Molecular diagnosis of SARS-CoV-2 in seminal fluid.

PURPOSE: Due to relevant repercussions on reproductive medicine, we aimed to evaluate feasibility of RT-PCR as a detection method of SARS-CoV-2 RNA in seminal fluid. METHODS: A qualitative determination of the RT-PCR assays in semen was performed through different approaches: (1) efficiency of RNA extraction from sperm and seminal plasma was determined using PRM1 and PRM2 mRNA and a heterologous system as control; (2) samples obtained by diluting viral preparation from a SARS-CoV-2 panel (virus cultured in Vero E6 cell lines) were tested; (3) viral presence in different fractions of seminal fluid (whole sample, seminal plasma and post-centrifugation pellet) was evaluated. Semen samples from mild and recovered COVID-19 subjects were collected by patients referring to the Infectious Disease Department of the Policlinico Umberto I Hospital - "Sapienza" University of Rome. Control subjects were recruited at the Laboratory of Seminology-Sperm Bank "Loredana Gandini'' of the same hospital. RESULTS: The control panel using viral preparations diluted in saline and seminal fluid showed the capability to detect viral RNA presence with Ct values depending on the initial viral concentration. All tested semen samples were negative for SARS-CoV-2, regardless of the nasopharyngeal swab result or seminal fluid fraction. CONCLUSION: These preliminary data show that RT-PCR for SARS-CoV-2 RNA testing appears to be a feasible method for the molecular diagnosis of SARS-CoV-2 in seminal fluid, supported by results of the control panel. The ability to detect SARS-CoV-2 in semen is extremely important for reproductive medicine, especially in assisted reproductive technology and sperm cryopreservation.

Evaluation of a rapid and sensitive RT-qPCR assay for the detection of Ebola Virus.

BACKGROUND: The 2013-2016 Ebola virus disease (EVD) outbreak showed a lack of diagnostic point-of-care methods. Currently, EBOV diagnosis relies on quantitative reverse-transcription-PCR (RT- qPCR), highly specific and sensitive, but requiring skilled personnel and well-equipped laboratories. In field settings, these factors and others, such as samples' time of collection and transportation, determine a prolonged turnaround-time to final results. In outbreak scenarios, a rapid and transportable method could eliminate issues of cohorting suspected and actual EVD patients for lack of diagnostic certainty. The aim of this study was the field evaluation of the new fast, easy-to-use and reliable RT-qPCR assay and platform for EBOV detection, developed in the framework of the EbolaMoDRAD project by CLONIT S.r.l. and STMicroelectronics S.r.l. STUDY DESIGN: We evaluated its performance during the outbreak and in further studies in the EVD laboratory at the Princess Christian Maternity Hospital (PCMH) in Freetown (Sierra Leone) run by Emergency NGO and the Italian National Institute for Infectious Diseases (INMI). The assay was tested on residual aliquots of clinical specimens from EBOV-positive or -negative patients (n=116, EVD prevalence 37%). RESULTS AND CONCLUSION: Overall, the test was very easy-to-use and the instrument was robust and reliable in field-settings. The sensitivity of the assay was 100% and the specificity was 98.63% (95%CI: 96.34-100.92%). The positive and negative predictive values were 97.73 (95%CI:94.77-100.68%) and 100%, respectively. The high sensitivity and specificity of this new assay indicate that it is promising for laboratory diagnosis, especially in resource-limited settings.

Transthyretin inhibition of amyloid beta aggregation and toxicity.

OBJECTIVES: The aim of this study was to investigate transthyretin (prealbumin) effects on Abeta25-35-induced cytotoxicity. DESIGN AND METHODS: In view of the well-recognized literature data demonstrating that Abeta25-35 fibrillar aggregates cause in vitro cytotoxicity to human red blood cells and apoptotic changes to SK-N-BE neuroblastoma cells in cultures (ultrastructural evidence), we tested transthyretin effects on these two experimental models. RESULTS: Incubation of Abeta25-35 with transthyretin (at transthyretin concentrations equal to CSF physiological levels) demonstrated both inhibition of red blood cells lysis and neutralization of SK-N-BE neuroblastoma cells ultrastructural apoptotic changes. Moreover, transthyretin was shown to be able to inhibit the formation of fibrillar macroaggregates of Abeta25-35. CONCLUSIONS: The findings imply that experimental systems investigating Abeta-induced cytotoxicity consider the protective interaction of transthyretin with Abeta; an interaction to be considered also in vivo in view of the fact that transthyretin immunoreactivity has been previously demonstrated in amyloid plaques of brains from Alzheimer's disease patients.

HCV infection by cell-to-cell transmission: choice or necessity?

In vitro models of HCV infection have allowed for the clarifying of molecules and mechanisms involved in the main steps of virus cell-entry. HCV entry and neutralization appear to be closely related. Neutralizing antibodies inhibit the E2-CD81 binding, therefore CD81 is considered to be a major target of immune response. The tight-junction proteins are also implicated in E2-binding to CD81 and successive steps of virus entry, in cooperation with several co-receptors, whose involvement has still to be elucidated. Increasing evidence has emphasized the importance of cell-to-cell HCV-transmission in chronic infection. This route for infection could favour virus-escape from host-neutralization though its CD81-dependency is still debated. The main reasons which have delayed our understanding of HCV-infection are here critically reviewed, as are the challenges faced by investigators in the field. A deeper insight into the different pathways involved could help to elucidate some crucial features of HCV infection mechanisms and disclose important implications in its pathogenesis, which could help in suggesting new targets for successful immune-prophylactic/therapeutic strategies.

Susceptibility of human lymphoblastoid B-cell line CE to HIV1 infection.

In this preliminary report, we provide evidence that the human B-lymphoblastoid cell line (LCL) CE, bone-marrow-derived, previously reported to be permissive to hepatitis C virus, is also permissive to HIV1 infection. HIV1 genomes were detectable in cell supernatants, virus RNA transcripts and proviral DNAs in cell extracts at different times post-infection. Therefore, we propose this LCL cell line as a tool for exploring the mutual interactions of the two viruses in double-infected cells.

Ultrastructural observations of viral particles within hepatitis C virus-infected human B lymphoblastoid cell line.

Hepatitis C virus (HCV)-infected TOFE cells, a human bone marrow-derived B-cell line, were studied by transmission electron microscopy (TEM). We reported the presence of virus-like particles, (VLPs) having a diameter of 45 nm on average, within the cytoplasm of HCV-infected cells and their absence in uninfected cells. The VLPs were mostly seen in dilated cisternae of endoplasmic reticulum (ER) and consisted of an electron-dense core and a membrane envelope with surface projections. In the HCV-infected TOFE cells, examination by TEM revealed tubular structures similar to those observed in liver cells of chimpanzees and humans infected by the virus. In some instances, the outer membrane of the particles was connected to the ER membrane, indicating possible virus budding into the cisternae. Rarely, coated vesicles containing a particle were seen. In most of the infected cells, enlarged cytoplasmic vacuoles filled with degenerative amorphous material were observed. The data suggest a flavivirus-like pattern of HCV morphogenesis in infected non-hepatic cells and identify lymphoblastoid TOFE cells as a valuable system for the study of the sequential steps of the infection process and the morphological effects produced by HCV infection.

Hepatitis C virus infection of a Vero cell clone displaying efficient virus-cell binding.

The susceptibility of Vero cells and derivative cell clones to hepatitis C virus (HCV) infection was assayed by qualitative and quantitative polymerase chain reaction (PCR)-based methods. Cell extracts from Vero cells inoculated with HCV were tested for the presence of both positive and negative strands of HCV RNA; in parallel, cell-free HCV genomes were assayed in culture supernatant fluids. Quantitation of genomic HCV RNA molecules in infected cells by competitive reverse transcription PCR (cRT-PCR) indicated that HCV replication was more efficient in a derivative clone (named clone 10) than in parental Vero cells or other clones under study. Analysis of HCV-binding to cell receptors, performed by cRT-PCR quantitation of viral particles adsorbed to the cell surface, demonstrated a 10-fold higher virus-binding level of clone 10 than that of parental Vero cells. The results shown here indicate that the Vero clone 10 may constitute an efficient model system for analysing early events in HCV infection as well as a source of virus for diagnostic and biotechnological applications.

Detection of a 5' UTR variation in the HCV genome after a long-term in vitro infection.

The TOFE lymphoid cell line from normal human bone marrow is susceptible to infection by a hepatitis C virus (HCV) serum strain. A sequence analysis of the 5' untranslated region (UTR) of HCV before and after long-term in vitro infection revealed one base substitution at position -158 (C > T) of the 5' UTR. We performed the direct sequencing of 5' UTR polymerase chain reaction-amplified sequences of the HCV genome: a) from the original serum-derived strain; b) from TOFE cell extracts 6 months post infection. This base substitution in the regulatory elements of the 5' UTR might be related to the ability of the virus to grow in cell culture.

Morphological modifications induced by HCV infection in the TOFE human lymphoblastoid cell line.

In this study, we analysed by transmission electron microscopy (TEM), sequential details of morphological modifications that accompanied viral morphogenesis in the lymphoblastoid cell line (LCL) TOFE infected in vitro with hepatitis C virus (HCV). As previously reported, we observed virus-like particles (VLPs) in cytoplasmic vesicles mainly located in the perinuclear region of infected cells. In this area, the Golgi apparatus and the endoplasmic reticulum (ER) appeared hyperplastic, remarkably enriched in vesicles and lysosomal structures. Furthermore, only in this perinuclear region, cytopathic-effect(CPE)-like changes seemed to originate, consisting in enlarged cytoplasmic vacuoles filled with degenerative amorphous material containing VLPs. Finally, the complete filling-up of the cytoplasm with these degenerative vacuoles, in addition to cellular lysis displayed by some cells, appeared as the possible terminal pattern of the infectious process. Our data suggest that in vitro HCV-infected TOFE cells undergo typical CPE-like changes that may be connected with virus replication.

TOFE human-B-cell-line-based adsorption-inhibition assay to detect HCV neutralizing antibodies.

We previously demonstrated that the human lymphoblastoid B-cell line (LCL) TOFE, derived from normal human bone marrow, is permissive to HCV infection. In this report we developed an in vitro HCV adsorption-inhibition assay based on TOFE cells, to reveal the presence of neutralizing antibodies in sera from acutely infected patients.

Quantitation of hepatitis C virus RNA production in two human bone marrow-derived B-cell lines infected in vitro.

The ability of hepatitis C virus (HCV) to replicate in two B-cell lines, CE and TOFE, derived from bone marrow of healthy subjects was compared using qualitative and quantitative molecular methods. The presence of intracellular negative-stranded HCV RNA (replicative intermediate) was investigated by nested polymerase chain reaction (PCR) in the infected cultures at different times after infection. The amounts of positive-stranded HCV RNA (genomic RNA copies) synthesized and released from cells one week after in vitro infection were determined by competitive PCR after reverse transcription of viral RNA for the 5' viral untranslated region. In both cell lines, HCV RNA replication took place, but the TOFE cell line appeared to be a more efficient virus producer than the CE cell line. The TOFE cell line could be a valuable and reliable tool for basic and clinical HCV studies.

Fusion of EBV with the surface of receptor-negative human hepatoma cell line Li7A permits virus penetration and infection.

Our preliminary data suggest that Epstein-Barr virus (EBV) is able to bind to and fuse with the surface membranes of hepatoma cell line Li7A. In order to obtain further evidence, we utilized the relief of rhodamine fluorescence to monitor whether fusion would also take place when Li7A cells were exposed to experimental conditions such as neutral or low pH. It is well known that for some viruses, protonation in the endosomal compartment is needed to trigger the fusion. We show, furthermore, that the rate and extent of fusion are not affected by pretreatment of the cells with agents known to elevate the lysosomal and ensodomal pH, such as chloroquine or NH4Cl (lysosomotropic agent). By indirect immunofluorescence assay, in addition, we confirmed the binding of the EBV to the Li7A cell surface membrane. We attempted finally to correlate the above processes with successful infection of Li7A cells by EBV detected using the polymerase chain reaction technique. In spite of the apparent lack of viral receptor CD21, these nonlymphoid cells appeared susceptible to EBV penetration and infection through fusion with the plasma membrane at the surface of the cells.

Detection of virus-like particles in liver biopsies from HCV-infected patients.

In order to directly ascertain the presence of HCV virus infection in livers of patients with HCV chronic hepatitis, we investigated, by transmission electron microscopy (TEM), liver biopsies from 2 adults and 4 children for the presence of virus-like particles (VLPs). The plasmas of these HCV-positive patients were HCV-RNA-positive, with high ALT values. In liver tissue samples examined, we were able to detect plus and minus strands of HCV RNA by strand-specific RT-PCR. Aggregates or single VLPs of about 45 nm in diameter were detectable in variable amounts in endoplasmic cisternae and in hepatocyte cytoplasms of infected patients. These results emphasize the relevance of performing TEM assays to confirm the diagnosis of HCV infection.

Molecular characterization and dynamics of hepatitis C virus replication in human fetal hepatocytes infected in vitro.

The molecular features of hepatitis C virus (HCV) replication in human fetal hepatocytes (HFHs) were addressed in this study. Using a competitive reverse-transcription polymerase chain reaction (RT-PCR) assay for the quantitation of HCV-RNA molecules, the highest level of viral replication was detected 30 days' postinfection. At this time point, viral particles of 41 to 45 nm in diameter accumulated in the cell cytoplasm. Their density in cell extracts and culture medium was distributed between heavy (1.180-1.360 g/cm3) and light fractions (1.105-1.050 g/cm3) of a sucrose gradient, while, in the serum inoculum, they had a positive fraction at 1.180 g/cm3. In infected HFHs, minus-strand HCV RNA was observed in fractions displaying a sedimentation coefficient of 28 S to 18 S, while plus-strand HCV RNA showed a peak restricted to the 21 S fraction; the HCV RNA of serum inoculum had a sedimentation coefficient of 38 to 40 S, which revealed the presence of HCV RNA of unique positive polarity. The 21 S RNA fraction of cell extracts was resistant to 20 minutes of RNase I digestion, while the same incubation time totally inactivated a comparable amount of HCV RNA purified from the serum inoculum, revealing the presence of completely and/or partially double-stranded HCV-RNA molecules in the infected cells. Detection in HFHs of replicative forms and replicative intermediates suggests that the dynamic profile of HCV replication in these cells is similar to that described in other flaviviruses.