Comprehensive response to Usutu virus following first isolation in blood donors in the Friuli Venezia Giulia region of Italy: Development of recombinant NS1-based serology and sensitivity to antiviral drugs.

Surveillance of Usutu virus is crucial to prevent future outbreaks both in Europe and in other countries currently naïve to the infection, such as the Americas. This goal remains difficult to achieve, notably because of the lack of large-scale cohort studies and the absence of commercially available diagnostic reagents for USUV. This work started with the first identification of USUV in a blood donor in the Friuli Venezia Giulia (FVG) Region in Northern-Eastern Italy, which is endemic for West Nile virus. Considering that only one IgG ELISA is commercially available, but none for IgM, a novel NS1 antigen based IgG/M ELISA has been developed. This assay tested successfully for the detection of Usutu virus in blood donors with the identification of a second case of transmission and high levels of exposure. Furthermore, two pan-flavivirus antiviral drugs, that we previously characterized to be inhibitors of other flavivirus infectivity, were successfully tested for inhibition of Usutu virus with inhibitory concentrations in the low micromolar range. To conclude, this work identifies North-Eastern Italy as endemic for Usutu virus with implications for the screening of transfusion blood. A novel NS1-based ELISA test has been implemented for the detection of IgM/G that will be of importance as a tool for the diagnosis and surveillance of Usutu virus infection. Finally, Usutu virus is shown to be sensitive to a class of promising pan-flavivirus drugs.

Comparative specificity and sensitivity of NS1-based serological assays for the detection of flavivirus immune response.

Flaviviruses are relevant animal and human pathogens of increasing importance worldwide. The similarities of the initial clinical symptoms and the serological cross-reactivity of viral structural antigens make a laboratory diagnosis of flavivirus infection problematic. The main aim of the present study was the comparative specificity and sensitivity analysis of the non-structural protein NS1 as an antigen to detect flavivirus antibodies in sera from exposed individuals. A strategy for the purification of native recombinant non-structural protein 1 of representative flaviviruses including tick-borne encephalitis, West Nile, Zika and dengue virus was developed. The immunological properties of the purified antigens were analyzed using sera of immunized mice and of infected individuals in comparison with standard commercial assays. Recombinant NS1 protein was confirmed as a valuable option for the detection of flavivirus antibodies with reduced cross-reactivity and high sensitivity offering additional advantages for the detection of vaccine breakthrough cases.

25-hydroxycholesterol reduces inflammation, viral load and cell death in ZIKV-infected U-87 MG glial cell line.

Zika virus (ZIKV) infection is a global health issue due to its worldwide diffusion and to the related effects on neural progenitor cells with severe consequences on developing brain as well as on the central nervous system of adults. Previous studies showed that ZIKV infection induces an increment of IL1B expression in the central nervous system and also in the blood. IL-1ß is a pro-inflammatory cytokine essential for cellular defence, tissue repair and neuroinflammation, a mechanism seen to be associated with several neuroinflammatory diseases. 25-hydroxycholesterol (25-HC) is a natural oxysterol, derived from hydroxylation of cholesterol, possessing important antiviral activity possibly correlated to its ability to alter host membrane structures. Furthermore, 25-HC is involved in the modulation of IL1B gene expression, being able to suppress IL-1ß driven inflammation probably by blocking the activation of the SREB proteins. In our study, we analysed the antiviral action of 25-HC in ZIKV-infected U-87 MG cells, also evaluating its impact on inflammation and cell death. We demonstrated that 25-HC is able to reduce inflammation and cell death caused by ZIKV infection and also to diminish intracellular ZIKV load in U-87 MG glial cell line. Considering its antiviral activity and its ability to penetrate blood-brain barrier, 25-HC could be proposed, based on our results and literature findings, as a potential anti-ZIKV agent.

Antiviral properties of blue laser in an in vitro model of HSV-1 infection.

Herpes simplex virus type-1 (HSV-1) is known to cause lifelong infections in humans. First infection is characterized by gingiva-stomatitis and pharyngitis, while virus reactivation causes recurrent herpes labialis with ulcerations on intraoral mucosa, mouth or external facial skin [1]. Laser therapy (LT), set at red and infrared wavelengths, has been reported as able to reduce HSV-1 recurrence and duration of herpetic sores [2]. Despite the blue wavelength already showed its efficacy in killing different strains of bacteria, it has never been tested on viruses [3].

Zika virus induces inflammasome activation in the glial cell line U87-MG.

In the last years, neurological complications related to Zika virus (ZIKV) infection have emerged as an important threat to public health worldwide. ZIKV infection has been associated to neurological disorders such as congenital microcephaly in newborns and Guillain-Barré syndrome, myelopathy and encephalitis in adults. ZIKV is characterized by neurotropism and neurovirulence. Several studies have identified microglial nodules, gliosis, neuronal and glial cells degeneration and necrosis in the brain of ZIKV infected infants, suggesting that ZIKV could play a role in these neurological disorders through neuroinflammation and microglial activation. Little information is available about neuroinflammation and ZIKV-related neurological disorders. Therefore, we investigated if ZIKV is able to infect a glial cell line (U87-MG) and how the glial cell line responds to this infection in terms of inflammation (IL-1ß, NLRP-3 and CASP-1), oxidative stress (SOD2 and HemeOX) and cell death. We observed a significant increase of ZIKV load in both cells and supernatants after 72 h, compared to 48 h of infection. We found that ZIKV infection induces an increase of IL-1ß, NLRP-3 and CASP-1 genes expression. Significant increase of IL-1ß and unchanged pro-IL-1ß protein levels have also been detected. Moreover, we observed SOD2 and HemeOX increased gene expression mainly after 72 h post ZIKV infection. Subsequently, we found a decrease of U87-MG cell viability, after both 48 h and 72 h of ZIKV infection. Our results show that U87-MG cells are susceptible to ZIKV infection. ZIKV is able to successfully replicate in infected cells causing oxidative stress, NLRP3 inflammasome activation and subsequent release of mature IL-1ß; this process culminates in cell death. Thus, considering the central role of neuroinflammation in neurological disorders, it is important to comprehend every aspect of this mechanism in order to better understand the pathogenesis of ZIKV infection and to identify possible strategies to fight the virus by rescuing cell death.

Persistent viremia and urine shedding of tick-borne encephalitis virus in an infected immunosuppressed patient from a new epidemic cluster in North-Eastern Italy.

A persistent tick-borne encephalitis virus infection in an immune-suppressed patient is presented. Such an unusual clinical case offers the unique chance of detecting persistent viremia associated to the erythrocyte fraction and shedding of the virus in the urine for more than six weeks. The infection occurred in a new area of the Friuli Venezia-Giulia region (North Eastern Italy) where two additional cases are also being reported.