Role of Caprine Herpesvirus – 1 (CpHV-1) in the innate immuno-evasion Interferon mediate.

Caprine Herpesvirus-1 (CpHV-1) is a member of Varicellovirus genus within Herpesvirus family. CpHV-1 is the responsible of a disease that causes respiratory symptoms, balanopostitis, vulvovaginitis and abortion in adult goats. It also affects young goats with a systemic disease [1]. Type I (IFN-a/ ) and type II (IFN g) interferons, are the first weapons of the host to fight against viral infections. Type I IFNs induce the expression of more than 100 Interferon Stimulated Genes (ISGs) to establish an antiviral state that limits viral replication and dissemination. [2] Type II IFN is produced by activated immune cells and leads to the production of a different subset of ISGs via a distinct signaling pathway. Many viruses are able to subvert both type I and type II IFN-mediated antiviral responses. Herpesviruses are able to evade the IFN response by targeting different transcriptions factors of the interferon (IFN) signaling pathway [3]. There are no reports in literature about the role of CpHV-1 in IFN antagonism. Aim of the work: Herein, we describe a work to address the possible role of CpHV-1 as modulator of the innate immune response interferon mediate. Materials and methods. To investigate whether CpHV-1 interferes with type I interferon production, we performed an IFN- Beta reporter assay, using a reporter plasmid that carries the IFN-Beta promoter driving the expression of a firefly luciferase gene. A renilla-luciferase reporter plasmid was used as control. For the activation of the pathway we used the constitutively active N-terminal (2CARD) domain of RIG-I. To evaluate the potential CpHV-1-mediated inhibition of IFN a/b signaling, we performed an ISRE54 reporter assay using a construct having an ISRE54 promoter driving the expression of firefly luciferase. A renilla-luciferase reporter plasmid was used as internal control. Results. Our results show that CpHV-1 infection strongly suppressed the activation of IFN-β promoter induced by RIGI 2 CARD domain. Moreover cells mock infected and treated with type I IFN showed a significant increase in luciferase expression, as expected compared with the cells that were not treated with type I IFN and were not infected. The cells infected with CpHV-1 and treated with type I IFN showed significantly reduced luciferase expression driven by the ISRE54 promoter. These results showed that CpHV-1 is a strong inhibitor of type I Interferon production and signaling pathways. [1] Tempesta M, Pratelli A, Greco G, Martella V, Buonavoglia C, 1999. Detection of caprine herpes virus 1 in the sacral ganglia of latently infected goats by polimerasi chain reactionj J Clin Microbiol 37, 1598-99. [2] Garcia-Sastre A, Biron CA (2006) Type 1 interferons and the virus-host relationship: a lesson in detente. Science 312: 879-882. [3] Afroz S, Brownlie R, Fodje M, van Drunen Littel-van den Hurk S. VP8, the Major Tegument Protein of Bovine Herpesvirus 1, Interacts with Cellular STAT1 and Inhibits Interferon Beta Signaling. J Virol. 2016 Apr 29; 90(10): 4889-904

Positive regulatory role of the e3 ubiquitin ligase TRIM65 downstream the IFNβ induction pathway.

Introduction: Viral infection triggers a fast and effective cellular response mediated primarily by the production of interferon β (IFNβ) that induces an anti-viral state through complex signal cascades. Therefore, the regulation of its induction and subsequent IFNβ signaling needs to be tightly controlled. There is growing evidence implicating the members of Tripartite-motif (TRIM) protein family of E3 ligases as critical players in this regulation. However, the exact role, mechanism of action, and the physiological relevance of their activity in vivo still remain poorly investigated. Previous work in our lab revealed that an unprecedented large number of TRIMs play critical roles as enhancers in the regulation of innate immune signaling pathways. Methods: To study the role of TRIM65 in innate immune signaling we have used luciferase assays, overexpression in A549 and 293T cells, transient knock down using siRNAs in 293T cells, TRIM65 CRISPR knock out cell lines, Western blots, RT-qPCR, PR8-GLuc antiviral assays and immunofluorescence. Results: Our recent studies focused on TRIM65 showed that this protein possesses antiviral activity comparable to TRIM25 in a PR8-GLuc antiviral assay. We have also demonstrated that its overexpression strongly increased not only the 2CARD-RIG-I- but also the IRF3-dependent activation of the INFβ and ISRE reporters. Consequently, IFNβ, ISG54 as well as other cytokines and ISGs mRNA levels are decreased in TRIM65 knock down and knock out (KO) cells upon infection compared to infected/treated control cells. Re-constitution assays on TRIM65 KO cells reverted the inhibition of IFNβ and ISG54 confirming the phenotype. Altogether, these data indicates a stimulatory role for TRIM65 downstream the interferon induction pathway. Since the E3 ubiquitin ligase activity of many TRIMs has been linked to their antiviral functions, we have identified IRF3 and IRF7 as TRIM65 interacting factors and putative substrates. Immunofluorescence experiments suggest that IRF3 binds TRIM65 in the nucleus and in vitro ubiquitination assays indicates that TRIM65 is able to ubiquitinate IRF3 in a RING-independent manner. Our current studies are focused on completely delineate the molecular mechanism by which TRIM65-mediated ubiquitination or TRIM65 E3 Ub ligase-independent function could regulate the response to viral infection. Conclusion: A better understanding of positive regulatory networks of the IFN response will provide new knowledge that will help to design more effective therapeutics

Live Visualization of Hemagglutinin Dynamics During Infection by Using a Novel Reporter Influenza A Virus

Live visualization of influenza A virus (IAV) structural proteins during viral infection in cells is highly sought objective to study different aspects of the viral replication cycle. To achieve this, we engineered an IAV to express a Tetra Cysteine tag (TC tag) from hemagglutinin (HA), which allows intracellular labeling of the engineered HA protein with biarsenic dyes and subsequent fluorescence detection. Using such constructs, we rescued a recombinant IAV with TC tag inserted in HA, in A/Puerto Rico/8/1934(H1N1) background (HA-TC). This recombinant HA-TC tag reporter IAV was replication-competent; however, as compared to wild type PR8 IAV, it was attenuated in multicycle replication. We confirmed expression of TC tag and biarsenical labeling of HA by immunofluorescence assay in cells infected with an HA-TC tag reporter IAV. Further, we used this reporter virus to visualize HA expression and translocation in IAV infected cells by live confocal imaging. We also tested the utility of the HA-TC IAV in testing chemical inhibitors of the HA translocation. Overall, HA-TC IAV is a versatile tool that will be useful for studying viral life cycle events, virus-host interactions, and anti-viral testing

Environmental impact assessments of integrated food and non-food production systems in Italy and Denmark

Given the environmental footprints of the conventional agriculture, it is imperative to test and validate alternative production systems, with lower environmental impacts to mitigate and adapt our production systems. In this study, we identified six production systems, four in Italy and two in Denmark, to assess the environmental footprint for comparison among the production systems and additionally with conventional production systems. SimaPro 8.4 software was used to carry out the life cycle impact assessment. Among other indicators, three significantly important indicators, namely global warming potential, acidification, and eutrophication, were used as the proxy for life cycle impact assessment. In Italy, the production systems compared were silvopastoral, organic, traditional, and conventional olive production systems, whereas in Denmark, combined food and energy production system was compared with the conventional wheat production system. Among the six production systems, conventional wheat production system in Denmark accounted for highest global warming potential, acidification, and eutrophication. In Italy, global warming potential was highest in traditional agroforestry and lowest in the silvopastoral system whereas acidification and eutrophication were lowest in the traditional production system with high acidification effects from the silvopastoral system. In Italy, machinery use contributed the highest greenhouse gas emissions in silvopastoral and organic production systems, while the large contribution to greenhouse gas emissions from fertilizer was recorded in the traditional and conventional production systems. In Denmark, the combined food and energy system had lower environmental impacts compared to the conventional wheat production system according to the three indicators. For both systems in Denmark, the main contribution to greenhouse gas emission was due to fertilizer and manure application. The study showed that integrated food and non-food systems are more environmentally friendly and less polluting compared to the conventional wheat production system in Denmark with use of chemical fertilizers and irrigation. The study can contribute to informed decision making by the land managers and policy makers for promotion of environmentally friendly food and non-food production practices, to meet the European Union targets of providing biomass-based materials and energy to contribute to the bio-based economy in Europe and beyond

Host-Specific NS5 Ubiquitination Determines Yellow Fever Virus Tropism

The recent yellow fever virus (YFV) epidemic in Brazil in 2017 and Zika virus (ZIKV) epidemic in 2015 serve to remind us of the importance of flaviviruses as emerging human pathogens. With the current global flavivirus threat, there is an urgent need for antivirals and vaccines to curb the spread of these viruses. However, the lack of suitable animal models limits the research questions that can be answered. A common trait of all flaviviruses studied thus far is their ability to antagonize interferon (IFN) signaling so as to enhance viral replication and dissemination. Previously, we reported that YFV NS5 requires the presence of type I IFN (IFN-α/β) for its engagement with human signal transducer and activator of transcription 2 (hSTAT2). In this manuscript, we report that like the NS5 proteins of ZIKV and dengue virus (DENV), YFV NS5 protein is able to bind hSTAT2 but not murine STAT2 (mSTAT2). Contrary to what has been demonstrated with ZIKV NS5 and DENV NS5, replacing mSTAT2 with hSTAT2 cannot rescue the YFV NS5-STAT2 interaction, as YFV NS5 is also unable to interact with hSTAT2 in murine cells. We show that the IFN-α/β-dependent ubiquitination of YFV NS5 that is required for STAT2 binding in human cells is absent in murine cells. In addition, we demonstrate that mSTAT2 restricts YFV replication in vivo These data serve as further impetus for the development of an immunocompetent mouse model that can serve as a disease model for multiple flaviviruses.IMPORTANCE Flaviviruses such as yellow fever virus (YFV), Zika virus (ZIKV), and dengue virus (DENV) are important human pathogens. A common flavivirus trait is the antagonism of interferon (IFN) signaling to enhance viral replication and spread. We report that like ZIKV NS5 and DENV NS5, YFV NS5 binds human STAT2 (hSTAT2) but not mouse STAT2 (mSTAT2), a type I IFN (IFN-α/β) pathway component. Additionally, we show that contrary to what has been demonstrated with ZIKV NS5 and DENV NS5, YFV NS5 is unable to interact with hSTAT2 in murine cells. We demonstrate that mSTAT2 restricts YFV replication in mice and that this correlates with a lack of IFN-α/β-induced YFV NS5 ubiquitination in murine cells. The lack of suitable animal models limits flavivirus pathogenesis, vaccine, and drug research. These data serve as further impetus for the development of an immunocompetent mouse model that can serve as a disease model for multiple flaviviruses

Antimicrobial Peptides and Physical Activity: A Great Hope against COVID 19

Antimicrobial peptides (AMPs), α- and β-defensins, possess antiviral properties. These AMPs achieve viral inhibition through different mechanisms of action. For example, they can: (i) bind directly to virions; (ii) bind to and modulate host cell-surface receptors, disrupting intracellular signaling; (iii) function as chemokines to augment and alter adaptive immune responses. Given their antiviral properties and the fact that the development of an effective coronavirus disease 2019 (COVID-19) treatment is an urgent public health priority, they and their derivatives are being explored as potential therapies against COVID-19. These explorations using various strategies, range from their direct interaction with the virus to using them as vaccine adjuvants. However, AMPs do not work in isolation, specifically in their role as potent immune modulators, where they interact with toll-like receptors (TLRs) and chemokine receptors. Both of these receptors have been shown to play roles in COVID-19 pathogenesis. In addition, it is known that a healthy lifestyle accompanied by controlled physical activity can represent a natural weapon against COVID-19. In competitive athletes, an increase in serum defensins has been shown to function as self-protection from the attack of microorganisms, consequently a controlled physical activity could act as a support to any therapies in fighting COVID-19. Therefore, including information on all these players' interactions would produce a complete picture of AMP-based therapies' response

Dengue virus co-opts UBR4 to degrade STAT2 and antagonize type I interferon signaling.

An estimated 50 million dengue virus (DENV) infections occur annually and more than forty percent of the human population is currently at risk of developing dengue fever (DF) or dengue hemorrhagic fever (DHF). Despite the prevalence and potential severity of DF and DHF, there are no approved vaccines or antiviral therapeutics available. An improved understanding of DENV immune evasion is pivotal for the rational development of anti-DENV therapeutics. Antagonism of type I interferon (IFN-I) signaling is a crucial mechanism of DENV immune evasion. DENV NS5 protein inhibits IFN-I signaling by mediating proteasome-dependent STAT2 degradation. Only proteolytically-processed NS5 can efficiently mediate STAT2 degradation, though both unprocessed and processed NS5 bind STAT2. Here we identify UBR4, a 600-kDa member of the N-recognin family, as an interacting partner of DENV NS5 that preferentially binds to processed NS5. Our results also demonstrate that DENV NS5 bridges STAT2 and UBR4. Furthermore, we show that UBR4 promotes DENV-mediated STAT2 degradation, and most importantly, that UBR4 is necessary for efficient viral replication in IFN-I competent cells. Our data underscore the importance of NS5-mediated STAT2 degradation in DENV replication and identify UBR4 as a host protein that is specifically exploited by DENV to inhibit IFN-I signaling via STAT2 degradation

Productivity and Economic Evaluation of Agroforestry Systems for Sustainable Production of Food and Non-Food Products

Agroforestry systems have multifunctional roles in enhancing agronomic productivity, co-production of diversity of food and non-food products and provision of ecosystem services. The knowledge of the performance of agroforestry systems compared with monoculture is scarce and scattered. Hence, the objective of the study was to analyze the agronomic productivity and economic viability of diverse agroforestry systems in Europe. A network of five agroforestry systems integrating arable crops, livestock and biomass trees was investigated to assess the range of agricultural products in each agroforestry system. Land Equivalent Ratio (LER) was used to measure the agronomic productivity, whereas gross margin was used as an indicator for economic viability assessment. LER values ranged from 1.36–2.00, indicating that agroforestry systems were more productive by 36–100% compared to monocultures. Agroforestry gross margin was lower in Denmark (€112 ha−1 year−1) compared to United Kingdom (€5083 ha−1 year−1) and the crop component yielded higher returns compared to negative returns from the tree component in agroforestry. Hence, the study provided robust field-based evidence on agronomic productivity and economic viability assessment of agroforestry systems in diverse contexts for informed decision making by land managers, advisory services, farmers and policymakers

Guinea worm wrap-up

Sudan has reported 21,433 cases of dracunculiasis in January-July 2002, which is 73% of the global total of cases reported for that period. Whereas 36% of 8,058 endemic villages reported in January-July 2001, 62% of 6,224 endemic villages reported during the same period of 2002. The latest update on the status of the program was discussed during the annual Program Review of the Guinea Worm Eradication Programs of Sudan, Ethiopia and Uganda, which was held in Nairobi, Kenya on September 30 \ue2\u20ac\u201c October 2. The percentage of known endemic villages with nylon filters in every household increased from 29% to 58% between 2001 and 2002, and over 7 million pipe filters were distributed in 2001. Health education talks by village volunteers have increased from 50% to 83% of endemic villages, and are increasingly supplemented by radio broadcasts in local languages. Abate usage is still limited in all but the northern states of the country