Detection and whole genome sequencing of murine norovirus in animal facility in Italy.

Viruses belonging to the genus Norovirus (NoV) of the family Caliciviridae are the major cause of acute viral gastroenteritis worldwide. NoVs are classified into 10 genogroups (GI-GX), and those belonging to the genogroup GV are able to infect several species of rodents. To evaluate the circulation of MNV among mice housed in an Italian facility, sampling was performed over two separate periods, in 2011, and 3 years later in 2014. During the two samplings, 75 fecal samples were collected from healthy mice housed in the animal facility and subjected to RT-PCR for viral detection. After the analysis, 41/75 animals (54.6%) resulted positive for the presence of MNV in feces. Nucleotide sequencing revealed the presence of two MNV variants co-circulating in both 2011 and 2014. One MNV strain was isolated on RAW264.7 cell line, and subjected to full genome sequencing. Our study showed that the murine noroviruses are widespread in the investigated animal facility, despite guidelines for animal care and maintenance. Full genome sequence analysis of the MNV strain described in this study showed a correlation with other strains circulating in Europe. Understanding the molecular epidemiology of this virus should give insight into its natural history and evolution in mice.

On the mechanism of tumor cell entry of aloe-emodin, a natural compound endowed with anticancer activity.

Aloe-emodin (1,8-dihydroxy-3-[hydroxymethyl]-anthraquinone), AE, is one of the active constituents of a number of plant species used in traditional medicine. We have previously identified, for the first time, AE as a new antitumor agent and shown that its selective in vitro and in vivo killing of neuroblastoma cells was promoted by a cell-specific drug uptake process. However, the molecular mechanism underlying the cell entry of AE has remained elusive as yet. In this report, we show that AE enters tumor cells via two of the five somatostatin receptors: SSTR2 and SSTR5. This observation was suggested by gene silencing, receptor competition, imaging and molecular modeling experiments. Furthermore, SSTR2 was expressed in all surgical neuroblastoma specimens we analyzed by immunohistochemistry. The above findings have strong implications for the clinical adoption of this natural anthraquinone molecule as an antitumor agent.

Detection and characterization of porcine caliciviruses in Italy.

Porcine noroviruses and sapoviruses have been sporadically reported in European countries, and more rarely in Italy. In this study, stools samples were collected from both asymptomatic and diarrheic pigs from northern Italy and were screened for caliciviruses by RT-PCR. Sapoviruses were detected frequently and were genetically related to both the GIII reference strain and the newly described porcine sapovirus genogroups. Porcine norovirus was detected in one asymptomatic pig (0.5%) and was genotyped as GII.11. This is the first detection of porcine norovirus in Italy.

Detection of serum antibodies to hepatitis E virus in domestic pigs in Italy using a recombinant swine HEV capsid protein.

BACKGROUND: The hepatitis E virus (HEV) has been detected in both humans and animals, particularly pigs, worldwide. Several evidences, including human infection following consumption of raw contaminated meat, suggest a zoonotic transmission of HEV. In Italy, large circulation of genotype 3 HEV has been reported in swine, and recent studies have confirmed the involvement of this genotype in autochthonous human cases. RESULT: In this study 111 sera collected from healthy pigs in two Italian regions were tested for anti-HEV IgG antibodies. For specific HEV antibody detection in swine, we developed ELISA and Western blotting methods, using a truncated capsid (ORF2) protein lacking the first 111 amino acids of a swine HEV genotype 3 strain. The ORF2-based ELISA revealed anti-HEV antibodies in 104 out of 111 pigs compared with 102 detected with a commercial ELISA kit. A lower number of sera reacted with the recombinant ORF2 protein in a Western blotting format (81/111). Using a Latent class analysis (LCA), the estimated sensitivities for ELISA-ORF2 and ELISA-kit tests were 0.961 and 0.936, respectively, whereas specificities were 0.599 and 0.475. The estimated sensitivity of Western blotting was 0.775, and the specificity was 0.944. CONCLUSIONS: The overall results confirm the high prevalence of HEV seropositive healthy pigs in Italy. Through comparisons with a commercial ELISA test, the swine genotype 3 HEV antigen produced in this study was proven suitable to detect anti-HEV antibodies in pig sera by both ELISA and Western Blotting.

Pattern of activation of human antigen presenting cells by genotype GII.4 norovirus virus-like particles.

BACKGROUND: Virus-like particles (VLPs) from an Italian GII.4 norovirus strain were used to investigate activation and maturation of circulating antigen presenting cells (APCs) of human origin. METHODS: Peripheral blood mononuclear cells (PBMCs) isolated from five healthy subjects were pulsed ex vivo with VLPs, and stained with a set of monoclonal antibodies (MAbs) for phenotypic analysis by flow cytometry. Cytokine release in cell supernatants was investigated by ELISA. RESULTS: Norovirus VLPs induced activation and maturation of circulating APCs derived from the five donors, as well as production of IL-6, IFN-γ and TNF-α cytokines. CONCLUSIONS: The present results suggest that VLPs can activate antigen presenting cells for an efficient induction of the adaptive immune response.

Zoonotic transmission of hepatitis E virus in industrialized countries.

Hepatitis E is an infectious viral disease with clinical and morphological features of acute hepatitis. The disease represents an important public health problem in developing countries, where it is often related to outbreaks mainly associated with consumption of contaminated water. During recent years, an increasing number of sporadic cases have also been described in industrialized countries. Besides humans, the hepatitis E virus (HEV) has also been identified in animals. In 1997, the virus was first detected in swine, and is now considered ubiquitous. Human and swine HEV strains from the same geographical region present a high level of nucleotide identity, and experimental infections have confirmed the cross-species transmission of swine strains to humans and of human strains to non-human primates. Studies on anti-HEV antibodies detection have demonstrated that people working in contact with swine or wild boar have a higher risk of infection than normal blood donors. In Japan and more recently in France, cases of hepatitis E have been associated with ingestion of uncooked meat from pigs, wild boar, or deer. The disease is currently considered an emerging zoonosis.

Identification of Targets to Redirect CAR T Cells in Glioblastoma and Colorectal Cancer: An Arduous Venture.

The chimeric antigen receptor (CAR) is an artificial molecule engineered to induce cytolytic T cell reactions in tumors. Generally, this molecule combines an extracellular single-chain variable fragment (scFv) able to recognize tumor-associated epitopes together with the intracellular signaling domains that are required for T cell activation. When expressed by T cells, the CAR enables the recognition and subsequent destruction of cancer cells expressing the complementary antigen on their surface. Although the clinical application for CAR T cells is currently limited to some hematological malignancies, researchers are trying to develop CAR T cell-based therapies for the treatment of solid tumors. However, while in the case of CD19, or other targets restricted to the hematopoietic compartment, the toxicity is limited and manageable, the scarcity of specific antigens expressed by solid tumors and not by healthy cells from vital organs makes the clinical development of CAR T cells in this context particularly challenging. Here we summarize relevant research and clinical trials conducted to redirect CAR T cells to surface antigens in solid tumors and cancer stem cells with a focus on colorectal cancer and glioblastoma. Finally, we will discuss current knowledge of altered glycosylation of CSCs and cancer cells and how these novel epitopes may help to target CAR T cell-based immunotherapy in the future.

Generation of Combinatorial Lentiviral Vectors Expressing Multiple Anti-Hepatitis C Virus shRNAs and Their Validation on a Novel HCV Replicon Double Reporter Cell Line.

Despite the introduction of directly acting antivirals (DAAs), for the treatment of hepatitis C virus (HCV) infection, their cost, patient compliance, and viral resistance are still important issues to be considered. Here, we describe the generation of a novel JFH1-based HCV subgenomic replicon double reporter cell line suitable for testing different antiviral drugs and therapeutic interventions. This cells line allowed a rapid and accurate quantification of cell growth/viability and HCV RNA replication, thus discriminating specific from unspecific antiviral effects caused by DAAs or cytotoxic compounds, respectively. By correlating cell number and virus replication, we could confirm the inhibitory effect on the latter of cell over confluency and characterize an array of lentiviral vectors expressing single, double, or triple cassettes containing different combinations of short hairpin (sh)RNAs, targeting both highly conserved viral genome sequences and cellular factors crucial for HCV replication. While all vectors were effective in reducing HCV replication, the ones targeting viral sequences displayed a stronger antiviral effect, without significant cytopathic effects. Such combinatorial platforms as well as the developed double reporter cell line might find application both in setting-up anti-HCV gene therapy approaches and in studies aimed at further dissecting the viral biology/pathogenesis of infection.

Virus like particles of GII.4 norovirus bind Toll Like Receptors 2 and 5.

Norovirus (NoV) is now recognized as a major cause of gastroenteritis outbreaks, worldwide. Norovirus replication mechanisms are still poorly understood, mainly because a reliable cell culture system is still lacking. The present study aims at understanding some aspects of the immune response against norovirus, and particularly the capacity of virus like particles (VLPs) from an Italian strain, belonging to the GII.4 genotype predominating worldwide, to interact with target cells via Toll Like Receptors (TLRs). The capacity of GII.4 NoV VLPs to interact and cause the activation of TLR2, 4 and 5 was studied in recombinant HEK cells. The results obtained show the ability of GII.4 NoV VLPs to induce activation of TLR2 and 5. The results on TLRs activation confirm that GII.4 NoV VLPs interact with TLR2 and 5, that may represent putative receptors and play a role in NoV infection of intestinal cells.

Adenovirus 36 and Obesity: An Overview.

There is an epidemic of obesity starting about 1980 in both developed and undeveloped countries definitely associated with multiple etiologies. About 670 million people worldwide are obese. The incidence of obesity has increased in all age groups, including children. Obesity causes numerous diseases and the interaction between genetic, metabolic, social, cultural and environmental factors are possible cofactors for the development of obesity. Evidence emerging over the last 20 years supports the hypothesis that viral infections may be associated with obesity in animals and humans. The most widely studied infectious agent possibly linked to obesity is adenovirus 36 (Adv36). Adv36 causes obesity in animals. In humans, Adv36 associates with obesity both in adults and children and the prevalence of Adv36 increases in relation to the body mass index. In vivo and in vitro studies have shown that the viral E4orf1 protein (early region 4 open reading frame 1, Adv) mediates the Adv36 effect including its adipogenic potential. The Adv36 infection should therefore be considered as a possible risk factor for obesity and could be a potential new therapeutic target in addition to an original way to understand the worldwide rise of the epidemic of obesity. Here, the data indicating a possible link between viral infection and obesity with a particular emphasis to the Adv36 will be reviewed.

Detection of hepatitis E virus in pork liver sausages.

Hepatitis E infection is regarded as an emerging public-health concern. The disease is normally self-limiting (mortality rate 1%), but chronic infections have recently been observed in transplanted patients. The etiological agent HEV is a small RNA virus infecting both humans and animals. In humans, the disease may be food-borne and pig is a main reservoir for zoonotic strains. In the present study, we evaluated the presence of HEV and swine fecal cross-contamination in pork liver sausages sold at a grocery store in Italy. HEV genome detection was performed by RT-qPCR, using harmonized protocols that included a process control (murine norovirus) and an internal amplification control. Swine fecal cross-contamination was assessed by determination of the ubiquitous porcine adenovirus. Overall, HEV genome belonging to genotype 3 was detected in both raw (10 out of 45 slices, 250 mg each, 22.2%) and dry (1 of 23 slices, 4.3%) liver sausages, but infectivity of the virus was not demonstrated. This pilot study fosters more investigations on HEV presence in pork-derived food, to assess the possible risk for the consumers.

Identification and Genotyping of Human Sapoviruses Collected from Sewage Water in Naples and Palermo, Italy, in 2011.

Human sapoviruses were identified in 15 (12.4 %) of 121 inlet sewage samples collected from wastewater treatment plants in Naples and Palermo, Italy, in 2011. All strains, except one GI.1, were genotyped as GI.2 by sequencing a capsid gene fragment. This is the first detection of sapovirus in wastewaters in Italy.

Viral and antibody HEV prevalence in swine at slaughterhouse in Italy.

Hepatitis E is an acute disease of humans caused by a small RNA virus, Hepatitis E virus (HEV). In recent years, an increasing number of autochthonous human infections have been reported in industrialized countries. Genotype 3 is the main HEV type circulating in swine, and is also reported in sporadic cases of hepatitis E in humans worldwide. To date one serotype has been described. We have conducted a survey to detect antibodies against HEV in 48 swine at a slaughterhouse in Northern Italy, using ELISA test. Mean seroprevalence in the studied animal group was 87.0%. Bile, liver and feces from the 48 animals were also collected, and HEV RNA was detected by nested reverse transcription-polymerase chain reaction, amplifying a fragment of the ORF2. HEV genome was most frequently detected in bile samples (51.1%), followed by feces (33.3%) and liver (20.8%). Thirty-one out of 48 studied pigs (64.6%) were positive for HEV RNA in at least one sample. Overall, HEV RNA was found at a statistically higher rate in the 3-4-month-old than in 9-10-month-old animals (95.0% vs. 42.9%). Genetic characterization of swine strains identified was performed by sequencing and database alignment. Phylogenetic analysis on the nucleotide sequences from 14 positive PCR products indicated that all strains belonged to genotype 3, clustering in two branches subtypes g3c and g3f.