with the human immunodeficiency virus (HIV). This review describes some of the most relevant cellular and molecular changes in the immune system that can be detected by FCM during HIV infection. Finally, it will be discussed how this technology has facilitated the understanding not only of the biology of the virus but also of the mechanisms that the immune system activates to fight HIV and is allowing to monitor the efficacy of antiretroviral therapy.

Antiretroviral therapy in macrophages: implication for HIV eradication.

HIV type-1 (HIV-1) accounts for more than 25 million deaths and nearly 40 million people are infected worldwide. A significant obstacle in clearing virus from infected individuals is latently infected viral reservoirs. Latent HIV-1 can emerge with recrudescence as a productive infection later in disease progression and could provide a source for the emergence of resistant HIV-1. It is widely recognized that macrophages represent a latently infected viral reservoir and are a significant and critical HIV-1 target cell in vivo. Macrophages can be divided into multiple subsets of macrophage-like cells, all of which are susceptible to HIV-1 infection, including dendritic cells, Langerhans cells, alveolar macrophages, mucosal macrophages and microglial cells. Current antiretroviral therapy (ART) often displays differential antiviral activity in macrophages relative to CD4(+) T-lymphocytes. Significant work has been performed to establish antiviral activity of many clinically approved ART in macrophages; however, a direct link between antiviral activity and specific mechanisms responsible for these antiviral effects are incompletely understood. This review identifies many understudied areas of research, along with topics for further research in the field of HIV therapy and eradication. Discussion focuses upon the known cellular pharmacology and antiviral activity of antiretroviral agents in macrophages and its relationship to latency, chronic HIV-1 infection and therapeutic strategies to eradicate systemic HIV-1 infection.

A novel adenovirus expressing Flt3 ligand enhances mucosal immunity by inducing mature nasopharyngeal-associated lymphoreticular tissue dendritic cell migration.

Previously, we showed that nasal administration of a naked cDNA plasmid expressing Flt3 ligand (FL) cDNA (pFL) enhanced CD4(+) Th2-type, cytokine-mediated mucosal immunity and increased lymphoid-type dendritic cell (DC) numbers. In this study, we investigated whether targeting nasopharyngeal-associated lymphoreticular tissue (NALT) DCs by a different delivery mode of FL, i.e., an adenovirus (Ad) serotype 5 vector expressing FL (Ad-FL), would provide Ag-specific humoral and cell-mediated mucosal immunity. Nasal immunization of mice with OVA plus Ad-FL as mucosal adjuvant elicited high levels of OVA-specific Ab responses in external secretions and plasma as well as significant levels of OVA-specific CD4(+) T cell proliferative responses and OVA-induced IFN-gamma and IL-4 production in NALT, cervical lymph nodes, and spleen. We also observed higher levels of OVA-specific CTL responses in the spleen and cervical lymph nodes of mice given nasal OVA plus Ad-FL than in mice receiving OVA plus control Ad. Notably, the number of CD11b(+)CD11c(+) DCs expressing high levels of costimulatory molecules was preferentially increased. These DCs migrated from the NALT to mucosal effector lymphoid tissues. Taken together, these results suggest that the use of Ad-FL as a nasal adjuvant preferentially induces mature-type NALT CD11b(+)CD11c(+) DCs that migrate to effector sites for subsequent CD4(+) Th1- and Th2-type cytokine-mediated, Ag-specific Ab and CTL responses.

Adenovirus-platelet interaction in blood causes virus sequestration to the reticuloendothelial system of the liver.

Intravenous (i.v.) delivery of recombinant adenovirus serotype 5 (Ad5) vectors for gene therapy is hindered by safety and efficacy problems. We have discovered a new pathway involved in unspecific Ad5 sequestration and degradation. After i.v. administration, Ad5 rapidly binds to circulating platelets, which causes their activation/aggregation and subsequent entrapment in liver sinusoids. Virus-platelet aggregates are taken up by Kupffer cells and degraded. Ad sequestration in organs can be reduced by platelet depletion prior to vector injection. Identification of this new sequestration mechanism and construction of vectors that avoid it could improve levels of target cell transduction at lower vector doses.

Sendai virus fusion protein mediates simultaneous induction of MHC class I/II-dependent mucosal and systemic immune responses via the nasopharyngeal-associated lymphoreticular tissue immune system.

Nasal administration of Ags using a novel hybrid Ag delivery vehicle composed of envelope glycoproteins of Sendai virus on the surface of liposome membranes (fusogenic liposome) efficiently delivered Ags to Ag-sampling M cells in nasopharyngeal-associated lymphoreticular tissue. Additionally, fusogenic liposomes also effectively delivered the Ags into epithelial cells and macrophages in nasopharyngeal-associated lymphoreticular tissue and nasal passages. In vitro Ag presentation assays clearly showed that fusogenic liposomes effectively presented encapsulated Ags via the MHC class II-dependent pathway of epithelial cells as well as macrophages. Fusogenic liposomes also have an adjuvant activity against mucosal epithelial cells to enhance MHC class II expression. According to these high delivery and adjuvant activities of fusogenic liposomes, nasal immunization with OVA-encapsulated fusogenic liposomes induced high levels of OVA-specific CD4(+) Th1 and Th2 cell responses. Furthermore, Ag-specific CTL responses and Ab productions were also elicited at both mucosal and systemic sites by nasal immunization with Ag-encapsulated fusogenic liposomes. These results indicate that fusogenic liposome is a versatile and effective system for the stimulation of Ag-specific immune responses at both mucosal and systemic compartments.

Comparison of cellular accumulation, tissue distribution, and anti-HIV activity of free and liposomal 2',3'-dideoxycytidine.

We have investigated the cellular accumulation, tissue distribution, and antihuman immunodeficiency virus activity of free dideoxycytidine (ddC) and liposomal ddC (L-ddC). We have found that L-ddC was more efficiently taken up than its free form by RAW 264.7 cells (a monocyte-macrophage cell line) (p < 0.01) while a comparable uptake was seen in U937 cells (a promonocytic cell line). In the rat, L-ddC accumulated preferentially in liver and spleen when injected intravenously (p < 0.01), and mostly in spleen when given intraperitoneally (p < 0.01). In contrast, free ddC was rapidly eliminated out of the body. Liposomal ddC showed a similar anti-HIV activity in comparison with free ddC in U937 cells. Given the fact that encapsulation of ddC in liposomes does not affect its anti-HIV activity but enhances its in vitro cellular accumulation and its in vivo distribution in reticuloendothelial system (RES) tissues, we conclude that ddC in liposomal formulation is a promising anti-HIV agent with a targeted action on the RES, which is considered a reservoir for dissemination of virus to other cells, tissues, and organs.