Juvenile, but Not Adult, Mice Display Increased Myeloid Recruitment and Extracellular Matrix Remodeling during Respiratory Syncytial Virus Infection.

Early life respiratory syncytial virus (RSV) infection has been linked to the onset of asthma. Despite this association, our knowledge of the progression of the initial viral infection is limited, and no safe or effective vaccine currently exists. Bronchioalveolar lavage, whole-lung cellular isolation, and gene expression analysis were performed on 3-wk- (juvenile) and 8-wk-old (adult) RSV-infected C57BL/6 mice to investigate age-related differences in immunologic responses; juvenile mice displayed a sustained myeloid infiltrate (including monocytes and neutrophils) with increased RNA expression of Ccl2, Ccl3, and Ccl4, when compared with adult mice, at 72 h postinfection. Juvenile mice demonstrated αSma expression (indicative of myofibroblast activity), increased hyaluronan deposition in the lung parenchyma (attributed to asthma progression), and a lack of CD64 upregulation on the surface of monocytes (which, in conjunction with serum amyloid P, is responsible for clearing residual hyaluronan and cellular debris). RSV infection of human airway epithelial cell, human lung fibroblast, and U937 monocyte cocultures (at air-liquid interface) displayed similar CCL expression and suggested matrix metalloproteinase-7 and MMP9 as possible extracellular matrix modifiers. These mouse data, in conjunction with our findings in human monocytes, suggest that the sustained influx of myeloid cells in the lungs of juvenile mice during acute RSV infection could potentiate extracellular matrix remodeling, facilitating conditions that support the development of asthma.

Human myoma tissue-based extracellular matrix models for testing the effects of irradiation on the HPV positive cells.

BACKGROUND: This study was designed to investigate the invasion of human papillomavirus (HPV) positive human cervical carcinoma cell lines in human leiomyoma-based extracellular matrices in vitro, and to test the suitability of the model for studying the irradiation effects on the cancer cell invasion. METHODS: HPV positive cervical carcinoma cell lines SiHa and CaSki, and HPV negative squamous cell carcinoma cell line HSC-3 were used. CaSki cells contain around 600 copies of HPV 16 virus in the genome, whereas SiHa have only 1-2 copies per cell. Cells were analyzed using two different human tumor derived extracellular matrix methods (3D myoma disc model, and Myogel Transwell invasion assay). Cultures were irradiated with 4 Gy. Myoma invasion area and the depth of invasion were measured with ImageJ 1.51j8 software. Statistical analyses were performed with SPSS Statistics (IBM SPSS® Statistics 25). RESULTS: All cells invaded through Myogel coated Transwell membranes and within myoma discs. In myoma discs, a difference in the invasion depth (p = 0.0001) but not in invasion area (p = 0.310) between the HPV positive cell lines was seen, since SiHa (less HPV) invaded slightly better than CaSki (more HPV). HSC-3 cells (HPV negative) invaded deepest (p = 0.048) than either of the HPV positive cell line cells. No difference was detected in the invasion area (p = 0.892) between HPV positive and HPV negative cells. The ionized radiation significantly reduced the invasion depth of HSC-3 (p = 0.008), SiHa (p = 0.0001) and CaSki (p = 0.005). No significant effect on the invasion area was detected in any of the cell lines. However, a significant difference was observed between SiHa and CaSki in the reduction of the invasion depth after radiation (p = 0.013) as the reduction was greater with SiHa than CaSki. CONCLUSIONS: Both solid and gelatinous human leiomyoma-based extracellular matrix models were suitable platforms to study the invasion of HPV positive cervical carcinoma cells in vitro. SiHa cells with less HPV copy number cells invaded slightly better and were slightly more sensitive to irradiation than CaSki cells with high HPV copy number. However, there was no drastic differences between the invasion properties of these carcinoma cells.

The Roles of Matricellular Proteins in Oncogenic Virus-Induced Cancers and Their Potential Utilities as Therapeutic Targets.

Matricellular proteins differ from other classical extracellular matrix proteins; for instance, they are transiently expressed as soluble proteins rather than being constitutively expressed in pathological conditions, such as acute viral infections. Accumulating studies have revealed that matricellular proteins, including osteopontin and tenascin-C, both of which interact with integrin heterodimers, are involved in inflammatory diseases, autoimmune disorders, and cancers. The concentrations of these matricellular proteins are elevated in the plasma of patients with certain types of cancers, indicating that they play important roles in oncogenesis. Chronic viral infections are associated with certain cancers, which are distinct from non-viral cancers. Viral oncogenes play critical roles in the development and progression of such cancers. It is vital to investigate the mechanisms of tumorigenesis and, particularly, the mechanism by which viral proteins induce tumor progression. Viral proteins have been shown to influence not only the viral-infected cancer cells, but also the stromal cells and matricellular proteins that constitute the extracellular matrix that surrounds tumor tissues. In this review, we summarize the recent progress on the involvement of matricellular proteins in oncogenic virus-induced cancers to elucidate the mechanism of oncogenesis and consider the possible role of matricellular proteins as therapeutic targets in virus-induced cancers.

Interaction of human papillomavirus type 16 particles with heparan sulfate and syndecan-1 molecules in the keratinocyte extracellular matrix plays an active role in infection.

Oncogenic human papillomaviruses (HPVs) attach predominantly to extracellular matrix (ECM) components during infection of cultured keratinocytes and in the rodent vaginal challenge model in vivo. However, the mechanism of virion transfer from the ECM to receptors that mediate entry into host cells has not been determined. In this work we strove to assess the role of heparan sulfate (HS) chains in HPV16 binding to the ECM and determine how HPV16 release from the ECM is regulated. We also assessed the extent to which capsids released from the ECM are infectious. We show that a large fraction of HPV16 particles binds to the ECM via HS chains, and that syndecan-1 (snd-1) molecules present in the ECM are involved in virus binding. Inhibiting the normal processing of snd-1 and HS molecules via matrix metalloproteinases and heparanase dramatically reduces virus release from the ECM, cellular uptake and infection. Conversely, exogenous heparinase activates each of these processes. We confirm that HPV16 released from the ECM is infectious in keratinocytes. Use of a specific inhibitor shows furin is not involved in HPV16 release from ECM attachment factors and corroborates other studies showing only the intracellular activity of furin is responsible for modulating HPV infectivity. These data suggest that our recently proposed model, describing the action of HS proteoglycan processing enzymes in releasing HPV16 from the cell surface in complex with the attachment factor snd-1, is also relevant to the release of HPV16 particles from the ECM to promote efficient infection of keratinocytes.

Cell entry of Lassa virus induces tyrosine phosphorylation of dystroglycan.

The extracellular matrix (ECM) receptor dystroglycan (DG) serves as a cellular receptor for the highly pathogenic arenavirus Lassa virus (LASV) that causes a haemorrhagic fever with high mortality in human. In the host cell, DG provides a molecular link between the ECM and the actin cytoskeleton via the adapter proteins utrophin or dystrophin. Here we investigated post-translational modifications of DG in the context of LASV cell entry. Using the tyrosine kinase inhibitor genistein, we found that tyrosine kinases are required for efficient internalization of virus particles, but not virus-receptor binding. Engagement of cellular DG by LASV envelope glycoprotein (LASV GP) in human epithelial cells induced tyrosine phosphorylation of the cytoplasmic domain of DG. LASV GP binding to DG further resulted in dissociation of the adapter protein utrophin from virus-bound DG. This virus-induced dissociation of utrophin was affected by genistein treatment, suggesting a role of receptor tyrosine phosphorylation in the process.

Infection spreading in tissue as a reaction-diffusion wave.

Viral infection develops in the organism due to virus replication inside infected cells and its transmission from infected to uninfected cells through the extracellular matrix or cell junctions. In this work, we model infection spreading in tissue with a delay reaction-diffusion system of equations for the concentrations of uninfected cells, infected cells and virus. We prove the wave existence, determine its speed of propagation and introduce a simplified one-equation model obtained from the complete model using a quasi-stationary approximation.

Origin of the chicken splenic reticular cells influences the effect of the infectious bursal disease virus on the extracellular matrix.

The effects of infectious bursal disease virus (IBDV) (strain F52/70) infection were studied by immunohistochemical methods on the splenic extracellular matrix (ECM). The major fibrillar components of the ECM, the type I and type III collagens and the main ECM organizing glycoproteins (laminin, tenascin and fibronectin) were monitored up to 11 days post-infection (d.p.i.). By 3 d.p.i., the collagens that form the basic scaffold of the antigen-trapping region of the spleen are destroyed, which is followed by deterioration of the glycoproteins. The ECM in the red pulp and the other regions of the white pulp (periarteriolar lymphatic sheath and germinal centre) seem to be normal. The reason for the significantly different pathological alterations in the ECM between the two regions of the spleen may be explained by the origin of the reticular cells. The reticular cells in the antigen-trapping zone and other splenic regions are of haemopoietic and mesenchymal origins, respectively. Possibly, the reticular cells of the haemopoietic origin are more susceptible for the IBDV infection than the mesenchymal ones. Development of the antigen-trapping, B-cell-dependent zone of the splenic white pulp precedes that of the periarteriolar lymphatic sheath and germinal centre, which suggests that this region may contribute to B-cell maturation. Damage of the ECM in the antigen-trapping zones results in impairment of tissue organization, which may contribute to the permanent immunosuppression.

Rhinovirus infection induces expression of airway remodelling factors in vitro and in vivo.

BACKGROUND AND OBJECTIVE: A hallmark of asthma is airway remodelling, which includes increased deposition of extracellular matrix (ECM) protein. Viral infections may promote the development of asthma and are the most common causes of asthma exacerbations. We evaluated whether rhinovirus (RV) infection induces airway remodelling, as assessed by ECM deposition. METHODS: Primary human bronchial epithelial cells and lung parenchymal fibroblasts were infected with RV-2 or RV-16, or treated with RV-16 RNA, imiquimod (Toll-like receptor (TLR) 7/8 agonist) or polyinosinic : polycytidylic acid (poly I : C) (activator of TLR 3, retinoic-acid-inducible protein I and melanoma-differentiated-associated gene 5). Changes in ECM proteins and their transcription were measured by ELISA and quantitative real-time PCR. In addition, gene expression for ECM proteins was assessed in a mouse model of RV infection. RESULTS: RV infection increased deposition of the ECM protein, perlecan, by human bronchial epithelial cells, and collagen V and matrix-bound vascular endothelial growth factor were increased in both human bronchial epithelial cell and fibroblast cultures. Purified RV-16 RNA, poly I : C and imiquimod induced similar increases in ECM deposition to those observed with RV-infected fibroblasts. However, only poly I : C induced ECM deposition by bronchial epithelial cells, suggesting that RV-induced ECM deposition is mediated through TLR. Furthermore, gene expression for fibronectin and collagen I was increased in lung homogenates of mice infected with RV-1b. CONCLUSIONS: RV infection and TLR ligands promote ECM deposition in isolated cell systems and RV induces ECM gene expression in vivo, thus demonstrating that RV has the potential to contribute to remodelling of the airways through induction of ECM deposition.

Repurposing of histone deacetylase inhibitors: A promising strategy to combat pulmonary fibrosis promoted by TGF-ß signalling in COVID-19 survivors.

Coronavirus disease 2019 (COVID-19) has rapidly spread around the world causing global public health emergency. In the last twenty years, we have witnessed several viral epidemics such as severe acute respiratory syndrome coronavirus (SARS-CoV), Influenza A virus subtype H1N1 and most recently Middle East respiratory syndrome coronavirus (MERS-CoV). There were tremendous efforts endeavoured globally by scientists to combat these viral diseases and now for SARS-CoV-2. Several drugs such as chloroquine, arbidol, remdesivir, favipiravir and dexamethasone are adopted for use against COVID-19 and currently clinical studies are underway to test their safety and efficacy for treating COVID-19 patients. As per World Health Organization reports, so far more than 16 million people are affected by COVID-19 with a recovery of close to 10 million and deaths at 600,000 globally. SARS-CoV-2 infection is reported to cause extensive pulmonary damages in affected people. Given the large number of recoveries, it is important to follow-up the recovered patients for apparent lung function abnormalities. In this review, we discuss our understanding about the development of long-term pulmonary abnormalities such as lung fibrosis observed in patients recovered from coronavirus infections (SARS-CoV and MERS-CoV) and probable epigenetic therapeutic strategy to prevent the development of similar pulmonary abnormalities in SARS-CoV-2 recovered patients. In this regard, we address the use of U.S. Food and Drug Administration (FDA) approved histone deacetylase (HDAC) inhibitors therapy to manage pulmonary fibrosis and their underlying molecular mechanisms in managing the pathologic processes in COVID-19 recovered patients.

Human influenza A virus causes myocardial and cardiac-specific conduction system infections associated with early inflammation and premature death.

AIMS: Human influenza A virus (hIAV) infection is associated with important cardiovascular complications, although cardiac infection pathophysiology is poorly understood. We aimed to study the ability of hIAV of different pathogenicity to infect the mouse heart, and establish the relationship between the infective capacity and the associated in vivo, cellular and molecular alterations. METHODS AND RESULTS: We evaluated lung and heart viral titres in mice infected with either one of several hIAV strains inoculated intranasally. 3D reconstructions of infected cardiac tissue were used to identify viral proteins inside mouse cardiomyocytes, Purkinje cells, and cardiac vessels. Viral replication was measured in mouse cultured cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were used to confirm infection and study underlying molecular alterations associated with the in vivo electrophysiological phenotype. Pathogenic and attenuated hIAV strains infected and replicated in cardiomyocytes, Purkinje cells, and hiPSC-CMs. The infection was also present in cardiac endothelial cells. Remarkably, lung viral titres did not statistically correlate with viral titres in the mouse heart. The highly pathogenic human recombinant virus PAmut showed faster replication, higher level of inflammatory cytokines in cardiac tissue and higher viral titres in cardiac HL-1 mouse cells and hiPSC-CMs compared with PB2mut-attenuated virus. Correspondingly, cardiac conduction alterations were especially pronounced in PAmut-infected mice, associated with high mortality rates, compared with PB2mut-infected animals. Consistently, connexin43 and NaV1.5 expression decreased acutely in hiPSC-CMs infected with PAmut virus. YEM1L protease also decreased more rapidly and to lower levels in PAmut-infected hiPSC-CMs compared with PB2mut-infected cells, consistent with mitochondrial dysfunction. Human IAV infection did not increase myocardial fibrosis at 4-day post-infection, although PAmut-infected mice showed an early increase in mRNAs expression of lysyl oxidase. CONCLUSION: Human IAV can infect the heart and cardiac-specific conduction system, which may contribute to cardiac complications and premature death.

Differential binding patterns to host cells associated with particles of several human alphapapillomavirus types.

The focus of this research was to compare the binding profiles of human papillomavirus (HPV) 11, 16, 18 and 45 virus-like particles (VLPs) to HaCaT cells and to the extracellular matrix (ECM) secreted by these cells. All four HPV types tested bind to a component(s) of the ECM. HPV11 VLP binding is blocked when the ECM is pretreated with an anti-laminin 5 (LN5) polyclonal antibody. A series of treatments utilizing heparins and heparinase revealed that HPV18 VLPs are dependent on heparan sulfates (HS) for binding to cells and ECM. HPV16 and HPV45 VLPs are dependent on HS for binding to HaCaT cells and dependent on both HS and LN5 for binding to ECM. These studies emphasize the need to study the binding characteristics of different HPV types before applying universal binding principles to all papillomaviruses.

Herpes simplex virus type 1 preferentially targets human colon carcinoma: role of extracellular matrix.

Viral therapy of cancer (viral oncolysis) is dependent on selective destruction of the tumor tissue compared with healthy tissues. Several factors, including receptor expression, extracellular components, and intracellular mechanisms, may influence viral oncolysis. In the present work, we studied the potential oncolytic activity of herpes simplex virus type 1 (HSV-1), using an organ culture system derived from colon carcinoma and healthy colon tissues of mouse and human origin. HSV-1 infected normal colons ex vivo at a very low efficiency, in contrast to high-efficiency infection of colon carcinoma tissue. In contrast, adenoviral and lentiviral vectors infected both tissues equally well. To investigate the mechanisms underlying the preferential affinity of HSV-1 for the carcinoma tissue, intracellular and extracellular factors were investigated. Two extracellular components, collagen and mucin molecules, were found to restrict HSV-1 infectivity in the healthy colon. The mucin layer of the healthy colon binds to HSV-1 and thereby blocks viral interaction with the epithelial cells of the tissue. In contrast, colon carcinomas express small amounts of collagen and mucin molecules and are thus permissive to HSV-1 infection. In agreement with the ex vivo system, HSV-1 injected into a mouse colon carcinoma in vivo significantly reduced the volume of the tumor. In conclusion, we describe a novel mechanism of viral selectivity for malignant tissues that is based on variance of the extracellular matrix between tumor and healthy tissues. These insights may facilitate new approaches to the application of HSV-1 as an oncolytic virus.

Integrin α3 is involved in non-enveloped hepatitis E virus infection.

Hepatitis E virus (HEV) causes acute and fulminant hepatitis worldwide. Although enveloped (e) and non-enveloped (ne) forms of HEV have been discovered, host factors involved in infection, including receptors, remain to be elucidated. Here, we identified integrin α3 (encoded by ITGA3), a protein that binds and responds to the extracellular matrix, as an essential host factor for HEV infection. Integrin α3 expression was lower in four HEV-non-permissive cell subclones than in an HEV-permissive subclone. ITGA3 knockout cells lost HEV permissibility, suggesting that integrin α3 is critical for HEV infection. Stable expression of integrin α3 in an HEV-non-permissive subclone provided permissibility only to infection by neHEV; expression of integrin α3 lacking the ectodomain did not. Direct interaction between neHEV and the integrin α3 ectodomain was confirmed by co-precipitation using a soluble integrin α3-Fc. These results strongly suggest that integrin α3 is a key molecule for cellular attachment and entry of neHEV.

Quantitative proteomics identify an association between extracellular matrix degradation and immunopathology of genotype VII Newcastle disease virus in the spleen in chickens.

Pathogenesis of genotype VII Newcastle disease virus (NDV) is characterized with remarkable immunopathology in the spleen in chickens. However, the mechanism for this unique pathological phenotype is not fully understood. Previous transcriptomics data showed that genotype VII NDV JS5/05 caused a greater downregulation of extracellular matrix (ECM) genes than genotype IV virus Herts/33 in the spleen. In this study, the role of ECM in pathology of genotype VII NDV was investigated using quantitative proteomics. Pathology studies showed that JS5/05 caused severe immunopathology characterized with remarkable necrosis in the spleen, whereas Herts/33 only induced mild pathological changes. The ECM was firstly enriched from the spleens and ECM proteins of different categories were identified by LC-MS/MS. Quantitative proteomic analysis showed that JS5/05 caused a significant disruption of ECM integrity and molecular composition compared to Herts/33. Particularly, JS5/05 induced a more remarkable collagen breakdown in the spleen compared to Herts/33. Moreover, matrix metalloproteinase (MMP)-13 and -14 were significantly upregulated by JS5/05 infection. KEGG pathway analysis suggested that differential regulation of ECM proteins by JS5/05 and Herts/33 may impact pathology through different pathways. Therefore, our results suggested that MMP upregulation and consequent ECM degradation contribute to immunopathology of genotype VII NDV in the spleen. SIGNIFICANCE: Pathogenesis of genotype VII NDV is characterized with severe immunopathology in the spleen in chickens. Elucidating the mechanism of this pathology phenotype is critical to understand pathogenesis of genotype VII NDV. Here, we present the proteomic data of an important non-cellular compartment, the extracellular matrix (ECM), in the spleen from chickens infected with genotype VII and IV NDVs. Our results suggest that significant upregulation of matrix metalloproteinases by genotype VII NDV and consequent disruption of ECM integrity and composition may be associated with immunopathology in the spleen. Moreover, ECM degradation, represented by collagen breakdown, is an important pathology event in the process of genotype VII NDV infection. Our study for the first time presents evidence of ECM regulation by NDV and adds ECM remodeling as a new manifestation for NDV pathology. Our findings also deepen the understanding of NDV pathogenesis.

Regulation of interferon signaling and HCV­RNA replication by extracellular matrix.

Although interferon (IFN)­based treatment of patients with chronic hepatitis C virus (HCV) infection is widely applied, treatment resistance is often observed in patients with advanced liver fibrosis. Given that the molecular mechanisms of IFN resistance in liver fibrosis remain elusive, the present study investigated the effects of extracellular matrix (ECM) on IFN signaling in hepatic cells. The native HuH­7 human hepatoma cell line and HuH­7 cells were stably transfected with full­length HCV­RNA fused with Renilla luciferase (OR6 cells) were cultured on ECM­coated dishes or non­coated plastic dishes (NDs), and treated with human IFN­α. In Huh­7 cells cultured on coated dishes, the IFN­stimulated response element (ISRE) luciferase activity was measured following ISRE plasmid transfection and the expression of IFN­stimulated genes (ISG) were significantly lower than those in cells cultured on NDs. In addition, after IFN­α treatment, the amount of HCV­RNA and viral protein produced by OR6 cells cultured on coated dishes was higher than that produced by cells cultured on NDs. When cells were treated with ß1­integrin­blocking antibody to disrupt the cell­matrix interaction, the ISRE luciferase activity was restored, and the protein expression of ISG was increased, while that of HCV proteins was suppressed. Treatment of cells with integrin­linked kinase (ILK) inhibitor or focal adhesion kinase (FAK) inhibitor restored the ISRE luciferase activity and expression of ISG proteins. These results suggested that ß1­integrin­mediated signals affected the IFN signaling and promoted HCV replication. Therefore, the accumulation of ECM in liver fibrosis may impair IFN signaling through ß1­integrin­mediated signaling involving ILK and FAK.

The location in cartilage of infectious retrovirus in cats infected with feline leukemia virus.

BACKGROUND: Previous studies have suggested that articular cartilage allografts were not likely to transmit infectious retrovirus since viral DNA could not be isolated from chondrocytes of infected individuals. However, the ability of the extracellular matrix of articular cartilage to harbor and transmit a retrovirus has not been examined. We hypothesized that articular cartilage fragments, but not isolated chondrocytes, from cats systemically infected with feline leukemia virus (FeLV) are capable of transmitting infectious retrovirus. METHODS: Fresh cartilage segments and chondrocytes isolated from cats systemically infected with feline leukemia virus were used in this study. Feline embryonic fibroblast cells were cocultured with segments of cartilage, isolated chondrocytes, or fragments of cortical bone from each infected cat. The FeLV p27 antigen was measured in the coculture media by enzyme-linked immunosorbent assay. In addition, FeLV proviral nucleic acids were quantified by real-time quantitative polymerase chain reaction with use of DNA extracted from feline embryonic fibroblast cell cocultures as well as isolated chondrocytes. Immunohistochemistry was used to assess for FeLV p27 antigen in both intact cartilage fragments and isolated chondrocytes. RESULTS: Feline embryonic fibroblast cells cocultured with cartilage fragments from each of the five FeLV-infected cats all demonstrated high levels of proviral DNA, indicating transmission of infective virus. In addition, media from all cocultures of feline embryonic fibroblast cells and chondral fragments became positive for p27 antigen, indicating active viral replication. In contrast, cocultures of feline embryonic fibroblast cells and isolated chondrocytes from all FeLV-infected cats were negative for proviral DNA and p27 antigen. Likewise, no proviral nucleic acids could be detected in isolated chondrocytes from any infected cats. Cocultures of feline embryonic fibroblast cells with cortical bone fragments were positive for proviral DNA and p27 antigen. Immunohistochemical staining of cartilage fragments from FeLV-infected cats demonstrated the presence of p27 antigen throughout the extracellular matrix, but the p27 antigen was not detected in isolated chondrocytes. CONCLUSIONS: Articular cartilage fragments can readily transmit infectious retrovirus, but isolated chondrocytes were likely not the source of the infectious virus because they did not harbor proviral DNA or p27 antigen.

Molecular Mechanisms of Human Papillomavirus Induced Skin Carcinogenesis.

Infection of the cutaneous skin with human papillomaviruses (HPV) of genus betapapillomavirus (ßHPV) is associated with the development of premalignant actinic keratoses and squamous cell carcinoma. Due to the higher viral loads of ßHPVs in actinic keratoses than in cancerous lesions, it is currently discussed that these viruses play a carcinogenic role in cancer initiation. In vitro assays performed to characterize the cell transforming activities of high-risk HPV types of genus alphapapillomavirus have markedly contributed to the present knowledge on their oncogenic functions. However, these assays failed to detect oncogenic functions of ßHPV early proteins. They were not suitable for investigations aiming to study the interactive role of ßHPV positive epidermis with mesenchymal cells and the extracellular matrix. This review focuses on ßHPV gene functions with special focus on oncogenic mechanisms that may be relevant for skin cancer development.

Infectious pancreatic necrosis virus infection of fish cell lines: Preliminary analysis of gene expressions related to extracellular matrix remodeling and immunity.

The pathogenic infectious pancreatic necrosis virus (IPNV) causes high economic losses in fish farming. This virus can modulate several cellular processes during infection, but little is known about the infection mechanism. To investigate gene activation in response to IPNV, CHSE/F and SHK-1 cell line were infected with a cytopathic Sp field isolate of IPNV, and the expression profiles of proinflammatory, antiviral cytokine, and extracellular matrix markers were analyzed. IPNV induced the production of perlecan, fibulin-1, matrix metalloproteinase-2, 14-3-3ß, interleukin-1ß, Mx1, and interferon regulatory factors-1, -3, and -9. Interestingly, IPNV-mediated activity was blocked by pharmacological inhibitors of the NF-κB signaling pathway. These results, together with in silico analyses showing the presence of several regulatory consensus-target motifs, suggest that IPNV regulates gene expressions in fish through the activation of several key transcription factors. Collectively, these data indicate that IPNV is a viral regulator of expression for extracellular-matrix and immune markers, even during early infection. Finally, this is the first report in fish to find IPNV modulating the activation of interleukin-1ß production primarily through the NF-κB pathway.

Chikungunya virus dissemination from the midgut of Aedes aegypti is associated with temporal basal lamina degradation during bloodmeal digestion.

In the mosquito, the midgut epithelium is the initial tissue to become infected with an arthropod-borne virus (arbovirus) that has been acquired from a vertebrate host along with a viremic bloodmeal. Following its replication in midgut epithelial cells, the virus needs to exit the midgut and infect secondary tissues including the salivary glands before it can be transmitted to another vertebrate host. The viral exit mechanism from the midgut, the midgut escape barrier (MEB), is poorly understood although it is an important determinant of mosquito vector competence for arboviruses. Using chikungunya virus (CHIKV) as a model in Aedes aegypti, we demonstrate that the basal lamina (BL) of the extracellular matrix (ECM) surrounding the midgut constitutes a potential barrier for the virus. The BL, predominantly consisting of collagen IV and laminin, becomes permissive during bloodmeal digestion in the midgut lumen. Bloodmeal digestion, BL permissiveness, and CHIKV dissemination are coincident with increased collagenase activity, diminished collagen IV abundance, and BL shredding in the midgut between 24-32 h post-bloodmeal. This indicates that there may be a window-of-opportunity during which the MEB in Ae. aegypti becomes permissive for CHIKV. Matrix metalloproteinases (MMPs) are the principal extracellular endopeptidases responsible for the degradation/remodeling of the ECM including the BL. We focused on Ae. aegypti (Ae)MMP1, which is expressed in midgut epithelial cells, is inducible upon bloodfeeding, and shows collagenase (gelatinase) activity. However, attempts to inhibit AeMMP activity in general or specifically that of AeMMP1 did not seem to affect its function nor produce an altered midgut escape phenotype. As an alternative, we silenced and overexpressed the Ae. aegypti tissue inhibitor of metalloproteinases (AeTIMP) in the mosquito midgut. AeTIMP was highly upregulated in the midgut during bloodmeal digestion and was able to inhibit MMP activity in vitro. Bloodmeal-inducible, midgut-specific overexpression of AeTIMP or its expression via a recombinant CHIKV significantly increased midgut dissemination rates of the virus. Possibly, AeTIMP overexpression affected BL degradation and/or restoration thereby increasing the midgut dissemination efficiency of the virus.

Extracellular matrix-remodeling metalloproteinases and infection of the central nervous system with retrovirus human T-lymphotropic virus type I (HTLV-I).

Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are involved in physiological processes and contribute to the phenotype of several pathological conditions associated with uncontrolled tissue degradation. In the central nervous system (CNS), MMPs are thought to play a role in cell migration and synaptic plasticity. We have investigated the expression, regulation and possible role of MMPs and TIMPs during infection of glial cells with human T-lymphotropic virus type I (HTLV-I), the causative agent of a progressive chronic myelopathy, TSP/HAM. The major alteration consists in a high increase in MMP-9 secretion and TIMP-2 mRNA expression. Cytokines TNF alpha and IL1 alpha, induced in glial cells during HTLV-I infection, promote the upregulation of MMP-9. In addition, cerebrospinal fluid from TSP/HAM patients contain high MMP-9 level. The exact role of dysregulated MMPs/TIMPs in the pathogenesis of TSP/HAM is not known; however, functions of these proteases in physiological processes should provide valuable clues. MMPs can affect the blood-brain barrier and the intercellular connectivity by degrading the extracellular matrix of endothelial and neural cells. They can be involved in autoimmunity by generating preformed specific peptides from myelin components. Finally, they can direct and prolong TNF activity in the CNS by converting its inactive precursor into active molecules.