Maurizio Mazzei

In situ PCR-associated immunohistochemistry identifies cell types harbouring the Maedi-Visna virus genome in tissue sections of sheep infected naturally

Maedi-Visna virus (MVV) is a non-oncogenic ovine lentivirus whose main targets are the lung, mammary gland, central nervous system and joints. Cells of the monocyte-macrophage lineage are the major viral target in vivo; other cell types are infected as well, as indicated by several studies, largely based on the examination of animals infected experimentally or on the in vitro infection of cultured cells. Aim of this study was to investigate the cell types harbouring the viral genome in lungs and mammary glands of animals infected naturally by using in situ PCR-associated immunohistochemistry. Several types of cells were infected: in the lung type I and II pneumocytes, interstitial and alveolar macrophages, endothelial cells and fibroblast-like cells. Epithelial cells, macrophages, endothelial cells and fibroblast-like cells were infected also in the mammary gland. These results indicate that the in situ PCR, a powerful technique which combines the high sensitivity of the conventional PCR with the ability to localise the cellular targets within a tissue, can be improved further by its association with the immunohistochemistry. This can be especially advantageous when the presence and localisation of the target sequence are investigated in the context of a tissue with its complex cellular organisation.

Presence of hepatitis E RNA in mussels used as bio-monitors of viral marine pollution

Mussels (Mytilus galloprovincialis), collected from a harvesting area approved by European Community Regulation, were transplanted to four polluted sites located in the Northwestern Mediterranean area (Tuscany). They were used as bio-monitors to test the quality of the marine water pollution. At different times after the transplantation, mussels were withdrawn and tested for presence of phages and enteric viruses by molecular tests. 52.4% of the transplanted mussel samples were positive for at least one enteric virus. Hepatitis A virus (HAV) was identified in each site (17/37; 45.9%). Three samples were positive for hepatitis E virus (HEV) (8.1%) and two (5.4%) for norovirus (NoV) genogroup I. Coliphages and RYC 2056 phages were detected in all sites, while HSP 40 phages were detected in three sites. Results demonstrate the ability of transplanted mussels in accumulating and retaining different species of enteric microorganisms. Their utility as bio-monitor organisms enables testing for viral marine pollution.

Infectivity of DWV Associated to Flower Pollen: Experimental Evidence of a Horizontal Transmission Route

Deformed wing virus (DWV) is a honeybee pathogen whose presence is generally associated with infestation of the colony by the mite Varroa destructor, leading to the onset of infections responsible for the collapse of the bee colony. DWV contaminates bee products such as royal jelly, bee-bread and honey stored within the infected hive. Outside the hive, DWV has been found in pollen loads collected directly from infected as well as uninfected forager bees. It has been shown that the introduction of virus-contaminated pollen into a DWV-free hive results in the production of virus-contaminated food, whose role in the development of infected bees from virus-free eggs has been experimentally demonstrated. The aim of this study was twofold: (i) to ascertain the presence of DWV on pollen collected directly from flowers visited by honeybees and then quantify the viral load and (ii) determine whether the virus associated with pollen is infective. The results of our investigation provide evidence that DWV is present on pollen sampled directly from visited flowers and that, following injection in individuals belonging to the pollinator species Apis mellifera, it is able to establish an active infection, as indicated by the presence of replicating virus in the head of the injected bees. We also provide the first indication that the pollinator species Osmia cornuta is susceptible to DWV infection.

Characterization of new Small Ruminant Lentivirus subtype B3 suggests animal trade within the Mediterranean Basin.

Small ruminant lentiviruses (SRLVs) represent a group of viruses infecting sheep and goats worldwide. Despite the high heterogeneity of genotype A strains, which cluster into as many as ten subtypes, genotype B was believed to be less complex and has, so far, been subdivided into only two subtypes. Here, we describe two novel full-length proviral sequences isolated from Sarda sheep in two Italian regions. Genome sequence as well as the main linear epitopes clearly placed this cluster into genotype B. However, owing to long-standing segregation of this sheep breed, the genetic distances that are clearly >15 % with respect to B1 and B2 subtypes suggest the designation of a novel subtype, B3. Moreover the close relationship with a gag sequence obtained from a Turkish sheep adds new evidence to historical data that suggest an anthropochorous dissemination of hosts (small ruminants) and their pathogens (SRLV) during the colonization of the Mediterranean from the Middle East.

Serologic and molecular survey for hepatitis E virus in wild boar (Sus scrofa) in Central Italy.

The aim of this study was to further investigate the role of wild boar (Sus scrofa) as a reservoir for hepatitis E virus (HEV). Sixty-four blood and faecal samples collected from wild boar hunted in Central Italy in 2011–2012 were examined by indirect enzyme-linked immunosorbent assay and RT-PCR analysis. Positive RT-PCR samples were further examined by nucleotide sequence determination and subsequent phylogenetic analysis. Thirty-six sera (56.2%) were positive for HEV-specific antibodies, and six (9.4%) faecal samples scored RT-PCR-positive results. Four animals were positive by both enzyme-linked immunosorbent assay and RT-PCR. Phylogenetic analysis showed that the detected wild boar–derived HEV sequences clustered within genotype 3, with similarity to sequences of human origin collected in a nearby area in 2012. Our data confirm that HEV is endemic in the wild boar population in the research area and that these wild animals could play an important role in the epidemiology of HEV infection.

Mucosal immunization against ovine lentivirus using PEI-DNA complexes and modified vaccinia Ankara encoding the gag and/or env genes

Sheep were immunized against Visna/Maedi virus (VMV) gag and/or env genes via the nasopharynx-associated lymphoid tissue (NALT) and lung using polyethylenimine (PEI)-DNA complexes and modified vaccinia Ankara, and challenged with live virus via the lung. env immunization enhanced humoral responses prior to but not after VMV challenge. Systemic T cell proliferative and cytotoxic responses were generally low, with the responses following single gag gene immunization being significantly depressed after challenge. A transient reduction in provirus load in the blood early after challenge was observed following env immunization, whilst the gag gene either alone or in combination with env resulted in significantly elevated provirus loads in lung. However, despite this, a significant reduction in lesion score was observed in animals immunized with the single gag gene at post-mortem. Inclusion of IFN-gamma in the immunization mixture in general had no significant effects. The results thus showed that protective effects against VMV-induced lesions can be induced following respiratory immunization with the single gag gene, though this was accompanied by an increased pulmonary provirus load.

Systemic DNA immunization against ovine lentivirus using particle-mediated epidermal delivery and modified vaccinia Ankara encoding the gag and/or env genes

To determine whether systemic immunization with plasmid DNA and virus vector against visna/maedi virus (VMV) would induce protective immune responses, sheep were immunized with VMV gag and/or env sequences using particle-mediated epidermal bombardment and injection of recombinant modified vaccinia Ankara. The results showed that immunization induced both humoral and cell-mediated responses prior to and after virus challenge. The vaccination protocol did not prevent infection, but immunization with the gag gene or a combination of gag and env genes resulted in significantly reduced provirus loads in blood and mediastinal lymph node, respectively. Provirus loads in lung and draining lymph node were unaffected, but p25 expression was undetectable in lungs of animals immunized with a combination of gag and env genes. Analysis of target tissues for lesions at post-mortem showed that immunization with the env gene caused a significant increase in lesion score, while the gag gene or a combination of gag and env genes had no effect. Inclusion of the ovine interferon-gamma gene in the initial priming mixture had minimal effect on immune responses, provirus load, or lesion development, although it resulted in a decreased p25 expression in the lung. The results thus show that systemic immunization with gag or a combination of gag and env genes reduces provirus load in blood and lymphoid tissue, respectively whereas env immunization has no effect on provirus load but increased lesion development.

Seroconversion against SU5 derived synthetic peptides in sheep experimentally infected with different SRLV genotypes.

Synthetic peptides were generated, corresponding to SU5 domain of envelope glycoprotein of Italian SRLV isolates It-561 and It-Pi1, belonging respectively to MVV- and CAEV-like genotypes. The peptides, encompassing an N-terminal variable and a C-terminal conserved antibody-binding site, were used in an ELISA assay to analyse the sera of two groups of sheep experimentally infected with these isolates. The kinetics and specificity of the humoral response to the homologous and heterologous antigen and the affinity maturation of the sera were evaluated. Seroconversion occurred between week 3 and 8. The response to SU5 antigen was mostly type-specific. The few broadly reacting sera may reflect the production of antibodies directed to the SU5 constant antibody-binding site. All sera underwent with time avidity maturation, resulting in the appearance of high affinity antibodies. This study suggests constant monitoring of the circulating viral variants to develop a panel of diagnostic peptides representative of local genotypes.

SEROLOGIC, MOLECULAR, AND PATHOLOGIC SURVEY OF PSEUDORABIES VIRUS INFECTION IN HUNTED WILD BOARS (SUS SCROFA) IN ITALY

Abstract To investigate pseudorabies-virus (PrV) –antibody and viral-DNA prevalence, we collected blood, nasal and genital swabs, and tonsillar and lymph-node tissue samples from 139 wild boars (Sus scrofa; 39 piglets, 30 juveniles, and 70 adults), during the hunting season of 2010–2011 in Tuscany, Central Italy. We performed immunohistochemistry with anti-PrV monoclonal antibodies on selected tissue samples. Forty-three of 139 (30.9%) boars were PrV-antibody positive and a 1,954–base-pair PrV-specific product was amplified from nine nasal (6.5%) and 26 genital (18.7%) swabs. Sequence analysis of PrV-positive PCR products revealed identity scores of 99–100% with Suid herpesvirus 1 strain Becker (JF797219) and confirmed the identification of PrV DNA in tested swabs. There was significantly higher antibody prevalence in adults than in juveniles and in piglets than in juveniles. The prevalence of viral DNA was significantly higher in genital swabs than in nasal specimens. The percentage of positive nasal swabs did not differ among age classes. Piglets had a higher percentage of PCR-positive genital swabs than juvenile and adult subjects (30.8% vs. 13.3% and 14.3%, respectively). Results confirmed that PrV infection is widespread in the wild boar population in the study area. The presence of anti-PrV antibodies and of the PrV virus in piglets could be related to vertical transmission of the virus. This hypothesis was also supported by a higher presence of viral genome in genital swabs than in nasal swabs. This field study supports the importance of vertical transmission of PrV, and the high prevalence of virus in genital swabs supports venereal transmission in adult feral boars.

Development and comparison of strain specific gag and pol real-time PCR assays for the detection of Visna/maedi virus

The aim of this study was the development of gag and pol dual labelled probe real-time PCR and RT PCR assays to quantify the proviral load and the transcripts of the British Visna/maedi virus EV1 strain. Primers and probes were chosen based on the consensus sequences of gag and pol clones representative of EV1 genetic variants. Both PCRs had a detection limit of 3 copies of target gene, with a linearity over 6 orders of magnitude. The performances of the two PCRs in vivo were evaluated and compared on a panel of DNAs extracted from blood of sheep infected experimentally with EV1. The pol assay detected in most cases lower numbers of viral molecules than gag assay, yielding some false negative results. The gag real-time RT PCR had a detection limit of 100 RNA molecules with a linearity over 5 orders of magnitude. This did not result in a lower performance of the RT PCR compared to the PCR in cells permissive for virus replication, which contain higher numbers of viral transcripts than proviral genomes. The real-time assays developed in this study, particularly the gag assay, provide a sensitive tool which can be used to quantify the viral load in experimental infections.