A molecular beacon, bead-based assay for the detection of nucleic acids by flow cytometry

Molecular beacons are dual-labelled probes that are typically used in real-time PCR assays, but have also been conjugated with solid matrices for use in microarrays or biosensors. We have developed a fluid array system using microsphere-conjugated molecular beacons and the flow cytometer for the specific, multiplexed detection of unlabelled nucleic acids in solution. For this array system, molecular beacons were conjugated with microspheres using a biotin-streptavidin linkage. A bridged conjugation method using streptavidin increased the signal-to-noise ratio, allowing for further discrimination of target quantitation. Using beads of different sizes and molecular beacons in two fluorophore colours, synthetic nucleic acid control sequences were specifically detected for three respiratory pathogens, including the SARS coronavirus in proof-of-concept experiments. Considering that routine flow cytometers are able to detect up to four fluorescent channels, this novel assay may allow for the specific multiplex detection of a nucleic acid panel in a single tube

Viral hemorrhagic fevers: advancing the level of treatment

The management of viral hemorrhagic fevers (VHFs) has mainly focused on strict infection control measures, while standard clinical interventions that are provided to patients with other life-threatening conditions are rarely offered to patients with VHFs. Despite its complexity, a proper clinical case management of VHFs is neither futile nor is it lacking in scientific rationale. Given that patient outcomes improve when treatment is started as soon as possible, development and implementation of protocols to promptly identify and treat patients in the earliest phases of diseases are urgently needed. Different pharmacological options have been proposed to manage patients and, as for other life-threatening conditions, advanced life support has been proved effective to address multiorgan failure. In addition, high throughput screening of small molecular libraries has emerged as a novel promising way to find new candidates drugs for VHFs therapy and a relevant number of new molecules are currently under investigation. Here we discuss the current knowledge about VHF clinical management to propose a way to step up the approach to VHFs beyond the mere application of infection control measures

Orthopoxvirus Seroprevalence in Cats and Veterinary Personnel in North-Eastern Italy in 2011

Orthopoxviruses (OPV) are emerging zoonotic pathogens, and an increasing number of human infections is currently reported in Europe and in other continents, warranting heightened attention on this topic. Following two OPV infections reported in veterinarians scratched by sick cats in 2005 and 2007 in North-Eastern-Italy, involving a previously undescribed OPV, a similar strain was isolated by a sick cat from the same territory in 2011, i.e., 6 years later, raising attention on OPV circulation in this region. A surveillance program was launched to assess the OPV seroprevalence among the veterinarians working in local veterinary clinics and in the local wild and domestic cat population; seroprevalence was 33.3% in veterinarians and 19.5% in cats. Seroprevalence in cats was unevenly distributed, peaking at 40% in the area where OPV-infected cats had been observed

Performance of rapid tests in the management of dengue fever imported cases in Lazio, Italy 2014-2019

Abstract Background In Italy, dengue virus is the most frequent agent of imported viral infections. The use of rapid diagnostic tests (RDTs) may be of help as a preliminary user-friendly quick assay to facilitate dengue diagnosis, as ordinary laboratory diagnosis of dengue fever may require special efforts in terms of tools availability, interpretation of results, and skilled personnel. The performance of RDTs, however, may vary according to different epidemiological and laboratory background. Methods We reviewed five years of laboratory records of two dengue RDT results (Colorimetric SD-Bioline Dengue-Duo-RDT and Fluorimetric SD-Biosensor-STANDARD-F-Dengue-RDT), able to detect viral NS1 antigen and specific IgM and IgG. Diagnostic parameters were calculated using as reference the results of molecular (RT-PCR) and serological (immunofluorescence, IFA) tests. Overall performance, calculated considering the final case definition, was included in the accuracy assessment of RDTs. Results The combined use of NS1 and IgM/IgG RDT for the detection of acute dengue cases resulted in an overall sensitivity and specificity of 87.2% and 97.9% for Colorimetric RDT, 96.2% and 96.2% for Fluorimetric RDT. NS1 was the most reliable marker of acute infection, while IgM resulted falsely positive in nine samples, including sera derived from 2 Zika and 4 non-arbovirus infected patients. Conclusions The inclusion of RDT in the diagnostic algorithm is of undeniable help in the prompt management and surveillance of dengue infection in non-endemic areas. Confirmatory tests are, however, necessary to rule in or rule out dengue fever diagnosis

Zika virus NS2A inhibits interferon signaling by degradation of STAT1 and STAT2

The Interferon (IFN) response is crucial to restrain pathogenic infections. Investigations into flavivirus-host interactions reported that the high virulence is linked to innate immune evasion. Zika Virus (ZIKV) has developed diversified strategies to evade the innate immune system. We report that the viral protein NS2A counteracts the IFN response by strongly suppressing the IFN signaling. NS2A targets transcription factors STAT1 and STAT2, to impede their nuclear localization, thereby suppressing the transcription of ISRE promoter and IFN-stimulated genes. We found that NS2A promotes degradation of STAT1 and STAT2. Treatment of NS2A transfected cells with MG132 restores the levels of both transcription factors, suggesting the involvement of the proteasome system. Given the impact that the IFN antagonism has on flavivirus virulence, the knowledge gained by characterizing the mechanism through which ZIKV evades the IFN response paves the ground for new strategies to attenuate the pathogenesis and to develop countermeasures against effective pharmacological targets

In vitro Evaluation of Antiviral Efficacy of a Standardized Hydroalcoholic Extract of Poplar Type Propolis Against SARS-CoV-2

Except for specific vaccines and monoclonal antibodies, effective prophylactic or post-exposure therapeutic treatments are currently limited for COVID-19. Propolis, a honeybee's product, has been suggested as a potential candidate for treatment of COVID-19 for its immunomodulatory properties and for its powerful activity against various types of viruses, including common coronaviruses. However, direct evidence regarding the antiviral activities of this product still remains poorly documented. VERO E6 and CALU3 cell lines were infected with SARS-CoV-2 and cultured in the presence of 12.5 or 25 mu g/ml of a standardized Hydroalcoholic Extract acronym (sHEP) of Eurasian poplar type propolis and analyzed for viral RNA transcription, for cell damage by optical and electron microscopy, and for virus infectivity by viral titration at 2, 24, 48, and 72 h post-infection. The three main components of sHEP, caffeic acid phenethyl ester, galangin, and pinocembrin, were tested for the antiviral power, either alone or in combination. On both cell lines, sHEP showed significant effects mainly on CALU3 up to 48 h, i.e., some protection from cytopathic effects and consistent reduction of infected cell number, fewer viral particles inside cellular vesicles, reduction of viral titration in supernatants, dramatic drop of N gene negative sense RNA synthesis, and lower concentration of E gene RNA in cell extracts. Interestingly, pre-treatment of cells with sHEP before virus inoculation induced these same effects described previously and was not able to block virus entry. When used in combination, the three main constituents of sHEP showed antiviral activity at the same levels of sHEP. sHEP has a remarkable ability to hinder the replication of SARS-CoV-2, to limit new cycles of infection, and to protect host cells against the cytopathic effect, albeit with rather variable results. However, sHEP do not block the virus entry into the cells. The antiviral activity observed with the three main components of sHEP used in combination highlights that the mechanism underlying the antiviral activity of sHEP is probably the result of a synergistic effect. These data add further emphasis on the possible therapeutic role of this special honeybee's product as an adjuvant to official treatments of COVID-19 patients for its direct antiviral activity

West Nile virus: the Italian national transplant network reaction to an alert in the north-eastern region, Italy 2011.

We report four cases of West Nile virus (WNV) transmission following a single multiorgan donation in north-eastern Italy. The transmissions were promptly detected by local transplant centres. The donor had been tested for WNV by nucleic acid amplification test (NAT) prior to transplantation and was negative. There were no detected errors in the nationally implemented WNV safety protocols

Analysis of HIV quasispecies and virological outcome of an HIV D+/R+ kidney-liver transplantation

Introduction Transplantation among HIV positive patients may be a valuable therapeutic intervention. This study involves an HIV D+/R+ kidney-liver transplantation, where PBMC-associated HIV quasispecies were analyzed in donor and transplant recipients (TR) prior to transplantation and thereafter, together with standard viral monitoring. Methods The donor was a 54 year of age HIV infected woman: kidney and liver recipients were two HIV infected men, aged 49 and 61. HIV quasispecies in PBMC was analyzed by ultra-deep sequencing of V3 env region. During TR follow-up, plasma HIV-1 RNA, HIV-1 DNA in PBMC, analysis of proviral integration sites and drug-resistance genotyping were performed. Other virological and immunological monitoring included CMV and EBV DNA quantification in blood and CD4 T cell counts. Results Donor and TR were all ART-HIV suppressed at transplantation. Thereafter, TR maintained a nearly suppressed HIV-1 viremia, but HIV-1 RNA blips and the increase of proviral integration sites in PBMC attested some residual HIV replication. A transient peak in HIV-1 DNA occurred in the liver recipient. No major changes of drug-resistance genotype were detected after transplantation. CMV and EBV transient reactivations were observed only in the kidney recipient, but did not require specific treatment. CD4 counts remained stable. No intermixed quasispecies between donor and TR was observed at transplantation or thereafter. Despite signs of viral evolution in TR, HIV genetic heterogeneity did not increase over the course of the months of follow up. Conclusions No evidence of HIV superinfection was observed in the donor nor in the recipients. The immunosuppressive treatment administrated to TR did not result in clinical relevant viral reactivations

household transmission and disease transmissibility of a large hav outbreak in lazio italy 2016 2017

Abstract A major outbreak of Hepatitis A Virus (HAV) has swept through Europe between mid-2016 and 2017, mainly within the community of men who have sex with men (MSM). Over the same period, about 1,000 outbreak-related cases of acute Hepatitis A (AHA) were recorded in Lazio region, Italy. We calibrated a Bayesian model to reconstruct likely transmission events within all 44 households where multiple infections were recorded, representing a total of 103 cases from the HAV outbreak in Lazio. Based on information on the observed times of symptom onset, we estimated for the probability distribution function of the HAV generation time and used it to compute the effective and instantaneous reproduction numbers for the considered outbreak from the overall epidemic curve (N = 998 cases). We estimated a mean generation time of 30.2 days (95%CI: 25.2-33.0) and an effective reproduction number of about 1.63 (95% CI: 1.35-1.94). Transmissibility peaked in January 2017, shortly before targeted awareness and vaccination campaigns were put in place by health authorities; however, transmission remained above the epidemic threshold until June 2017. Within households, children (0-15) and young adults (16-30) infected preferentially individuals of the same age class, whereas transmission within older age groups was substantially homogeneous. These results suggest that the implemented interventions were able to slow down HAV transmission, but not to bring it rapidly to a halt. According to our estimates of the HAV transmissibility, about 50% of the at-risk persons should be immunized to prevent similar outbreaks in the future. Our results also indicate spillover from community transmission to household members, suggesting the opportunity of vaccinating household contacts of cases to prevent further spread of the epidemics