Detection and quantification of EBV, HHV-6 and CMV DNA in the gastrointestinal tract of HIV-positive patients.

Human herpes viruses (HHVs) have been frequently detected in the gastrointestinal (GI) tract and may contribute to the development of gastric cancer. In the present study, the detection rate and viral load of Epstein Barr virus (EBV), HHV-6 and Cytomegalovirus (CMV) were assessed in the GI tract of human immunodeficiency virus (HIV) positive patients and of uninfected patients. The analysis revealed a significantly higher detection rate of EBV and HHV-6 in HIV-infected individuals than in uninfected subjects (88.5 vs 63%; p = 0.03). Moreover, EBV DNA load was significantly higher in the stomach of HIV patients than in controls. These data suggest that the HIV infection status may increase the persistence of these viruses in the GI compartment. Intriguingly, CMV DNA was undetectable in all biopsy specimens analyzed.

Type III interferon (IFN-lambda) antagonizes the antiviral activity of interferon-alpha in vitro.

Type III interferons (IFN-lambda) are the most recently discovered members of IFN family. Synergism between different IFN types is well established, but for type I and type III IFNs no conclusive evidence has been reported so far. Possible synergism/antagonism between IFN-alpha and IFN-lambda in the inhibition of virus replication (EMCV, WNV lineage 1 and 2, CHIKV and HSV-1), and in the activation of intracellular pathways of IFN response (MxA and 2'-5' OAS) was evaluated in different cell lines (Vero E6, A549 and Wish cells). The antiviral potency of IFN-lambda1 and -l2 was lower than that of IFN-alpha. When IFN-alpha and -lambda were used together, the Combination Index (CI) for virus inhibition was greater than 1 virtually for all virus/host cell systems, indicating antagonistic effect. Antagonism between IFN-alpha and -l was also observed for the induction of mRNA for both MxA and 2'-5'OAS. Elucidating the interplay between IFN-alpha and -lambda may help to better understand innate defence mechanisms against viral infections, including the molecular mechanisms underlying the influence of IL-28B polymorphisms in the response to HCV and other viral infections.

The prevalence of antibodies to human herpesvirus 8 and hepatitis B virus in patients in two hospitals in Tanzania.

The aim of this study was to determine the seroprevalence of human herpesvirus 8 (HHV-8) and the immunization status for hepatitis B virus (HBV) infection in febrile patients in two districts of the United Republic of Tanzania. Between February and March 2007, blood samples were collected in Pemba Island and Tosamaganga from 336 outpatients and sent to the Virology Laboratory in Rome (Italy) for testing. HHV-8 DNA and HBV-DNA were amplified by two in-house molecular methods, anti-HHV-8 antibody titers were determined by an immunofluorescence assay (IFA), and anti-HCV, HBsAg, anti-HBs, and anti-HBc were evaluated by microplate enzyme immunoassay (MEIA). The seroprevalence of HHV-8 was 30.7% (96/313). In Pemba Island, the prevalence was lower than in Tosamaganga (14.4% vs. 46.3%). A higher prevalence of low titers of HHV-8 IgG (<1:80, 81%) was found among those under 5 years of age. HHV-8 DNA was detected in six seropositive patients (6.7%). The prevalence of HBsAg, anti-HBs, and anti-HBc was 4.3%, 37.6%, and 29.3%, respectively. Out of 277 patients, 70 had had a previous infection (25.3%). One case of occult hepatitis was found. The cover of hepatitis B vaccination was higher among children born after 2002 (66.7%) than in patients born before 2002. HHV-8 infection is endemic in Tanzania and the seroprevalence rate was higher in the mainland than on Pemba Island. The 3.9% percentage of HBsAg in children younger than 4 years of age suggests that increased efforts are required in order to achieve universal and compulsory immunization of children against HBV.

A case of dengue type 3 virus infection imported from Africa to Italy, October 2009.

In October 2009, a traveller returning from Africa to Italy was hospitalised with symptoms suggestive of a haemorrhagic fever of unknown origin. The patient was immediately placed in a special biocontainment unit until laboratory investigations confirmed the infection to be caused by a dengue serotype 3 virus. This case reasserts the importance of returning travellers as sentinels of unknown outbreaks occurring in other countries, and highlights how the initial symptoms of dengue fever resemble those of other haemorrhagic fevers, hence the importance of prompt isolation of patients until a final diagnosis is reached.

SARS-CoV-2 infection does not induce HIV viral escape in the central nervous system: A case series.

We report two cases of HIV positive patients with SARS-CoV-2 infection and a recent diagnosis of opportunistic infections of central nervous system (CNS). We investigated the potential impact of coinfection with SARS-CoV-2 on HIV replication in CNS.

Activation of interferon response genes and of plasmacytoid dendritic cells in HIV-1 positive subjects with GB virus C co-infection.

GB virus C (GBV-C) coinfection has a protective role in Human Immunodeficiency Virus (HIV) infection, and increases the duration of suppression of HIV-1 viremia in patients under Highly Active Anti-Retroviral Therapy (HAART). Since innate antiviral response may be involved in the protection, we analyzed the possible role of GBV-C as activator of innate immunity. To this aim, we measured the extent of activation of the interferon (IFN) system and of circulating Dendritic Cells (DC) in vivo, and the ability of GBV-C to activate these functions in vitro. Activation of IFN system and of circulating DC was compared in GBV-positive and -negative HIV-1 co-infected patients with HAART-driven suppression of HIV-1 viremia. Endogenous levels of IFN-gamma and RNA-dependent protein kinase (PKR) mRNA were significantly higher in peripheral blood mononuclear cells (PBMC) from GBV-C-positive when compared to GBV-C-negative patients. IFN-gamma expression was correlated with all the Interferon response genes (IRGs) and with GBV-C viremia. The frequency of circulating plasmacytoid DC (pDC) expressing the CD80 activation marker was increased in GBV-C-positive patients, and was correlated with GBV-C viral load. In vitro experiments indicated that GBV-C is able to induce IFN-gamma expression in PBMC. In addition, in PBMC cultures GBV-C induced an increase of CD80 expression by pDC, that was reduced by antibody to IFN-gamma. Our data indicate that in HIV-positive patients GBV-C coinfection promotes the activation of IFN-gamma and downstream IRG expression, as well as with the activation/maturation of circulating pDC. GBV-C-driven IFN-gamma activation is, at least in part, responsible for the increased maturation of pDC. This crosstalk may suggest a role for GBV-C coinfection in boosting the innate antiviral response to HIV infection.

Occupational transmission of an Orthopoxvirus infection during an outbreak in a colony of Macaca tonkeana in Lazio Region, Italy, 2015.

Orthopoxviruses spill over from animal reservoirs to accidental hosts, sometimes causing human infections. We describe the surveillance and infection control measures undertaken during an outbreak due to an Orthopoxvirus occurred in January 2015 in a colony of Macaca tonkeana in the province of Rieti, Latio, Italy, which caused a human asymptomatic infection. According to the epidemiological investigation, the human transmission occurred after an unprotected exposure. The contacts among wild, captive and domestic animals and humans, together with decreased immunity against Orthopoxviruses in the community, may put animal handlers at risk of infection, especially after the cessation of smallpox vaccination. To reduce these threats, standard precautions including respiratory hygiene and transmission-based precautions should be carefully applied also in veterinary medicine.

EBOLA Ag K-SeT rapid test: field evaluation in Sierra Leone.

OBJECTIVES: Efficient interruption of Ebola virus disease (EVD) transmission chains critically depends on reliable and fast laboratory diagnosis. We evaluated the performance of the EBOLA Virus Antigen Detection K-SeT (EBOLA Ag K-SeT), a new rapid diagnostic antigen test in field settings. METHODS: The study was conducted in a field laboratory located in Freetown (Sierra Leone) by the Italian National Institute for Infectious Diseases 'L. Spallanzani' and the EMERGENCY Onlus NGO. The EBOLA Ag K-SeT was tested on 210 residual plasma samples (EVD prevalence 50%) from patients hospitalized at the EMERGENCY Ebola treatment center in Goderich (Freetown), comparing the results with quantitative real-time PCR. RESULTS: Overall, the sensitivity of EBOLA Ag K-SeT was 88.6% (95% confidence interval (CI), 82.5-94.7), and the corresponding specificity was 98.1% (95% CI, 95.5-100.7). The positive and negative predictive values were 97.9% (95% CI, 95.0-100.8) and 89.6% (95% CI, 84-95.2), respectively. The sensitivity strongly increased up to 98.7% (95% CI, 96.1-101.2) for those samples with high virus load (≥6.2 log RNA copies/mL). CONCLUSIONS: Our results suggest that EBOLA Ag K-SeT could represent a new effective diagnostic tool for EVD, meeting a need for resource-poor settings and rapid diagnosis for individuals with suspected EVD.

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.

Ebola virus infection induces autoimmunity against dsDNA and HSP60.

Ebola virus (EBOV) survivors are affected by a variety of serious illnesses of unknown origin for years after viral clearance from the circulation. Identifying the causes of these persistent illnesses is paramount to develop appropriate therapeutic protocols. In this study, using mouse and non-human primates which survived EBOV challenge, ELISA, western blot, mass spectrometry and flow cytometry were used to screen for autoantibodies, identify their main targets, investigate the mechanism behind their induction and monitor autoantibodies accumulation in various tissues. In infected mice and NHP, polyclonal B cell activation and autoantigens secretion induced autoantibodies against dsDNA and heat shock protein 60 as well as antibody accumulation in tissues associated with long-term clinical manifestations in humans. Finally, the presence of these autoantibodies was confirmed in human EBOV survivors. Overall, this study supports the concept that autoimmunity is a causative parameter that contributes to the various illnesses observed in EBOV survivors.

Longitudinal characterization of dysfunctional T cell-activation during human acute Ebola infection.

Data on immune responses during human Ebola virus disease (EVD) are scanty, due to limitations imposed by biosafety requirements and logistics. A sustained activation of T-cells was recently described but functional studies during the acute phase of human EVD are still missing. Aim of this work was to evaluate the kinetics and functionality of T-cell subsets, as well as the expression of activation, autophagy, apoptosis and exhaustion markers during the acute phase of EVD until recovery. Two EVD patients admitted to the Italian National Institute for Infectious Diseases, Lazzaro Spallanzani, were sampled sequentially from soon after symptom onset until recovery and analyzed by flow cytometry and ELISpot assay. An early and sustained decrease of CD4 T-cells was seen in both patients, with an inversion of the CD4/CD8 ratio that was reverted during the recovery period. In parallel with the CD4 T-cell depletion, a massive T-cell activation occurred and was associated with autophagic/apoptotic phenotype, enhanced expression of the exhaustion marker PD-1 and impaired IFN-gamma production. The immunological impairment was accompanied by EBV reactivation. The association of an early and sustained dysfunctional T-cell activation in parallel to an overall CD4 T-cell decline may represent a previously unknown critical point of Ebola virus (EBOV)-induced immune subversion. The recent observation of late occurrence of EBOV-associated neurological disease highlights the importance to monitor the immuno-competence recovery at discharge as a tool to evaluate the risk of late sequelae associated with resumption of EBOV replication. Further studies are required to define the molecular mechanisms of EVD-driven activation/exhaustion and depletion of T-cells.

Unidirectional budding of HIV-1 at the site of cell-to-cell contact is associated with co-polarization of intercellular adhesion molecules and HIV-1 viral matrix protein.

OBJECTIVES: To explore the possibility that HIV-1 budding and cellular adhesion molecules co-polarize at cell-to-cell contact sites. To investigate the incorporation of host-cell-derived adhesion molecules into HIV-1. METHODS: The cellular sites involved in HIV-1 budding were examined by transmission electron microscopy. Single and double immunocytochemistry staining was used to evaluate the cellular distribution of the viral matrix protein and adhesion molecules. Quantitative flow cytometry was used to measure the cellular expression of adhesion molecules. An immunocapture technique was used to measure the presence of cell-derived proteins on HIV-1. The captured virus was measured by a p24 antigen assay. The infectivity of virus captured by monoclonal antibodies was tested by measuring the virus antigen yield in supernatants after the addition of sensitive cells. RESULTS: Released and budding HIV-1 was mainly localized at the cell-to-cell contact regions. This feature was consistent with a polarized staining for the virus matrix protein p18 at cell-to-cell contact regions. Intercellular adhesion molecules (ICAM)-1 in HIV-1-infected cells were polarized on both isolated cells and syncytia, co-localizing with HIV-1 matrix protein. HIV-1 incorporated all the adhesion molecules expressed by the host cells, although without quantitative correlation with their cellular expression. CONCLUSIONS: HIV-1 is released at cell-to-cell membrane contact sites. Both ICAM-1 and virus matrix protein co-polarized on isolated cells and syncytia at the sites involved in the recruitment of uninfected cells. The impressive concentration of ICAM at cell sites where most virions are released may account for the acquisition of these membrane proteins by the HIV-1 progeny, and may be important for the cell-mediated spread.

Effects of IFN alpha on late stages of HIV-1 replication cycle.

IFN alpha causes a modest reduction of HIV-1 expression in chronically infected monocytoid U937 cells. However, the ratio between cell-associated and shed viral p24 antigen is altered, being the cell-associated fraction dose-dependently enhanced by IFN. Furthermore, a significant decrease of infectivity of both cell-associated and shed material is observed. Transmission electron microscopy of IFN-treated cells revealed virus assembly being strongly inhibited, with the production of morphologically altered (tear-drop shaped) virus particles. Proteolytic processing of gag proteins appeared to be normal in IFN-treated cultures. However, virions shed from IFN-treated cells showed a markedly reduced incorporation of virus-specific gp120 and cell-derived ICAM-1 by the virus envelope. Additionally, these particles showed a significantly decreased ability to become bound to CD4+ target cells, accounting for, at least in part, the observed decrease of infectivity. Taken together, the data suggest that, in chronically infected cells, IFN alpha can affect late stages of HIV-1 replication, by inhibiting virus assembly and release, and by reducing the infectivity of shed virions. The latter effect seems to be due, at least in part, to altered incorporation of surface glycoproteins and defective particle formation. The relationship between impaired gp120 incorporation and altered morphogenesis of HIV-1 virions is under investigation.

Coordinate induction of interferon alpha and gamma by recombinant HIV-1 glycoprotein 120.

Similarly to HIV-infected cells, recombinant HIV-1 glycoprotein 120 induces acid-labile interferon production in peripheral blood mononuclear cells from healthy donors. Acid lability of this interferon is due to the presence of both IFN-alpha and -gamma molecules. In fact, although not revealed by neutralization of antiviral activity with antibody to IFN-gamma, the presence of IFN-gamma was shown both immunoenzymatically and by detection of specific mRNA in gp120-stimulated cells. The source of IFN-gamma appears to be a T cell present in the CD4-enriched subpopulation. Cultures treated with monoclonal antibodies to the ICAM-1 and LFA-1 adhesion molecules showed an impaired release of both IFN types after gp120 stimulation, suggesting a crucial role of cell-to-cell interactions in the process leading to IFN production. Our data suggest that the HIV envelope glycoprotein could be responsible for the induction of endogenous IFN-alpha and -gamma observed in AIDS patients.

HIV type 1 grown on interferon gamma-treated U937 cells shows selective increase in virion-associated intercellular adhesion molecule 1 and HLA-DR and enhanced infectivity for CD4-negative cells.

Cellular adhesion molecules, such as ICAM-1, -2, and -3; LFA-1; and HLA class I and II are incorporated into HIV-1 virions during budding from infected cells. These virion-associated molecules can be involved in the adsorption to susceptible cells displaying the corresponding counterligands. A number of cytokines have been shown to upregulate the cellular expression of adhesion molecules, such as ICAM-1 and HLA-DR. In this study we investigated the effects of IFN-gamma on the incorporation of ICAM-1, LFA-1, and HLA-DR into mature HIV-1 progeny from chronically infected cells. The ability of such virus progeny to infect either CD4-positive or -negative cells was also investigated. The results indicate that IFN-gamma stimulates the expression of ICAM-1 and of HLA-DR on HIV-1-infected cells, whereas LFA-1 expression is unaffected. The same modifications were also observed on virus progeny, because specific MAbs to ICAM-1 and HLA-DR captured infectious HIV-1 from IFN-treated cells with higher efficiency as compared to virus from control cells, whereas virus binding to anti LFA-1 MAb was unchanged. Moreover, the HIV-1 progeny released from IFN-treated cells showed an increased ability to bind to and to infect CD4-negative cells, whereas the infectivity was basically unchanged for CD4-positive cells. Our results suggest that cytokines, as well as other soluble factors, may expand the host cell range of HIV-1, possibly through modifications of the cell-derived surface molecules on the virions.(ABSTRACT TRUNCATED AT 250 WORDS)

The European network of Biosafety-Level-4 laboratories: enhancing European preparedness for new health threats.

Emerging and re-emerging infections and possible bioterrorism acts will continue to challenge both the medical community and civilian populations worldwide, urging health authorities to respond rapidly and effectively. Established in 2005, the European Community (EC)-funded European Network of Biosafety-Level-4 laboratories (Euronet-P4), which brings together the laboratories in Porton Down, London, Hamburg, Marburg, Solna, Lyon and Rome, seeks to increase international collaboration in the areas of high containment laboratory biosafety and viral diagnostic capability, to strengthen Europe's capacity to respond to an infectious disease emergency, and to offer assistance to countries not equipped with such costly facilities. Network partners have agreed on a common strategy to fill the gaps identified in the field of risk group-4 agents' laboratory diagnosis, namely the lack of standardization and of reference samples. The network has received a further 3-year funding, to offer assistance to external laboratories, and to start the planning of field activities.

Demographic indicators and risk of infection with human herpesvirus type 8 in Central Italy.

BACKGROUND: A cross-sectional study was conducted in Latium and Abruzzo Regions (Central Italy) to estimate the prevalence of infection with human herpesvirus type 8 (HHV-8) and the association between demographic indicators and risk of HHV-8 infection. PATIENTS AND METHODS: Sera from 416 healthy individuals (>or=45 years of age), originally recruited in a multicentric case-control study on classic Kaposi's sarcoma (KS), were tested for antibodies against HHV-8. The association between demographic indicators (i.e., urban/rural residence, occupation) and HHV-8 seropositivity was assessed by means of multiple logistic regression (MLR) odds ratios (OR) and 95% confidence intervals (CI), adjusted for age and occupation. RESULTS: Overall, 20.4% of the study participants had antibodies against HHV-8, 23.2% of the men and 17.0% of the women (p = 0.15). HHV-8 seropositivity rates significantly increased with age (p = 0.01), from 10.0% in those under 65 years of age to 24.9% in 75 years or older (MLROR = 2.4). By multivariate analysis, a significantly 2-fold higher risk of HHV-8 was found in individuals living in rural areas, as compared to those living in metropolitan/urban areas (MLR-OR = 2.0, 95% CI: 1.1-3.5), and in farmers, as compared to white collars (MLR-OR = 2.1, 95% CI: 1.1-4.1). CONCLUSIONS: The study findings suggest that demographic factors such as age, urban/rural residence, and occupation are associated with HHV-8 seropositivity among adult individuals living in central Italy.