COVID-19 and Its Impact on Tourism Industry.

The current COVID-19 pandemic is leading to significant changes in terms of people's economic behavior, which will inevitably impact the tourism industry and tourism activity both worldwide and in tourism host countries. Immediate control measures, such as necessary restrictions on travel, avoiding physical contact, social distancing, as well as tourists' and patients' changes in priority making, have vanished interest in traveling away from the place of usual residence and seeking to receive tourism services. COVID-19 pandemic has caused immediate impacts across the whole spectrum of economic and social activity. The duration and intensity of the arising malfunction in tourism are not yet known; thus, it is too early to make any assessments of the financial losses that will be recorded on an annual basis. However, an initial approach is necessary in order to assess the range of to date impacts, aiming at a critical appraisal of the current situation. It will mainly help in making the appropriate pandemic management plan in the tourism industry.

LIM-domain proteins, LIMD1, Ajuba, and WTIP are required for microRNA-mediated gene silencing.

In recent years there have been major advances with respect to the identification of the protein components and mechanisms of microRNA (miRNA) mediated silencing. However, the complete and precise repertoire of components and mechanism(s) of action remain to be fully elucidated. Herein we reveal the identification of a family of three LIM domain-containing proteins, LIMD1, Ajuba and WTIP (Ajuba LIM proteins) as novel mammalian processing body (P-body) components, which highlight a novel mechanism of miRNA-mediated gene silencing. Furthermore, we reveal that LIMD1, Ajuba, and WTIP bind to Ago1/2, RCK, Dcp2, and eIF4E in vivo, that they are required for miRNA-mediated, but not siRNA-mediated gene silencing and that all three proteins bind to the mRNA 5' m(7)GTP cap-protein complex. Mechanistically, we propose the Ajuba LIM proteins interact with the m(7)GTP cap structure via a specific interaction with eIF4E that prevents 4EBP1 and eIF4G interaction. In addition, these LIM-domain proteins facilitate miRNA-mediated gene silencing by acting as an essential molecular link between the translationally inhibited eIF4E-m(7)GTP-5(')cap and Ago1/2 within the miRISC complex attached to the 3'-UTR of mRNA, creating an inhibitory closed-loop complex.

miR-132 regulates antiviral innate immunity through suppression of the p300 transcriptional co-activator.

MicroRNAs are small, non-coding RNAs that negatively regulate gene expression. It has been proposed that microRNAs could function in the regulation of innate immunity, but this has not been demonstrated for viral infection. Here we test this hypothesis using the human pathogenic virus Kaposi's sarcoma-associated herpesvirus (KSHV) and one of its putative natural cellular targets, primary lymphatic endothelial cells (LECs). We show that an early antiviral microRNA response (6 h post-infection) includes expression of microRNAs that enhance viral gene expression. In particular, the CREB-induced miR-132 microRNA is highly upregulated after infection and has a negative effect on the expression of interferon-stimulated genes, facilitating viral replication. We show a similar function for miR-132 during infection of monocytes with herpes simplex virus-1 (HSV-1) and human cytomegalovirus (HCMV). miR-132 regulates innate antiviral immunity by inhibiting expression of the p300 transcriptional co-activator. p300 is downregulated early after KSHV infection, and inhibition of miR-132 induction restores p300 expression. Furthermore, p300 regulates miR-132 levels, revealing a dynamic equilibrium between miR-132 and p300. By targeting p300, rather than a transcription factor or signalling protein, miR-132 has a broad role in the regulation of antiviral immunity.

KSHV-encoded miRNAs target MAF to induce endothelial cell reprogramming.

Kaposi sarcoma herpesvirus (KSHV) induces transcriptional reprogramming of endothelial cells. In particular, KSHV-infected lymphatic endothelial cells (LECs) show an up-regulation of genes associated with blood vessel endothelial cells (BECs). Consequently, KSHV-infected tumor cells in Kaposi sarcoma are poorly differentiated endothelial cells, expressing markers of both LECs and BECs. MicroRNAs (miRNAs) are short noncoding RNA molecules that act post-transcriptionally to negatively regulate gene expression. Here we validate expression of the KSHV-encoded miRNAs in Kaposi sarcoma lesions and demonstrate that these miRNAs contribute to viral-induced reprogramming by silencing the cellular transcription factor MAF (musculoaponeurotic fibrosarcoma oncogene homolog). MAF is expressed in LECs but not in BECs. We identify a novel role for MAF as a transcriptional repressor, preventing expression of BEC-specific genes, thereby maintaining the differentiation status of LECs. These findings demonstrate that viral miRNAs could influence the differentiation status of infected cells, and thereby contribute to KSHV-induced oncogenesis.

KSHV manipulates Notch signaling by DLL4 and JAG1 to alter cell cycle genes in lymphatic endothelia.

Increased expression of Notch signaling pathway components is observed in Kaposi sarcoma (KS) but the mechanism underlying the manipulation of the canonical Notch pathway by the causative agent of KS, Kaposi sarcoma herpesvirus (KSHV), has not been fully elucidated. Here, we describe the mechanism through which KSHV directly modulates the expression of the Notch ligands JAG1 and DLL4 in lymphatic endothelial cells. Expression of KSHV-encoded vFLIP induces JAG1 through an NFkappaB-dependent mechanism, while vGPCR upregulates DLL4 through a mechanism dependent on ERK. Both vFLIP and vGPCR instigate functional Notch signalling through NOTCH4. Gene expression profiling showed that JAG1- or DLL4-stimulated signaling results in the suppression of genes associated with the cell cycle in adjacent lymphatic endothelial cells, indicating a role for Notch signaling in inducing cellular quiescence in these cells. Upregulation of JAG1 and DLL4 by KSHV could therefore alter the expression of cell cycle components in neighbouring uninfected cells during latent and lytic phases of viral infection, influencing cellular quiescence and plasticity. In addition, differences in signaling potency between these ligands suggest a possible complementary role for JAG1 and DLL4 in the context of KS.

The CD8 and CD4 T-cell response against Kaposi's sarcoma-associated herpesvirus is skewed towards early and late lytic antigens.

Kaposi's sarcoma-associated herpesvirus (KSHV) is causally related to Kaposi's sarcoma (KS), the most common malignancy in untreated individuals with HIV/AIDS. The adaptive T-cell immune response against KSHV has not been fully characterized. To achieve a better understanding of the antigenic repertoire of the CD8 and CD4 T-cell responses against KSHV, we constructed a library of lentiviral expression vectors each coding for one of 31 individual KSHV open reading frames (ORFs). We used these to transduce monocyte-derived dendritic cells (moDCs) isolated from 14 KSHV-seropositive (12 HIV-positive) and 7 KSHV-seronegative (4 HIV-positive) individuals. moDCs were transduced with up to 3 KSHV ORFs simultaneously (ORFs grouped according to their expression during the viral life cycle). Transduced moDCs naturally process the KSHV genes and present the resulting antigens in the context of MHC class I and II. Transduced moDCs were cultured with purified autologous T cells and the CD8 and CD4 T-cell proliferative responses to each KSHV ORF (or group) was assessed using a CFSE dye-based assay. Two pools of early lytic KSHV genes ([ORF8/ORF49/ORF61] and [ORF59/ORF65/K4.1]) were frequently-recognized targets of both CD8 and CD4 T cells from KSHV seropositive individuals. One pool of late lytic KSHV genes ([ORF28/ORF36/ORF37]) was a frequently-recognized CD8 target and another pool of late genes ([ORF33/K1/K8.1]) was a frequently-recognized CD4 target. We report that both the CD8 and CD4 T-cell responses against KSHV are skewed towards genes expressed in the early and late phases of the viral lytic cycle, and identify some previously unknown targets of these responses. This knowledge will be important to future immunological investigations into KSHV and may eventually lead to the development of better immunotherapies for KSHV-related diseases.

Toll-like receptor 4 mediates innate immunity to Kaposi sarcoma herpesvirus.

The involvement of Toll-like receptor 4 (TLR4) in immunity against human herpesviruses has not been previously demonstrated. We show that infection of endothelial cells with Kaposi sarcoma herpesvirus (KSHV), a human oncogenic virus, leads to rapid suppression of TLR4 expression. This is a mechanism of immune escape as TLR4 mediates innate immunity against KSHV. In vitro, cells lacking TLR4 are more susceptible to KSHV infection, whereas activation of TLR4 protects cells from infection. In vivo, HIV-1-infected individuals carrying a mutant TLR4 allele appear more likely to have multicentric Castleman's disease, a lymphoproliferation associated with enhanced KSHV replication. ERK activation by KSHV structural proteins and the KSHV-encoded vGPCR plays a key role in the TLR4 downregulation, whereas the KSHV vIRF1 also contributes to this effect. Our findings reveal a role for TLR4 in innate immunity against herpesviruses and suggest the potential use of TLR4 agonists for the treatment of KSHV-related neoplasms.

Associations between Burkitt lymphoma among children in Malawi and infection with HIV, EBV and malaria: results from a case-control study.

BACKGROUND: Burkitt lymphoma, a childhood cancer common in parts of sub-Saharan Africa, has been associated with Epstein Barr Virus (EBV) and malaria, but its association with human immunodeficiency virus (HIV) is not clear. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a case-control study of Burkitt lymphoma among children (aged < or = 15 years) admitted to the pediatric oncology unit in Blantyre, Malawi between July 2005 and July 2006. Cases were 148 children diagnosed with Burkitt lymphoma and controls were 104 children admitted with non-malignant conditions or cancers other than hematological malignancies and Kaposi sarcoma. Interviews were conducted and serological samples tested for antibodies against HIV, EBV and malaria. Odds ratios for Burkitt lymphoma were estimated using unconditional logistic regression adjusting for sex, age, and residential district. Cases had a mean age of 7.1 years and 60% were male. Cases were more likely than controls to be HIV positive (Odds ratio (OR)) = 12.4, 95% Confidence Interval (CI) 1.3 to 116.2, p = 0.03). ORs for Burkitt lymphoma increased with increasing antibody titers against EBV (p = 0.001) and malaria (p = 0.01). Among HIV negative participants, cases were thirteen times more likely than controls to have raised levels of both EBV and malaria antibodies (OR = 13.2; 95% CI 3.8 to 46.6; p = 0.001). Reported use of mosquito nets was associated with a lower risk of Burkitt lymphoma (OR = 0.2, 95% CI, 0.03 to 0.9, p = 0.04). CONCLUSIONS: Our findings support prior evidence that EBV and malaria act jointly in the pathogenesis of Burkitt lymphoma, suggesting that malaria prevention may decrease the risk of Burkitt lymphoma. HIV may also play a role in the etiology of this childhood tumor.

Kaposi's sarcoma-associated herpesvirus-encoded interleukin-6 and G-protein-coupled receptor regulate angiopoietin-2 expression in lymphatic endothelial cells.

Kaposi's sarcoma (KS) is caused by Kaposi's sarcoma-associated herpesvirus (KSHV) and consists of proliferating spindle cells, which are related to lymphatic endothelial cells (LEC). Angiopoietin-2 (Ang2) is a secreted proangiogenic and lymphangiogenic molecule. Here, we show the expression of Ang2 protein in KS and confirm that KSHV infection up-regulates Ang2 in LEC. We show that a paracrine mechanism contributes to this up-regulation. A lentiviral library of individual KSHV-encoding genes, comprising the majority of known latent genes and a selection of lytic viral genes, was constructed to investigate the underlying mechanism of this up-regulation. Two lytic genes, viral interleukin-6 (vIL6) and viral G-protein-coupled receptor (vGPCR), up-regulated Ang2 expression in LEC. Both vIL6 and vGPCR are expressed in KSHV-infected LEC and caused up-regulation of Ang2 in a paracrine manner. KSHV, vIL6, and vGPCR up-regulated Ang2 through the mitogen-activated protein kinase (MAPK) pathway. Gene expression microarray analysis identified several other angiogenic molecules affected by KSHV, including the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) axis, which is also affected by vIL6 and vGPCR in LEC, and matrix metalloproteinases, which could act in concert with Ang2 to contribute to KS development. These findings support the paracrine and autocrine roles of the lytic KSHV-encoded proteins, vIL6 and vGPCR, in KS pathogenesis and identify Ang2 as a potential therapeutic target for this neoplasm.

Kaposi sarcoma as a model of oncogenesis and cancer treatment.

Kaposi sarcoma is the most common cancer among HIV-infected individuals and one of the most common cancers in sub-Saharan Africa. Kaposi sarcoma lesions are highly vascularized, and comprised of spindle-shaped tumor cells. Kaposi sarcoma herpesvirus is etiologically linked to Kaposi sarcoma development and encodes genes that contribute to cellular transformation, evasion of apoptosis, aberrant angiogenesis and an inflammatory tumor microenvironment. The study of Kaposi sarcoma herpesvirus-driven malignancies has provided a model of oncogenesis and identified some of the key steps and, therefore, therapeutic targets of Kaposi sarcoma development. However, current Kaposi sarcoma treatments are not specific and rely on reconstitution of the immune system and systemic administration of cytotoxic agents. Recent studies have demonstrated that mechanism-based therapeutics, such as vascular endothelial growth factor A or mammalian target of rapamycin inhibitors, are promising therapeutic approaches bridging basic research with clinical practice.

Kaposi sarcoma herpesvirus-encoded vFLIP and vIRF1 regulate antigen presentation in lymphatic endothelial cells.

Kaposi sarcoma-associated herpesvirus (KSHV) is etiologically linked to Kaposi sarcoma (KS), a tumor genetically akin to lymphatic endothelial cells (LECs). We obtained the immune transcriptional signature of KS and used KSHV-infected LECs (KLECs) as an in vitro model to determine the effects of KSHV on transcription and expression of genes involved in immunity. The antigen presentation, interferon (IFN) response, and cytokine transcriptomes of KLECs resemble those of KS. Transcription of genes involved in class I presentation is increased in KS and after infection of LECs, but MHC-I and ICAM-1 surface expression are down-regulated in KLECs. Inhibition of IFN induction of MHC-I transcription indicates that KSHV regulates MHC-I transcription. We show that MHC-I transcription is regulated by the KSHV-encoded viral FLICE inhibitory protein (vFLIP) and by viral IFN regulatory factor 1 (vIRF1). vFLIP up-regulates MHC-I and ICAM-1 through activation of NF-kappaB and stimulates T-cell proliferation, revealing a mechanism to prevent uncontrolled viral dissemination. In contrast, vIRF1 inhibits basal and IFN- and vFLIP-induced MHC-I transcription and surface expression through its interaction with the transcriptional coactivator p300, contributing to immune evasion. We propose that regulation of MHC-I by vFLIP and vIRF1 plays a crucial role in the host-pathogen equilibrium.

Isolation and characterization of the Bactrocera oleae genes orthologous to the sex determining Sex-lethal and doublesex genes of Drosophila melanogaster.

Here we report the isolation and characterization of the olive fruit fly Bactrocera oleae genes orthologous to the Drosophila melanogaster sex-determining genes Sex-lethal (Sxl) and doublesex (dsx). Fragments of the Sxl and dsx orthologous were isolated with RT-PCR. Genomic and cDNA clones were then obtained by screening a genomic library and separate male and female cDNA adult libraries using the RT-PCR products as probes in both cases. B. oleae Sxl gene (BoSxl) expresses the same pattern of transcripts which encode for a single common polypeptide in both male and female flies. The gene shares a high degree of similarity in sequence and expression to its Ceratitis capitata orthologous and does not appear to play a key regulatory role in the sex-determining cascade. B. oleae dsx gene (Bodsx) expands in a chromosomal region of more than 50 kb, with 6 exons-5 introns, producing different sex-specific mRNAs, according to the Drosophila model. The cDNA sequences are almost identical to the gene orthologous of Bactrocera tryoni. Four repeat elements identical to the D. melanogaster TRA/TRA-2 binding sites have been found in the untranslated region of the female-specific exon 4, predicting a common regulatory splicing mechanism in all studied species of Diptera.

Kaposi sarcoma herpesvirus-induced cellular reprogramming contributes to the lymphatic endothelial gene expression in Kaposi sarcoma.

The biology of Kaposi sarcoma is poorly understood because the dominant cell type in Kaposi sarcoma lesions is not known. We show by gene expression microarrays that neoplastic cells of Kaposi sarcoma are closely related to lymphatic endothelial cells (LECs) and that Kaposi sarcoma herpesvirus (KSHV) infects both LECs and blood vascular endothelial cells (BECs) in vitro. The gene expression microarray profiles of infected LECs and BECs show that KSHV induces transcriptional reprogramming of both cell types. The lymphangiogenic molecules VEGF-D and angiopoietin-2 were elevated in the plasma of individuals with acquired immune deficiency syndrome and Kaposi sarcoma. These data show that the gene expression profile of Kaposi sarcoma resembles that of LECs, that KSHV induces a transcriptional drift in both LECs and BECs and that lymphangiogenic molecules are involved in the pathogenesis of Kaposi sarcoma.

Short- and long-term effects of highly active antiretroviral therapy on Kaposi sarcoma-associated herpesvirus immune responses and viraemia.

OBJECTIVE: To investigate the effect of highly active antiretroviral therapy (HAART) on Kaposi sarcoma-associated herpesvirus (KSHV) DNA load, anti-KSHV antibody responses and KSHV-specific CD8 T cell responses in HIV-infected individuals over a 2 year period. DESIGN: Prospective study of 27 HIV-infected antiretroviral therapy-naive individuals, with (n = 4) and without KS (n = 23), before HAART and at 3-month intervals, during treatment with HAART. METHODS: Sequential blood samples were collected for anti-KSHV antibody detection, KSHV DNA load in peripheral blood mononuclear cells (PBMC) and plasma, HIV Gag-specific and KSHV-specific CD8 T cell responses, HIV-1 plasma RNA load and CD4 and CD8 T cell counts. RESULTS: KSHV DNA in PBMC and plasma became less detectable over time during HAART, in particular after 12 months. KSHV DNA was undetectable in plasma after 24 months on HAART. Anti-KSHV lytic, but not latent, antibody levels increased within 12 months of treatment. KSHV-specific CD8 T cell responses were absent prior to HAART but became detectable in some patients within 6 months of starting treatment, and continued to increase thereafter. CONCLUSIONS: HAART (both protease inhibitor-based and non-nucleoside reverse transcriptase inhibitor-based antiretroviral combinations) is associated with immune reconstitution to KSHV and with undetectable KSHV viraemia. However, this restoration is apparent (in particular) only after a relatively long (> 24 months) period of treatment. These immune responses could contribute to the decreased incidence of KS during HAART, but it is unlikely to be a complete explanation for the often rapid resolution of KS when HAART is started.

Turnover-based in vitro selection and evolution of biocatalysts from a fully synthetic antibody library.

This report describes the selection of highly efficient antibody catalysts by combining chemical selection from a synthetic library with directed in vitro protein evolution. Evolution started from a naive antibody library displayed on phage made from fully synthetic, antibody-encoding genes (the Human Combinatorial Antibody Library; HuCAL-scFv). HuCAL-scFv was screened by direct selection for catalytic antibodies exhibiting phosphatase turnover. The substrate used was an aryl phosphate, which is spontaneously transformed into an electrophilic trapping reagent after cleavage. Chemical selection identified an efficient biocatalyst that then served as a template for error-prone PCR (epPCR) to generate randomized repertoires that were subjected to further selection cycles. The resulting superior catalysts displayed cumulative mutations throughout the protein sequence; the ten-fold improvement of their catalytic proficiencies (>10(10) M(-1)) resulted from increased kcat values, thus demonstrating direct selection for turnover. The strategy described here makes the search for new catalysts independent of the immune system and the antibody framework.