Recombinant Envelope-Proteins with Mutations in the Conserved Fusion Loop Allow Specific Serological Diagnosis of Dengue-Infections

Dengue virus (DENV) is a mosquito-borne flavivirus and a major international public health concern in many tropical and sub-tropical areas worldwide. DENV is divided into four major serotypes, and infection with one serotype leads to immunity against the same, but not the other serotypes. The specific diagnosis of DENV-infections via antibody-detection is problematic due to the high degree of cross-reactivity displayed by antibodies against related flaviviruses, such as West Nile virus (WNV), Yellow Fever virus (YFV) or Tick-borne encephalitis virus (TBEV). Especially in areas where several flaviviruses co-circulate or in the context of vaccination e.g. against YFV or TBEV, this severely complicates diagnosis and surveillance. Most flavivirus cross-reactive antibodies are produced against the highly conserved fusion loop (FL) domain in the viral envelope (E) protein. We generated insect-cell derived recombinant E-proteins of the four DENV-serotypes which contain point mutations in the FL domain. By using specific mixtures of these mutant antigens, cross-reactivity against heterologous flaviviruses was strongly reduced, enabling sensitive and specific diagnosis of the DENV-infected serum samples in IgG and IgM-measurements. These results have indications for the development of serological DENV-tests with improved specificity

Modeling viral infectious diseases and development of antiviral therapies using human induced pluripotent stem cell-derived systems

The recent biotechnology breakthrough of cell reprogramming and generation of induced pluripotent stem cells (iPSCs), which has revolutionized the approaches to study the mechanisms of human diseases and to test new drugs, can be exploited to generate patient-specific models for the investigation of host-pathogen interactions and to develop new antimicrobial and antiviral therapies. Applications of iPSC technology to the study of viral infections in humans have included in vitro modeling of viral infections of neural, liver, and cardiac cells; modeling of human genetic susceptibility to severe viral infectious diseases, such as encephalitis and severe influenza; genetic engineering and genome editing of patient-specific iPSC-derived cells to confer antiviral resistance

West Nile virus infection in individuals with pre-existing Usutu virus immunity, northern Italy, 2018

In 2018, there was a large West Nile virus (WNV) outbreak in northern Italy. We observed five atypical cases of WNV infection that were characterised by the presence of WNV RNA and WNV IgG at the time of diagnosis, but no IgM response during follow-up. Neutralisation assays demonstrated pre-existing Usutu virus immunity in all patients. Besides challenging diagnosis, the immunological crosstalk between the two viruses warrants further investigation on possible cross-protection or infection enhancement effects

126. Evaluation of Replication Competent and Replication Incompetent Adenovirus-Mediated Toxicity in Human Adrenocortical Cells

Objectives: Experimental studies indicate that adenoviruses have a natural tropism for the adrenal gland, thus, the systemic use of adenoviral vectors might be associated with side effects due to adrenal gland infection. In this study, human ACC cells were used to assess the toxicity of replication competent adenovirus type 5 (Ad5) and replication deficient E1-/E3- adenoviral vectors. Methods: To test the susceptibility of human ACC cells (SW13 and NCI-H295R) to adenoviral infection, expression of coxsackie and adenovirus receptor(CAR) and integrins was evaluated in ACC cells as well as in normal human adrenocortical tissues and in benign and malignant adrenocortical tumor samples by quantitative real-time RT-PCR. ACC cells were infected with Ad5 and tested for virus production at different time points pi. Recombinant E1-/E3- Ad5 vector expressing green fluorescent protein (Ad5-EGFP) was used to test adenovirus infectivity by fluorescent microscopy and FACS analysis. To assess the toxicity of replication competent and replication incompetent adenoviral vectors in the adrenal gland, Ad5, Ad5-EGFP and Ad5-HSV-TK were employed. In this regard, at different time points pi, we examined the effect of adenoviral infection on gene expression profile by DNA microarray analysis and on cell growth by proliferation assay, cell cycle analysis, and apoptosis test. The influence of adenoviral infection on steroid hormone production was also analyzed. Results: CAR expression was demonstrated in ACC cells and in normal and neoplastic adrenocortical tissues. Both ACC cells demonstrated productive Ad5 replication and efficient Ad5-EGFP transduction. Time- and dose-dependent induction of cell death was found only in Ad5-infected ACC cells, whereas Ad5- EGFP and Ad5-HSV-TK had no apparent effect on cell proliferation, cell cycle, and cell death as compared to uninfected control. ACC cells did not show marked alterations of gene expression after Ad5-EGFP infection, as demonstrated by microarray analysis in time course infection experiments. In the early phase of infection, genes involved in cell proliferation, stress and innate immune response were transiently upregulated. Moreover, expression of genes encoding steroidogenic enzymes was modulated toward cortisol hypersecretion. With regard to steroidogenesis, ACC cells infected with Ad5 showed decreased basal steroid hormone production in the early phase pi, followed by increased steroid hormone release at 72 h pi. At variance, Ad5-EGFP markedly induced cortisol and estradiol production, but not aldosterone production, at all time points pi. Conclusions: These results, which provide insight into the host response following adenoviral infection of ACC cells, contribute to the understanding of the adrenal involvement during natural adenovirus infection and vector administration for gene therapy

Infection dynamics in a traveller with persistent shedding of Zika virus RNA in semen for six months after returning from Haiti to Italy, January 2016

We describe the dynamics of Zika virus (ZIKV) infection in a man in his early 40s who developed fever and rash after returning from Haiti to Italy, in January 2016. Follow-up laboratory testing demonstrated detectable ZIKV RNA in plasma up to day 9 after symptom onset and in urine and saliva up to days 15 and 47, respectively. Notably, persistent shedding of ZIKV RNA was demonstrated in semen, still detectable at 181 days after onset. This article is copyright of The Authors, 2016

Report of two cases of influenza virus A/H1N1v and B co-infection during the 2010/2011 epidemics in the Italian Veneto Region

From October 2010 to April 2011, in the Italian Veneto Region, 1403 hospitalized patients were tested for influenza virus infection by specific real time RT-PCR. Overall, 327 samples were positive for either influenza A (75%) or B (25%) viruses. Among these positive patients two resulted co-infected by A/H1N1v and B viruses. Even though co-infection with both influenza A and B viruses appears to be a rare event, it occurs naturally and may play a role in epidemiology and pathogenicity. In the present study the two co-infected patients were a transplant recipient immunocompromised adult and a child displaying a severe respiratory illness. The co-infection was confirmed by inoculation of the nasopharyngeal swabs in MDCK.2 cells, followed by immunofluorescence and real time RT-PCR assays. Moreover, in the case of the adult patient, the immune system response against both viruses was assayed by hemoagglutination inhibition test against reference influenza virus strains. Both patients fully recovered from infection, without significant differences with mono-infected patients

Isolation of infectious Zika virus from saliva and prolonged viral RNA shedding in a traveller returning from the Dominican Republic to Italy, January 2016

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Genome sequence analysis of the first human West Nile virus isolated in Italy in 2009.

In 2009, six new human cases of West Nile neuroinvasive disease (WNND) were identified in Veneto region, following the six cases already reported in 2008. A human West Nile virus (WNV) isolate was obtained for the first time from an asymptomatic blood donor. Whole genome sequence of the human WNV isolate showed close phylogenetic relatedness to the Italy-1998-WNV strain and to other WNV strains recently isolated in Europe, with the new acquisition of the NS3-Thr249Pro mutation, a trait associated with avian virulence, increased virus transmission, and the occurrence of outbreaks in humans

Clinical and virological findings in the ongoing outbreak of West Nile virus Livenza strain in northern Italy, July to September 2012.

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Human cases of West Nile Virus infection in north-eastern Italy, 15 June to 15 November 2010.

In 2010, for the third consecutive year, human cases of West Nile virus (WNV) infection, including three confirmed cases of neuroinvasive disease and three confirmed cases of West Nile fever, were identified in north-eastern Italy. While in 2008 and 2009 all human cases of WNV disease were recorded in the south of the Veneto region, cases of WNV disease in 2010 additionally occurred in two relatively small northern areas of Veneto, located outside those with WNV circulation in the previous years. WNV IgG antibody prevalence in blood donors resident in Veneto was estimated as ranging from 3.2 per 1,000 in areas not affected by cases of WNV disease to 33.3 per 1,000 in a highly affected area of the Rovigo province. No further autochthonous human cases of WNV disease were notified in Italy in 2010. The recurrence of human cases of WNV infection for the third consecutive year strongly suggests WNV has become endemic in north-eastern Italy