The HAV transmission generally occurs through faecal-oral transmission, contaminated water or food, poor sanitary conditions or poor hygiene. Bulgaria is considered a country in transition between an intermediate and a low endemicity profile with significant number of cases and cyclic outbreaks. A phylogenetic, comparative analysis was applied to genotype I Bulgarian sequences collected from 2011 to 2023 to investigate the HAV circulation, the molecular epidemiology, the phylogenetic relationships respect to foreign sequences and gene flows between different Bulgarian towns. Five different HAV IA and IB VP1-2A dataset were investigated through phylogenetic analysis. The sequence alignments were conducted through MAFFT, under the Galaxy platform, followed by manual editing with Bioedit. Bayesian phylogeny was obtained with BEAST, the gene flow migration analysis in Bulgaria was explored through Mac Clade. Genotyping of the HAV sequences from Bulgaria revealed two main supported clades corresponding to the IA (n = 115) and IB (n = 120) subtypes. Phylogenetic analysis in IA highlighted a major cluster with strains strictly related to the frozen berries European outbreak. Other minor clusters indicated the simultaneous circulation of different variants. In HAV IB a major cluster of very similar sequences was reported, confirming the low variability. The Bayesian tree indicated intermixing between Bulgarian and foreign strains both in IA and IB. Time scaled phylogeny revealed that HAV IA began circulating in Bulgaria before the genotype IB, confirming previous data. For HAV genotype IA the gene flow analysis indicated bidirectional exchanges involving Sofia and Samokov (each 3.3

We investigated SARS-CoV-2 variants circulating, from November 2020 to March 2022, among military and civilian personnel at an Air Force airport in Italy in order to classify viral isolates in a potential hotspot for virus spread. Positive samples were subjected to Next-Generation Sequencing (NGS) of the whole viral genome and Sanger sequencing of the spike coding region. Phylogenetic analysis classified viral isolates and traced their evolutionary relationships. Clusters were identified using 70% cut-off. Sequencing methods yielded comparable results in terms of variant classification. In 2020 and 2021, we identified several variants, including B.1.258 (4/67), B.1.177 (9/67), Alpha (B.1.1.7, 9/67), Gamma (P.1.1, 4/67), and Delta (4/67). In 2022, only Omicron and its sub-lineage variants were observed (37/67). SARS-CoV-2 isolates were screened to detect naturally occurring resistance in genomic regions, the target of new therapies, comparing them to the Wuhan Hu-1 reference strain. Interestingly, 2/30 non-Omicron isolates carried the G15S 3CLpro substitution responsible for reduced susceptibility to protease inhibitors. On the other hand, Omicron isolates carried unusual substitutions A1803V, D1809N, and A949T on PLpro, and the D216N on 3CLpro. Finally, the P323L substitution on RdRp coding regions was not associated with the mutational pattern related to polymerase inhibitor resistance. This study highlights the importance of continuous genomic surveillance to monitor SARS-CoV-2 evolution in the general population, as well as in restricted communities.

HBV/HCV co-infection is common in HIV-1-infected prisoners. To investigate the characteristics of HIV co-infections, and to evaluate the molecular heterogeneity of HIV, HBV and HCV in prisoners, we carried-out a multicenter cross-sectional study, including 65 HIV-1-infected inmates enrolled in 5 Italian detention centers during the period 2017–2019. HIV-1 subtyping showed that 77.1% of inmates were infected with B subtype and 22.9% with non-B subtypes. Italian nationals were all infected with subtype B (93.1%), except two individuals, one infected with the recombinant form CRF72_BF1, and the other with the HIV-1 sub-subtype A6, both previously not identified in inmates of Italian nationality. Non-Italian nationals were infected with subtype B (52.6%), CRFs (36.8%) and sub-subtypes A1 and A3 (5.2%). HIV variants carrying resistance mutations to NRTI, NNRTI, PI and InSTI were found in 7 inmates, 4 of which were never exposed to the relevant classes of drugs associated with these mutations. HBV and/or HCV co-infections markers were found in 49/65 (75.4%) inmates, while 27/65 (41.5%) showed markers of both HBV and HCV coinfection. Further, Italian nationals showed a significant higher presence of HCV markers as compared to non-Italian nationals ( p = 0.0001). Finally, HCV phylogenetic analysis performed in 18 inmates revealed the presence of HCV subtypes 1a, 3a, 4d (66.6%, 16.7% and 16.7%, respectively). Our data suggest the need to monitor HIV, HBV and HCV infections in prisons in order to prevent spreading of these viruses both in jails and in the general population, and to implement effective public health programs that limit the circulation of different genetic forms as well as of viral variants with mutations conferring resistance to treatment.

BackgroundMost SARS-CoV-2 rapid antigen detection tests (RADTs) validation studies have been performed on specimens from COVID-19 patients and negative controls or from mostly symptomatic individuals. Herein we evaluated the diagnostic accuracy of AFIAS COVID-19 Ag, hereinafter denominated as AFIAS, during a COVID-19 screening program surveillance testing conducted among personnel of an Italian military airport.MethodsNasopharyngeal swabs (NPSs) were collected from study participants and were analysed by both AFIAS and RT-PCR assay. A questionnaire collecting demographic and exposure data were administered to all participants. AFIAS accuracy parameters including Cohen's kappa (K) were determined.ResultsOverall, from November 2020 to April 2021, 1294 (NPSs) were collected from 1183 participants (88.6% males, 11.4% females; mean age were 41.3, median age 42). Forty-nine NPSs (3.78%) were positive by RT-PCR, while 54 NPSs were positive by AFIAS. Overall baseline sensitivity, specificity, positive and negative predictive values were 0.633, 0.981, 0.574, 0.985, respectively and K was 0.585 (moderate). AFIAS sensitivity tended to be higher for NPSs with higher viral load. A higher sensitivity (0.944) compared to the overall baseline sensitivity (0.633) was also found for NPSs from participants with COVID-19 compatible symptoms, for which K was 0.891 (almost perfect). Instead, AFIAS sensitivity was quite poor for NPSs from asymptomatic participants. Most false negative NPSs in this group had moderate viral load.ConclusionOverall, AFIAS showed high specificity but only moderate sensitivity, mainly because of the high proportion of asymptomatic participants. However, AFIAS showed good sensitivity for NPSs with high viral load and nearly optimal accuracy parameters for NPSs from participants with COVID-19 compatible symptoms. Thus, taking into consideration its performance features, this test can be useful for COVID-19 case identification and management as well as for infection control.

Iron supplementation improves the erythropoiesis-stimulating agents’ (ESAs) response in chemotherapy-related anemia. The primary aim of our study is to assess the efficacy of sucrosomial iron, a new oral iron formulation, in cancer patients with chemotherapy-induced anemia treated with ESAs. The secondary objectives included the efficacy into two subgroups of patients (iron replete and functional iron deficiency) between the two study arms, safety and the effect on transfusion need. In this randomized, multicentre, open-label, phase III clinical trial, 60 cancer patients were enrolled. Each patient was randomly assigned (1:1) to receive 12 weeks of oral sucrosomial iron at the dose of 30 mg daily in combination with ESAs or no supplementation to ESA treatment. The endpoint considered for efficacy was the proportion of patients achieving complete hematological response at 12 weeks (increase in Hb > 2 g/dL from baseline, without RBC transfusions in the previous 28 days or achieving Hb ≥ 12 g/dL). There was a statistically significant association between oral sucrosomial iron supplementation in combination with ESAs and the achievement of a complete hematological response. This response was achieved within 12 weeks by 31% of patients in the control group and by 52% of patients supplemented with oral sucrosomial iron. A trend of greater response in sucrosomial iron arm was found in both subgroups. No difference was observed about safety and transfusion need. Sucrosomial iron is well tolerated and its combination with ESAs improves the hematological response in cancer patients with chemotherapy-related anemia. This study has been reviewed by the Institutional Ethics Committee of the IRCCS Policlinico San Matteo Foundation, Pavia, Italy (28/04/2015; prot. N. 20,150,002,059), and by the Institutional Ethics Committee of the other Italian oncological centers involved in this study.

The genetic diversity of Hepatitis A Virus (HAV) circulating in the Campania Region in years 2015–2018 was investigated through the monitoring of sentinel bivalve shellfish and water matrices. Overall, 463 water samples (71 sewage samples, 353 coastal discharge waters, and 39 seawaters samples), and 746 bivalve shellfish samples were analyzed. Positivity for HAV was detected in 20/71 sewage samples, 14/353 coastal discharge waters, 5/39 seawaters, and 102/746 bivalve shellfish. Sixty-one of the positive samples were successfully sequenced and were characterized as genotype IA (n = 50) and IB (n = 11). The prevalent strain circulating in 2015 in both bivalves and waters was the IA strain responsible for the outbreak occurring around the same time in the Naples area. This variant was no longer identified in subsequent years (2017–2018) when, instead, appeared two of the IA variants of the multistate outbreak affecting men who have sex with men (MSM), VRD_521_2016, and RIVM-HAV16–090, with the former prevailing in both shellfish and water environments. HAV IB isolates were detected over the years in shellfish and in water matrices, but not in clinical samples, suggesting that this genotype had been circulating silently. An integrated surveillance system (environment/food/clinical cases) can be a useful tool to monitor changes in viral variants in the population, as well as an early warning system.

Hepatitis C virus subtype 1b (HCV1b) is still the most prevalent subtype worldwide, with massive expansion due to poor health care standards, such as blood transfusion and iatrogenic procedures. Despite safe and effective new direct antiviral agents (DAA), treatment success can depend on resistance-associated substitutions (RASs) carried in target genomic regions. Herein we investigated transmission clusters and RASs among isolates from HCV1b positive subjects in the Calabria Region. Forty-one NS5B and twenty-two NS5A sequences were obtained by Sanger sequencing. Phylogenetic analysis was performed using the maximum likelihood method and resistance substitutions were analyzed with the Geno2pheno tool. Phylogenetic analysis showed sixteen statistically supported clusters, with twelve containing Italian sequences mixed with foreign HCV1b isolates and four monophyletic clusters including only sequences from Calabria. Interestingly, HCV1b spread has been maintained by sporadic infections in geographically limited areas and by dental treatment or surgical intervention in the metropolitan area. The L159F NS5B RAS was found in 15 isolates and in particular 8/15 also showed the C316N substitution. The Y93H and L31M NS5A RASs were detected in three and one isolates, respectively. The A92T NS5A RAS was found in one isolate. Overall, frequencies of detected NS5B and NS5A RASs were 36.6% and 22.7%, respectively. For the eradication of infection, improved screening policies should be considered and the prevalence of natural RASs carried on viral strains.

BACKGROUND:Evaluation of clinical performance of the anti-hepatitis C virus (HCV) rapid tests were carried out mostly in chronic hepatitis C patients and in individuals at high risk of HCV infection.METHODS:The aim of this study was to evaluate the performance of OraQuick and Wantai rapid tests on archived serum samples from 1408 individuals (mean age 46, range 18-90; 65% female) recruited with a systematic sampling procedure during a general population survey.RESULTS:The analysis of samples by Ortho HCV 3.0 ELISA and Cobas Taqman HCV RNA assays resulted in 69 anti-HCV antibody positive sera, including 42 HCV RNA positive (group 1) and 27 HCV RNA negative (group 2) samples. The performance of rapid tests was evaluated on the 69 anti-HCV positive (group 1+2) and 206 (OraQuick) and 198 (Wantai) anti-HCV negative sera, randomly selected from the 1339 anti-HCV negative samples. The OraQuick and Wantai rapid assays showed a sensitivity in group 1 of 92.9% and 90.5%, respectively. The sensitivity in group 2 was 40.7% and 51.9%, respectively. The anti-HCV antibodies signal/cutoff mean value was the only parameter that statistically differed between group 1 and group 2 individuals (P<0.0001). Further, 3 (OraQuick) and 4 samples (Wantai) from group 1, with very low HCV RNA level (<25 UI/mL), were misdiagnosed by rapid assays as false negative.CONCLUSIONS:The proportion of infections with low level of viremia and the risk associated with rapid assay failure remained to be carefully estimated in general population.

BACKGROUND:The routes of hepatitis E virus (HEV) transmission have still not been fully clarified. Here, we evaluated the possibility of sexual transmission of HEV, which remains a highly disputed issue.MATERIALS AND METHODS:Hepatitis E virus sexual transmission risk was assessed by comparing the prevalence of HEV infection in a sample of 196 Italian men who have sex with men (MSM) involved in a multi-country hepatitis A virus (HAV) outbreak, and in 3,912 Italian male blood donors selected from the same regions and provinces as the MSM. Selection of study of participants was motivated by the fact that HEV prevalence among Italian blood donors has been found to vary enormously between different geographical areas.RESULTS:Anti-HEV IgG prevalence was 14.8% and 5.6% in blood donors and MSM, respectively. Adjusted anti-HEV IgG prevalence was significantly lower in MSM than in blood donors (odds ratio [OR], 0.40; 95% confidence interval [CI]: 0.22-0.75; p<0.01), among residents in northern (OR, 0.45; 95% CI: 0.37-0.55; p<0.01) and southern (OR, 0.45; 95% CI: 0.35-0.58; p <0.01) Italy than among residents in Central Italy, while the prevalence was significantly higher in participants over 50 years of age than in those under 50 years of age (OR, 1.83; 95% CI: 1.48-2.27; p<0.01).DISCUSSION:Our findings suggest that sexual intercourse does not have a relevant role in HEV transmission. In particular, sexual transmission of HEV is unlikely to influence the prevalence of HEV infection at population level.

From April to October 2017, 27 cases of Hepatitis A (HA), 22 male and 5 female, were reported in Cosenza (South Italy). The median age of cases was 32 years (range 3-49 years). Out of 21 male adults, 14 were identified as men who have sex with men (MSM). Phylogenetic analysis was conducted in 15 cases and revealed two distinct sequences of genotype IA linking to clusters recognised in MSM in other European countries in 2016; genotype IB was recognized in only 2 cases. The report confirms that HA is an emerging issue among MSM. As suggested by the WHO, in countries with low HAV circulation, vaccination programmes should be tailored on local epidemiological patterns to prevent outbreaks among high risk groups and eventual spill-over of the infection into the general population.

The purpose of this study was to analyze sequences of hepatitis A virus (HAV) Ia and Ib genotypes from Bulgarian patients to investigate the molecular epidemiology of HAV genotype I during the years 2012 to 2014. Around 105 serum samples were collected by the Department of Virology of the National Center of Infectious and Parasitic Diseases in Bulgaria. The sequenced region encompassed the VP1/2A region of HAV genome. The sequences obtained from the samples were 103. For the phylogenetic analyses, 5 datasets were built to investigate the viral gene in/out flow among distinct HAV subpopulations in different geographic areas and to build a Bayesian dated tree, Bayesian phylogenetic and migration pattern analyses were performed. HAV Ib Bulgarian sequences mostly grouped into a single clade. This indicates that the Bulgarian epidemic is partially compartmentalized. It originated from a limited number of viruses and then spread through fecal-oral local transmission. HAV Ia Bulgarian sequences were intermixed with European sequences, suggesting that an Ia epidemic is not restricted to Bulgaria but can affect other European countries. The time-scaled phylogeny reconstruction showed the root of the tree dating in 2008 for genotype Ib and in 1999 for genotype Ia with a second epidemic entrance in 2003. The Bayesian skyline plot for genotype Ib showed a slow but continuous growth, sustained by fecal-oral route transmission. For genotype Ia, there was an exponential growth followed by a plateau, which suggests better infection control. Bidirectional viral flow for Ib genotype, involving different Bulgarian areas, was observed, whereas a unidirectional flow from Sofia to Ihtiman for genotype Ia was highlighted, suggesting the fecal-oral transmission route for Ia.

In Italy, the incidence of hepatitis A has progressively declined over the last 30 years, though not homogeneously throughout the country. In Campania, Southern Italy, high annual incidence rates have been reported and several periodic outbreaks have occurred. To investigate the phylogenetic and epidemiologic relationships among HAV strains circulating in Campania over the period 1997-2015, 87 hepatitis A cases were investigated. The most frequent risk factor was the consumption of raw/undercooked shellfish (75/87, 86.2%). During 1997-2002 most viral strains were subtype IA (16/23, 70%); the phylogenetic pattern suggests that the incidence peaks observed in 2000-2001 had likely been caused by multiple strains. During a large 2004 outbreak, almost all viral variants were subtype IB (38/41, 93%); most of them (22/38, 58%) were recognized to be one of two main strains (differing for just a single nucleotide), the remaining sequences were strictly related variants. In 2014/2015, only IA strains were observed; two phylogenetically related but distinct strains were responsible, respectively, for a small cluster in 2014 and an outbreak in 2015. In each outbreak, several strains unrelated to those responsible for most cases were detected in a minority of patients, documenting a background of sporadic cases occurring even in the course of outbreaks; some of them proved to be identical to strains detected 11-14 years previously. Overall, the data suggest that several related and unrelated HAV strains have endemically circulated over the last 15 years in Campania, with some strains gaining epidemic transmission likely because of a local combination of multiple factors, including inadequate waste water purification and dietary habits.

Background: Hepatitis A virus (HAV) infection is endemic in Eastern European and Balkan region countries. In 2012, Bulgaria showed the highest rate (67.13 cases per 100,000) in Europe. Nevertheless, HAV genotypes and strains circulating in this country have never been described. The present study reports the molecular characterization of HAV from 105 patients from Bulgaria.Methods: Anti-HAV IgM positive serum samples collected in 2012-2014 from different towns and villages in Bulgaria were analysed by nested RT-PCR, sequencing of the VP1/2A region and phylogenetic analysis; the results were analysed together with patient and geographical data.Results: Phylogenetic analysis revealed two main sequence groups corresponding to the IA (78/105, 74%) and IB (27/105, 26%) sub-genotypes. In the IA group, a major and a minor cluster were observed (62 and 16 sequences, respectively). Most sequences from the major cluster (44/62, 71%) belonged to either of two strains, termed "strain 1" and "strain 2", differing only for a single specific nucleotide; the remaining sequences (18/62, 29%) showed few (1 to 4) nucleotide variations respect to strain 1 and 2. Strain 2 is identical to the strain previously responsible for an outbreak in the Czech Republic in 2008 and a large multi-country European outbreak caused by contaminated mixed frozen berries in 2013. Most sequences of the IA minor cluster and the IB group were detected in large/medium centers (LMCs). Overall, sequences from the IA major cluster were more frequent in small centers (SCs), but strain 1 and strain 2 showed an opposite relative frequency in SCs and LMCs (strain 1 more frequent in SCs, strain 2 in LMCs).Conclusions: Genotype IA predominated in Bulgaria in 2012-2014 and phylogenetic analysis identified a major cluster of highly related or identical IA sequences, representing 59% of the analysed cases; these isolates were mostly detected in SCs, in which HAV shows higher endemicity than in LMCs. The distribution of viral sequences suggests the existence of some differences between the transmission routes in SCs and LMCs. Molecular characterization of an increased number of isolates from Bulgaria, regularly collected over time, will be useful to explore specific transmission routes and plan appropriate preventing measures.

Coinfection of blood-borne hepatitis B and hepatitis C viruses (HBV and HCV, respectively) in human immunodeficiency virus type 1 (HIV-1)-positive individuals frequently occurs in inmate population and peculiar viral strains and patterns of virological markers may be observed.Plasma from 69 HIV-1-positive inmates was obtained from 7 clinical centers connected with correctional centers in different towns in Italy. HIV, HBV, and HCV markers were tested by commercial assays. Virus genotyping was carried out by sequencing the protease and reverse transcriptase-encoding region (PR-RT region) for HIV and a region encompassing the NS5B gene for HCV and subsequent phylogenetic analysis.Twelve over 14 HIV-subtyped inmates were infected with HIV-1 subtype B strains. The 2 non-B strains belonged to subtype G and CRF02_AG, in an Italian and a Gambian patient, respectively. Variants carrying the K103N and Y181C resistance mutations to non-nucleoside reverse transcriptase inhibitors (NNRTIs) were found in 2 out of 9 patients naive for combined antiretroviral therapy (cART) (22.2%). Most HIV-positive patients (92.8%) showed evidence of past or present HBV and/or HCV infection. Prevalence of HBV and HCV was 81.2% for both viruses, whereas prevalence of HBV/HCV coinfection was 69.6%. A significantly higher presence of HCV infection was found in Italians [odds ratio (OR) 11.0; interval 1.7-80.9] and in drug users (OR 27.8; interval 4.9-186.0). HCV subtypes were determined in 42 HCV or HBV/HCV-coinfected individuals. HCV subtypes 1a, 3a, 4d, and 1b were found in 42.9%, 40.5%, 14.3%, and 2.4% of inmates, respectively. Low titers of HBV DNA in HBV DNA positive subjects precluded HBV subtyping.The high prevalence of HBV and HCV coinfections in HIV-infected inmates, as well as the heterogeneity of HIV and HCV subtypes suggest the need to adopt systematic controls in prisons to monitor both the burden and the genetic forms of blood-borne viral infections, in order to apply targeted therapeutic interventions.

The detection of baseline resistance mutations to new direct-acting antivirals (DAAs) in HCV chronically infected treatment-naïve patients could be important for their management and outcome prevision. In this study, we investigated the presence of mutations, which have been previously reported to be associated with resistance to DAAs in HCV polymerase (NS5B) and HCV protease (NS3) regions, in sera of treatment-naïve patients.

Circulation of HCV genotype 2 has been described in European Countries where numerous subtypes and unclassified HCV 2 lineages have been reported. In Italy, subtype 1b is the most prevalent, followed by genotype 2. In the present study, phylogeny of HCV 2c was investigated. The phylogeny of HCV 2c isolated from 54 Italian patients in the Calabria region (Southern Italy) was investigated by analyzing a fragment of the NS5B gene. Patients came from 5 metropolitan areas and a small village (Sersale). These areas were geographically dispersed throughout the entire region. A Bayesian coalescent-based framework was used to estimate origin and spreading of HCV 2c in this region. Phylogenetic analysis showed that 28 Italian sequences were intermixed with foreign HCV 2c reference sequences and grouped into 3 major clades: A, B, and C. Nineteen inter-clade sequences were associated uniquely with surgery as risk factor for HCV acquisition. By contrast, a sub-cluster within clade B was associated with blood transfusion. Moreover, sequences from Sersale village grouped in the Italian sub-cluster and were intermixed with 10 sequences from metropolitan areas. The three isolates with the longest branch came from Sersale and belonged to patients who had glass syringes as risk factor. HCV 2c isolates from the Calabria region shared a common ancestor whose origin was traced back to 1889. Our results suggest that, after its introduction – possibly as a result of population movements between Italy and African Countries during Italian colonialism – HCV 2c spread through multiple risk factors, not including intravenous drug use. So, transmission chains followed a pathway different from other European Countries. Although HCV incidence is decreasing, these ways are still ongoing, possibly justifying stability in the relative prevalence of HCV 2c.

BACKGROUND:In a 1996 survey, prevalence of hepatitis C virus antibodies (anti-HCV) in a southern Italian town was 12.6%. AIMS:To identify changes in the epidemiology of hepatitis C virus (HCV) infection. METHODS:Anti-HCV, HCV-RNA (PCR, detection limit 15 IU/mL), HCV genotype (Innolipa). Were performed in a random 1:4 systematic sample of the general population. Multiple logistic regression analysis was used to estimate factors independently associated with the likelihood of anti-HCV positivity. RESULTS:Of 1012 subjects, 58 (5.7%) were anti-HCV-positive, compared to 12.6% 14 years earlier. Prevalence was 0.4% in individuals <30 years old and 31.8% in those ≥ 70 years old. Among 139 HCV-negative in 1996 re-sampled in 2010, only one had seroconverted (incidence: 0.05 × 100 persons/year). Alanine transaminase levels were elevated in 8 (13.8%). HCV-RNA was detected by PCR in 46.5% anti-HCV-positive subjects. In 2010 59% were genotype 2-infected, in 1996 50.7% genotype 1-infected. Previous use of non-disposable glass syringes was a strong independent predictor (OR 3.2; CI 95%=1.4-7.3). CONCLUSION:Epidemiology of HCV infection in an endemic area of south Italy has changed over 14 years, now largely confined to the oldest age group; this seems to be due to the disappearance of its past main mode of transmission, namely the use of glass syringes.

Hepatitis C Virus (HCV) genotype 4 predominates in Middle East and Central Africa countries. Recently, it has become also prevalent in Southern European countries where it is thought to have been introduced through immigration and the movement of intravenous drug users. In Italy, the prevalence of genotype 4 is particularly high (4.5%) in Southern regions, such as Calabria, and reaches values of 8.4% in specific areas where there appears to be endemic circulation of this genotype. In the present study, the phylogeny of HCV subtype 4d isolated from 19 Italian patients in Calabria was investigated by analysing a fragment of the NS5B viral genomic region. A Bayesian coalescent-based framework was used to estimate origin and spread of the HCV 4d in this area. The mean evolutionary rate HCV 4d NS5B sequences was estimated using a dataset of sequences sampled at known times and a relaxed clock constant model that best fitted the data. By using a Bayesian coalescent method, the Italian 4d isolates collected in Calabria were found to share a common ancestor with reference 4d isolates whose origin was traced back to 1940s. The genotype 4d epidemic in Southern Italy was maintained in a steady non-expanding phase until the late 1970s after that it grew exponentially up to 1990s probably sustained by the vast increase of unsafe blood transfusions and the spread of illicit intravenous drug users. J. Med. Virol. 84:16131619, 2012. (c) 2012 Wiley Periodicals, Inc.

Virus-like particles (VLPs) are excellent tools for vaccines against pathogens and tumors. They can accommodate foreign polypeptides whose incorporation efficiency and immunogenicity however decrease strongly with the increase of their size. We recently described the CD8+ T cell immune response against a small foreign antigen (i.e., the 98 amino acid long human papilloma virus E7 protein) incorporated in human immunodeficiency virus (HIV)-1 based VLPs as product of fusion with an HIV-1 Nef mutant (Nefmut). Here, we extended our previous investigations by testing the antigenic/immunogenic properties of Nefmut-based VLPs incorporating much larger heterologous products, i.e., human hepatitis C virus (HCV) NS3 and influenza virus NP proteins, which are composed of 630 and 498 amino acids, respectively. We observed a remarkable cross-presentation of HCV NS3 in dendritic cells challenged with Nefmut–NS3 VLPs, as detected using a NS3 specific CD8+ T cell clone as well as PBMCs from HCV infected patients. On the other hand, when injected in mice, Nefmut–NP VLPs elicited strong anti-NP CD8+ T cell and CTL immune responses. In addition, we revealed the ability of Nefmut incorporated in VLPs to activate and mature primary human immature dendritic cells (iDCs). This phenomenon correlated with the activation of Src tyrosine kinase-related intracellular signaling, and can be transmitted from VLP-challenged to bystander iDCs. Overall, these results prove that Nefmut-based VLPs represent a rather flexible platform for the design of innovative CD8+ T cell vaccines.

BACKGROUND:Infections with hepatitis C virus (HCV) progress to chronic phase in 80% of patients. To date, the effect produced by HCV on the expression of microRNAs (miRs) involved in the interferon-β (IFN-β) antiviral pathway has not been explored in details. Thus, we compared the expression profile of 24 selected miRs in IFN-β-treated Huh-7 cells and in three different clones of Huh-7 cells carrying a self-replicating HCV RNA which express all viral proteins (HCV replicon system).METHODS:The expression profile of 24 selected miRs in IFN-β-treated Huh-7 cells and in HCV replicon 21-5 clone with respect to Huh-7 parental cells was analysed by real-time PCR. To exclude clone specific variations, the level of 16 out of 24 miRs, found to be modulated in 21-5 clone, was evaluated in two other HCV replicon clones, 22-6 and 21-7. Prediction of target genes of 3 miRs, confirmed in all HCV clones, was performed by means of miRGator program. The gene dataset obtained from microarray analysis of HCV clones was farther used to validate target prediction.RESULTS:The expression profile revealed that 16 out of 24 miRs were modulated in HCV replicon clone 21-5. Analysis in HCV replicon clones 22-6 and 21-7 indicated that 3 out of 16 miRs, (miR-128a, miR-196a and miR-142-3p) were modulated in a concerted fashion in all three HCV clones. Microarray analysis revealed that 37 out of 1981 genes, predicted targets of the 3 miRs, showed an inverse expression relationship with the corresponding miR in HCV clones, as expected for true targets. Classification of the 37 genes by Panther System indicated that the dataset contains genes involved in biological processes that sustain HCV replication and/or in pathways potentially implicated in the control of antiviral response by HCV infection.CONCLUSIONS:The present findings reveal that 3 IFN-β-regulated miRs and 37 genes, which are likely their functional targets, were commonly modulated by HCV in three replicon clones. The future use of miR inhibitors or mimics and/or siRNAs might be useful for the development of diagnostic and therapeutic strategies aimed at the recovering of protective innate responses in HCV infections.