Type dependent patterns of human adenovirus persistence in human T-lymphocyte cell lines.

Disseminated adenovirus infections cause significant mortality in stem cell transplanted patients and are suspected to originate from asymptomatic adenovirus persistence ("latency") in lymphocytes. The infection of three human T-lymphocyte lines (Jurkat, PM1, and CEM) with human adenovirus types of species A (HAdV-A31), B (HAdV-B3, -B11), and C (HAdV-C2, -C5) was investigated for 150 days in order to establish in vitro models for adenovirus persistence. HAdV-C5 persisted with continuous production of infectious virus progeny (about 10(7) TCID50 /ml) in PM1 cells. More than 100 copies of HAdV-C5-DNA per cell were detected by real-time PCR but hexon immunostaining showed that only 7.5% of the cells were infected ("carrier state infection"). Coxsackie and adenovirus receptor (CAR) expression was decreased in comparison to mock infected cultures suggesting selection of a semi-permissive subpopulation of PM-1 cells. By contrast, latency of HAdV-DNA (10(-3) -10(-4) copies/cell) without production of infectious virus progeny was observed in HAdV-C2 infection of PM1 and Jurkat, HAdV-A31 infection of PM1, and HAdV-B3 infection of Jurkat cells. In addition, transcription of E1A, DNA polymerase and hexon mRNA was not detected by RT-PCR suggesting an equivalent of clinical "HAdV latency." Persistence of HAdV-DNA was not observed in abortive infections of PM1 cells with HAdV-B3 and -B11 and in productive, lytical infections of Jurkat cells with HAdV-C5, HAdV-B11, and HAdV-A31. In conclusion, lytic and persistent infections with and without production of infectious virus were observed depending on the type of adenovirus. Genetic determinants for viral persistence may be investigated using these newly established infection models.

Porcine corneal cell culture models for studying epidemic keratoconjunctivitis.

PURPOSE: Epidemic keratoconjunctivitis (EKC) is a severe ocular infection caused by a few types (8, 19a [relabeled as 64 recently], 37, 53, and 54) of human adenoviruses (HAdVs). HAdVs are known for their strong host species specificity that limits studying HAdV virulence and pathophysiology in animal models. METHODS: A HAdV infection model of primary porcine corneal epithelial cells (PPCE) and primary porcine corneal keratocytes (PPCK) was established and compared to primary human corneal epithelial cells (PHCE) and primary human corneal keratocytes (PHCK). Induction of interleukin-8 (IL-8) messenger RNA (mRNA), HAdV DNA replication, and the release of infectious virus progeny by the EKC-associated type HAdV-D37 and the non-EKC-associated type HAdV-D22 were studied. RESULTS: PPCE and PPCK morphology and the expression of α2,3-linked sialic acid, the main receptor of EKC-associated HAdV types, were akin to human corneal cells (PHCE and PHCK). Induction of IL-8 mRNA was observed as early as 8 h after HAdV infection. Induction of IL-8 mRNA by HAdV-D37 infection was significantly higher (p≤0.001) than by HAdV-D22 infection in PPCE, PPCK, PHCE, and PHCK. Detection of HAdV-DNA replication, release of infectious virus progeny, and the development of cytopathic effect indicated that PPCE and PPCK were fully permissive for HAdV-D37 and HAdV-D22 replication as were the human corneal cells (PHCE and PHCK). Infectious virus titers after HAdV-D37 infection (1.0 × 10(5) TCID50/ml) were significantly higher (p=0.001) than after HAdV-D22 infection (1.8 × 10(4) TCID50/ml) in PPCE, PHCE, and PHCK but not significantly different in PPCK. CONCLUSIONS: Primary porcine epithelial cells and keratocytes are nonhuman corneal cell culture models fully permissive for HAdV infection. The models hold promise for studying the virulence and pathophysiology of EKC-associated adenovirus types compared to other adenovirus types.

Induction of a broad spectrum of inflammation-related genes by Coxsackievirus B3 requires Interleukin-1 signaling.

UNLABELLED: Coxsackievirus B3 (CVB3) is a major cause of acute and chronic forms of myocarditis. Previously, direct viral injury and post-infectious autoimmune response were suspected as main pathogenetic mechanisms. However, induction of pro-inflammatory cytokines may be crucial for pathogenesis in spite of host protein shut off caused by CVB3 replication. We investigated the global expression profile of pro-inflammatory genes induced by acute and persistent (carrier state) CVB3 infection in human fibroblast cell cultures with DNA microarrays, quantitative RT-PCR and ELISA. Rapid induction of a typical spectrum of about 30 inflammation-related genes (e.g., PTGS2, CCL2, IL-1ß, IL-6, IL-8, CSF2, MMP-1, MMP-3, and MMP-15) suggested an essential, autocrine role of IL-1. This hypothesis was confirmed by over-expression of IL-1RI, which resulted in a cytokine response upon CVB3 infection in HEK 293 cells otherwise refractory to CVB3-caused gene expression. Blocking IL-1 receptor type I (IL-1RI)-signaling during CVB3 infection with an IL-1 receptor antagonist (IL-1ra) as well as knockdown of IL-1RI using siRNA abrogated cytokine response in human fibroblasts. Both IL-1α and IL-1ß are relevant for the induction of inflammation-related genes during CVB3 infection as shown by neutralization experiments. Paracrine effects of IL-1 on the subset of non-infected cells in carrier state infected fibroblast cultures enhanced induction of inflammation-related genes. CONCLUSIONS: A broad spectrum of inflammatory cytokines was induced by CVB3 replication via a pathway that requires IL-1 signaling. Our results suggest that IL-1ra may be used as a therapeutic agent to limit inflammation and tissue destruction in myocarditis.

DNA polymerase theta up-regulation is associated with poor survival in breast cancer, perturbs DNA replication, and promotes genetic instability.

"Replicative stress" is one of the main factors underlying neoplasia from its early stages. Genes involved in DNA synthesis may therefore represent an underexplored source of potential prognostic markers for cancer. To this aim, we generated gene expression profiles from two independent cohorts (France, n=206; United Kingdom, n=117) of patients with previously untreated primary breast cancers. We report here that among the 13 human nuclear DNA polymerase genes, DNA Polymerase (POLQ) is the only one significantly up-regulated in breast cancer compared with normal breast tissues. Importantly, POLQ up-regulation significantly correlates with poor clinical outcome (4.3-fold increased risk of death in patients with high POLQ expression), and this correlation is independent of Cyclin E expression or the number of positive nodes, which are currently considered as markers for poor outcome. POLQ expression provides thus an additional indicator for the survival outcome of patients with high Cyclin E tumor expression or high number of positive lymph nodes. Furthermore, to decipher the molecular consequences of POLQ up-regulation in breast cancer, we generated human MRC5-SV cell lines that stably overexpress POLQ. Strong POLQ expression was directly associated with defective DNA replication fork progression and chromosomal damage. Therefore, POLQ overexpression may be a promising genetic instability and prognostic marker for breast cancer.

Broad-spectrum antiviral activity of the deubiquitinase inhibitor HBX against human adenoviruses

BACKGROUND: Human adenoviral (HAdV) infections are usually mild and self-limited, however, some infections from species A, B, C, D and E, can cause severe illnesses, which have raised public health concerns over the past few years. Current available antiviral therapies have limited efficacy and severe toxicity; therefore, finding new targets for specific anti-adenoviral drug design is urgently needed. Our previous work showed that the small molecule compound, HBX, inhibits HAdV type 5 (species C, HAdV-C5) replication and oncogenic transformation through inhibition of the cellular pro-viral factor ubiquitin-specific protease 7 (USP7). Here, we have tested the ability of HBX to inhibit other HAdV species, as well as different clinical isolates that are the cause of severe infections. METHODS: We treated HAdV-infected A549 cells with different concentrations of HBX and analysed the antiviral efficacy of the drug by determining the half maximal inhibitory concentration (IC50) necessary to decrease both viral genome copies and virus progeny production at different time points after infection. RESULTS: In addition to its effect on HAdV-C5, HBX was able to significantly inhibit virus genome replication and progeny release of all adenovirus types tested, with the exception of types 12 and 31, from species A. Of note, clinical isolates were more sensitive to HBX treatment than their prototype strains. CONCLUSIONS: These results point to HBX as a promising broad-spectrum anti-adenoviral drug, opening new opportunities to prevent severe adenoviral infections and to improve their treatment.

Effective Apical Infection of Differentiated Human Bronchial Epithelial Cells and Induction of Proinflammatory Chemokines by the Highly Pneumotropic Human Adenovirus Type 14p1.

BACKGROUND: Only a few pneumotropic types of the human adenoviruses (e.g. type B14p1) cause severe lower respiratory tract infections like pneumonia and acute respiratory distress syndrome (ARDS) even in immunocompetent patients. By contrast, many other human adenovirus (HAdV) types (e.g. HAdV-C5) are associated mainly with upper respiratory tract infections. This is in accordance with a highly physiological cell culture system consisting of differentiated primary human bronchial epithelial cells which are little susceptible for apical HAdV-C5 infections. OBJECTIVE AND METHODS: We hypothesized that a pneumotropic and highly pathogenic HAdV type infects differentiated human bronchial epithelial cells efficiently from the apical surface and also induces proinflammatory cytokines in order to establish ARDS and pneumonia. Therefore, the apical infection of differentiated primary human bronchial epithelial cells with the pneumotropic and virulent type HAdV-B14p1 was investigated in comparison to the less pneumotropic HAdV-C5 as a control. RESULTS: Binding of HAdV-B14p1 to the apical surface of differentiated human bronchial epithelial cells and subsequent internalization of HAdV DNA was 10 fold higher (p<0.01) compared to the less-pneumotropic HAdV-C5 one hour after infection. Overall, the replication cycle of HAdV-B14p1 following apical infection and including apical release of infectious virus progeny was about 1000-fold more effective compared to the non-pneumotropic HAdV-C5 (p<0.001). HAdV-B14p1 infected cells expressed desmoglein 2 (DSG2), which has been described as potential receptor for HAdV-B14p1. Moreover, HAdV-B14p1 induced proinflammatory chemokines IP-10 and I-Tac as potential virulence factors. Interestingly, IP-10 has already been described as a marker for severe respiratory infections e.g. by influenza virus A H5N1. CONCLUSIONS: The efficient "apical to apical" replication cycle of HAdV-B14p1 can promote endobronchial dissemination of the infection from the upper to the lower respiratory tract. Simultaneous induction of proinflammatory cytokines probably contributes to the high virulence of HAdV-B14p1.

Targeting species D adenoviruses replication to counteract the epidemic keratoconjunctivitis.

Human adenoviruses are non-enveloped DNA viruses causing various infections; their pathogenicity varies dependent on virus species and type. Although acute infections can sometimes take severe courses, they are rarely fatal in immune-competent individuals. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are hyperacute and highly contagious infections of the eye caused by human adenovirus types within species D. Currently there is no causal treatment available to counteract these diseases effectively. The E2B region of the adenovirus genome encodes for the viral DNA polymerase, which is required for adenoviral DNA replication. Here we propose novel model systems to test this viral key factor, DNA polymerase, as a putative target for the development of efficient antiviral therapy based on RNA interference. Using our model cell lines we found that different small interfering RNAs mediate significant suppression (up to 90%) of expression levels of viral DNA polymerase upon transfection. Moreover, permanent expression of short hairpin RNA based on the most effective small interfering RNA led to a highly significant, more than tenfold reduction in replication for different human group D adenoviruses involved in ocular infections.

Artesunate derived from traditional Chinese medicine induces DNA damage and repair.

Artesunate is a semisynthetic derivative from artemisinin, a natural product from the Chinese herb Artemisia annua L. It exerts antimalarial activity, and, additionally, artemisinin and its derivatives are active against cancer cells. The active moiety is an endoperoxide bridge. Its cleavage leads to the formation of reactive oxygen species and carbon-centered radicals. These highly reactive molecules target several proteins in Plasmodia, which is thought to result in killing of the microorganism. DNA damage induced by artemisinins has not yet been described. Here, we show that artesunate induces apoptosis and necrosis. It also induces DNA breakage in a dose-dependent manner as shown by single-cell gel electrophoresis. This genotoxic effect was confirmed by measuring the level of gamma-H2AX, which is considered to be an indication of DNA double-strand breaks (DSB). Polymerase beta-deficient cells were more sensitive than the wild-type to artesunate, indicating that the drug induces DNA damage that is repaired by base excision repair. irs1 and VC8 cells defective in homologous recombination (HR) due to inactivation of XRCC2 and BRCA2, respectively, were more sensitive to artesunate than the corresponding wild-type. This was also true for XR-V15B cells defective in nonhomologous end-joining (NHEJ) due to inactivation of Ku80. The data indicate that DSBs induced by artesunate are repaired by the HR and NHEJ pathways. They suggest that DNA damage induced by artesunate contributes to its therapeutic effect against cancer cells.