24The lung homogenate proved to be positive for both influenza type A (gene M) and H1N1pdm real time RT- 25PCRs. Virus isolation (A/Sw/It/116114/2010) was obtained from both SPF CEE and Caco-2 cells. 26Phylogenetic analysis showed that all of the genes of A/Sw/It/116114/2010, with the exception of 27 neuraminidase (NA), belonged to the H1N1pdm cluster. The NA was closely related to two H1N2 double 73multiplex PCR and PCR assays respectively, as previously described (Calsamiglia et al., 1999; Ouardani et 74 al., 1999; Suarez et al., 1994). 92were made using ClustalW and maximum parsimony phylogenetic trees were created using MEGA4 93 (Tamura et al., 2007). Each tree is a consensus of 500 bootstrap replicates. 94The identification as a novel reassortant strain was confirmed by full length sequencing of HA and NA genes 95 performed directly on lung homogenates. 106The presence of influenza A and the H1N1pdm was detected on AF and Caco-2 CS by real time RT-PCRs. 107A multiplex RT-PCR specific for subtype determination was further used to subtype both AF and CS that

SummaryHepatitis E virus (HEV) is a zoonotic pathogen with a worldwide distribution, and infects several mammalian species, including pigs and wild boars, which are recognized as its natural reservoirs. The virus causes a usually self-limiting liver disease with a mortality rate generally below 1%, although mortality rates of 15%-25% have been recorded in pregnant woman. Chronic infections can also occur. The prevalence of HEV has been extensively studied in wild boars and pigs in northern Italy, where intensive pig herds are predominantly located. In contrast, few data have been collected in south-central Italy, where small pig herds are surrounded by large regional parks populated with heterogeneous wild fauna. In this study, 291 liver samples from wild boars caught in south-central Italy were analysed with the molecular detection of viral RNA. Our results confirm the circulation of HEV in these animals, with a mean prevalence of 13.7% (40 of 291). A nucleotide sequence analysis showed that the HEV strains were highly conserved within the same geographic areas. The wild boar HEV strains belonged to the HEV-3c subtype, which is frequently described in wild boars, and to an uncommon undefined subtype (HEV-3j-like).The viral prevalence detected is concerning because it could represent a potential risk to hunters, meat workers and consumers of wild boar liver and derivative products. The hypothesized inter-species transmission of HEV to pigs and the possibility that the virus maintains its virulence in the environment and the meat chain also present potential risks to human health, and warrant further investigations in the near future.

BackgroundMiddle East respiratory syndrome coronavirus (MERS-CoV), which belongs to beta group of coronavirus, can infect multiple host species and causes severe diseases in humans. Multiple surveillance and phylogenetic studies suggest a bat origin. In this study, we describe the detection and full genome characterization of two CoVs closely related to MERS-CoV from two Italian bats, Pipistrellus kuhlii and Hypsugo savii.MethodsPool of viscera were tested by a pan-coronavirus RT-PCR. Virus isolation was attempted by inoculation in different cell lines. Full genome sequencing was performed using the Ion Torrent platform and phylogenetic trees were performed using IQtree software. Similarity plots of CoV clade c genomes were generated by using SSE v1.2. The three dimensional macromolecular structure (3DMMS) of the receptor binding domain (RBD) in the S protein was predicted by sequence-homology method using the protein data bank (PDB).ResultsBoth samples resulted positive to the pan-coronavirus RT-PCR (IT-batCoVs) and their genome organization showed identical pattern of MERS CoV. Phylogenetic analysis showed a monophyletic group placed in the Beta2c clade formed by MERS-CoV sequences originating from humans and camels and bat-related sequences from Africa, Italy and China. The comparison of the secondary and 3DMMS of the RBD of IT-batCoVs with MERS, HKU4 and HKU5 bat sequences showed two aa deletions located in a region corresponding to the external subdomain of MERS-RBD in IT-batCoV and HKU5 RBDs.ConclusionsThis study reported two beta CoVs closely related to MERS that were obtained from two bats belonging to two commonly recorded species in Italy (P. kuhlii and H. savii). The analysis of the RBD showed similar structure in IT-batCoVs and HKU5 respect to HKU4 sequences. Since the RBD domain of HKU4 but not HKU5 can bind to the human DPP4 receptor for MERS-CoV, it is possible to suggest also for IT-batCoVs the absence of DPP4-binding potential. More surveillance studies are needed to better investigate the potential intermediate hosts that may play a role in the interspecies transmission of known and currently unknown coronaviruses with particular attention to the S protein and the receptor specificity and binding affinity.

The aim of the study was to evaluate the mRNA expression of four relevant ABC-transporter genes [MDR1 (P-glycoprotein; Pgp), MRP1, MRP4, and MRP5] in HIV-positive individuals failing treatment and analyze the association between the levels of their expression and viral load, CD4 cell count, and therapeutic history. Ninety-eight HIV-positive samples and 20 samples from healthy donors were analyzed, retrospectively. Peripheral blood mononuclear cells (PBMCs) from HIV1-positive individuals were collected at the time of virological failure. Expression of mRNA of Pgp, MRP1, MRP4, and MRP5 in PBMCs was evaluated by real-time PCR. A high inter-individual variability was observed in both HIV-positive individuals and healthy donors but the expression levels of all mRNA analyzed were significantly higher in the HIV-infected group (P < 0.05). A weak but significant inverse correlation was observed between CD4 cell counts and expression levels of MRP4 and MRP5. Comparison of mRNA expression between individuals with different therapeutic histories showed that expression of MRP4 and MRP5 genes in patients who were both protease inhibitor (PI) and non-nucleoside reverse transcriptase inhibitor (NNRTI)-experienced was significantly higher than in patients who were PI experienced but NNRTI-naïve. In conclusion, the mRNA expression of Pgp, MRP1, MRP4, and MRP5 varies among HIV-infected patients and healthy donors but is significantly higher in HIV-positive patients than in donors. The expression of MRP4 and MRP5 seems to correlate with CD4 cell counts. The same protein seems to be overexpressed in patients receiving NNRTIs.

Highly pathogenic (HP) and low pathogenic (LP) avian influenza viruses (AIVs) belonging to H5 and H7 subtypes have been found to be associated with human infection as the result of direct transmission from infected poultry. Human infections by AIVs can cause mild or subclinical disease, and serosurveys are believed to represent an important tool to identify risk of zoonotic transmission. Therefore, we sought to examine Italian poultry workers exposed during LPAI and HPAI outbreaks with the aim of assessing serologic evidence of infection with H5 and H7 AIVs. From December 2008 to June 2010 serum samples were collected from 188 poultry workers and 379 nonexposed controls in Northern Italy. The hemagglutination inhibition (HI) assay using horse red blood cells (RBCs) and a microneutralization (MN)-enzyme-linked immunosorbent assay test were used to analyze human sera for antibodies against the following H5 and H7 LPAI viruses: A/Dk/It/4445/07(H5N2); A/Ty/It/2369/09(H5N7); A/Ty/It/218-193/ 10; A/Ck/It/3775/99(H7N1); A/Ty/It/214845/03(H7N3); and A/Dk/It/332145/09(H7N3). Since previous studies identified low antibody titer to AIVs in people exposed to infected poultry, a cutoff titer of > or = 1:10 was chosen for both serologic assays. Only HI-positive results confirmed by MN assay were considered positive for presence of specific antibodies. The Fisher exact test was used to analyze differences in seroprevalence between poultry workers and control groups, with the significance level set at P < 0.05. MN results showed a proportion of H7-seropositive poultry workers (6/188, i.e., 3.2%), significantly higher than that of controls (0/379), whereas no MN-positive result was obtained against three H5 LPAI subtypes recently identified in Italy. In conclusion, the survey indicated that assessing seroprevalence can be an important tool in risk assessment and health,surveillance of poultry workers.