Project

An epizootiological survey of bats as reservoirs of emerging zoonotic viruses in Italy: implications for public health and biological conservation

Goal: Bats are universally recognised as main reservoir for emerging and potentially zoonotic viruses. Poor eco-epidemiological data are available on the circulation of the most important zoonotic viruses in the Italian bat population. We will undertake passive and active surveillance in Italian bats mainly targeted on the detection of coronaviruses (CoVs), lyssaviruses (LYSVs) and orthoreoviruses (MRVs). The occurrence of selected emerging viral infections relevant for either bat conservation or public health will also be investigated. This project aims to cover both human health and biological conservation issues by enhancing survey for major viral infections in bats.

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Davide Lelli
added 2 research items
Interest in bat-related viruses has increased considerably during the last decade, leading to the discovery of a rising number of new viruses in several bat species. Poxviridae are a large, diverse family of DNA viruses that can infect a wide range of vertebrates and invertebrates. To date, only a few documented detections of poxviruses have been described in bat populations on three different continents (America, Africa, and Australia). These viruses are phylogenetically dissimilar and have diverse clinical impacts on their hosts. Herein, we report the isolation, nearly complete genome sequencing, and annotation of a novel poxvirus detected from an insectivorous bat (Hypsugo savii) in Northern Italy. The virus is tentatively named Hypsugopoxvirus (HYPV) after the bat species from which it was isolated. The nearly complete genome size is 166,600 nt and it encodes 161 genes. Genome analyses suggest that HYPV belongs to the Chordopoxvirinae subfamily, with the highest nucleotide identity (85%) to Eptesipoxvirus (EPTV) detected from a microbat Eptesicus fuscus in WA, USA, in 2011. To date, HYPV represents the first poxvirus detected in bats in Europe; thus, its viral ecology and disease associations should be investigated further.
Diagnosis of West Nile virus (WNV) infections is often difficult due to the extensive antigenic cross-reactivity among flaviviruses, especially in geographic regions where two or more of these viruses are present causing sequential infections. The purpose of this study was to characterize a panel of monoclonal antibodies (MAbs) produced against WNV to verify their applicability in WNV diagnosis and in mapping epitope targets of neutralizing MAbs. Six MAbs were produced and characterized by isotyping, virus-neutralization, western blotting and MAb-epitope competition. The MAb reactivity against various WNVs belonging to lineage 1 and 2 and other related flaviviruses was also evaluated. The molecular basis of epitopes recognized by neutralizing MAbs was defined through the selection and sequencing of MAb escape mutants. Competitive binding assays between MAbs and experimental equine and chicken sera were designed to identify specific MAb reaction to epitopes with high immunogenicity. All MAbs showed stronger reactivity with all WNVs tested and good competition for antigen binding in ELISA tests with WNV-positive equine and chicken sera. Four MAbs (3B2, 3D6, 4D3, 1C3) resulted specific for WNV, while two MAbs (2A8, 4G9) showed cross-reaction with Usutu virus. Three MAbs (3B2, 3D6, 4D3) showed neutralizing activity. Sequence analysis of 3B2 and 3D6 escape mutants showed an amino acid change at E307 (Lys → Glu) in the E protein gene, whereas 4D3 variants identified mutations encoding amino acid changed at E276 (Ser → Ile) or E278 (Thr → Ile). 3B2 and 3D6 mapped to a region on the lateral surface of domain III of E protein, which is known to be a specific and strong neutralizing epitope for WNV, while MAb 4D3 recognized a novel specific neutralizing epitope on domain II of E protein that has not previously been described with WNV MAbs. MAbs generated in this study can be applied to various analytical methods for virological and serological WNV diagnosis. A novel WNV-specific and neutralizing MAb (4D3) directed against the unknown epitope on domain II of E protein can be useful to better understand the role of E protein epitopes involved in the mechanism of WNV neutralization.
Davide Lelli
added 4 research items
Summary This study describes the isolation and molecular characterization of Mammalian orthoreovirus (MRV) in microbats. Faecal samples and dead individuals available from rehabilitation centres or collected from known roost sites were virologically tested. In total, 112 carcasses of bats found dead, and 44 faecal samples were analysed. Nineteen viral strains were isolated by in vitro cell culture from faecal and tissue samples of different bat species (Pipistrellus khulii, Tadarida teniotis, Rhinolophus hipposideros and Vespertilio murinus), and they were morphologically identified as reoviruses by negative staining electron microscopy observation. The definitive assignment of all isolates to MRV was confirmed by RT-PCR assays targeting the L1 gene. Through a multiplex RT-PCR assay targeting the S1 gene, we typed 15 of 19 isolates as MRV type 3. Partial L1 (416 bp) and complete S1 (1416 bp) sequences of the isolates were analysed and compared with those of reference strains obtained from GenBank, belonging to the three serotypes. Molecular analysis of the S1 gene revealed that the amino acid residues associated with neurotropism (198-204NLAIRLP, 249I, 350D and 419E) were highly conserved among the Italian bat strains. These results suggest that potentially neurotropic MRV type 3 strains are widespread among Italian bats. Furthermore, the identification of MRV type 3 in bat species such as Pipistrellus Khulii, which is common in urban areas and known for its close contact with humans, underlines the need for vigilance.
Davide Lelli
added 2 research items
BACKGROUND Bats are being increasingly recognized as reservoir hosts of highly pathogenic and zoonotic emerging viruses in tropical regions but little is known on human-pathogenic viruses that may be present in bats in Europe. A renewed interest in Mammalian Orthoreoviruses (MRVs) has recently grown since new viruses related to Bat-MRVs detected in Europe have been identified in humans and pigs with gastroenteritis. Here, we report the results of MRVs surveillance in bats in Italy and the full-genome characterization of representative isolates belonging to different serotypes. METHODS During the period 2009-2015 bat samples were collected in Northern Italy from rehabilitation centres or from known roost sites. Virus isolation and identification were performed using cells culture, electron microscopy and PCRs for L1 and S1 genes. Selected representative bat-MRVs were fully sequenced by NGS approach. Phylogenetic and molecular analyses were performed. Virus neutralization (VNT) was carried out for serotype identification. RESULTS A total of 394 faecal and tissue samples belonging to 8 different bat species were analysed. 27 MRVs (6.9%) were isolated on cell culture and for 21 of them it was succeeded to type as MRVs type 3 by PCR targeting the S1 gene. Representative strains denominated BatMRV1-191797/IT2011, BatMRV2-5515/3/IT2012 and BatMRV3-5515/2/IT2012, belonging to serotype 1, 2 and 3 respectively, were selected and fully sequenced. Bat-MRVs appeared genetically widely differentiated. Particularly, BatMRV3-5515/2/IT2012 showed high similarity with T3/Bat/Germany/342/08 detected in Germany and SL-MRV01 detected from a child with acute gastroenteritis in Slovenia. BatMRV1-IT2011 was a reassortant strain of serotype 1, never identified in bats before, with the S1 gene similar to T1/bovine/Maryland/Clone23/59 and C/bovine/Indiana/MRV00304/2014 and the other segments similar to MRVs of different host, origin and serotype. CONCLUSIONS This research provides evidence that insectivorous bats carry a wide variety of MRVs with members of the type 3 most represented. This finding extends the current knowledge on bat-MRVs, and stresses the importance to continue and improve the MRV surveillance in bats and other mammals even through the development and standardization of specific diagnostic tools.
A renewed interest in mammalian orthoreoviruses (MRVs) has emerged since new viruses related to bat MRV type 3, detected in Europe, were identified in humans and pigs with gastroenteritis. This study reports the isolation and characterization of a novel reassortant MRV from the lesser horseshoe bat (Rhinolophus hipposideros). The isolate, here designated BatMRV1-IT2011, was first identified by electron microscopy and confirmed using PCR and virus-neutralization tests. The full genome sequence was obtained by next-generation sequencing. Molecular and antigenic characterizations revealed that BatMRV1-IT2011 belonged to serotype 1, which had not previously been identified in bats. Phylogenetic and recombination detection program analyses suggested that BatMRV1-IT2011 was a reassortant strain containing an S1 genome segment similar to those of MRV T1/bovine/Maryland/Clone23/59 and C/bovine/ Indiana/MRV00304/2014, while other segments were more similar to MRVs of different hosts, origins and serotypes. The presence of neutralizing antibodies against MRVs has also been investigated in animals (dogs, pigs, bovines and horses). Preliminary results suggested that MRVs are widespread in animals and that infections containing multiple serotypes, including MRVs of serotype 1 with an S1 gene similar to BatMRV1-IT2011, are common. This paper extends the current knowledge of MRVs and stresses the importance to continue and improve MRV surveillance in bats and other mammals through the development and standardization of specific diagnostic tools.
Davide Lelli
added 4 research items
Publication Preview
GenBank Accession Numbers KU194659-KU194666 represent sequences from 8 segments of Mammalian orthoreovirus 3 T3/Pipistrellus_kuhlii/Italy/5515-2/2012. ##Assembly-Data-START## Assembly Method :: Lasergene v. 12 Sequencing Technology :: IonTorrent ##Assembly-Data-END##
Publication Preview
GenBank Accession Numbers KU194659-KU194666 represent sequences from 8 segments of Mammalian orthoreovirus 3 T3/Pipistrellus_kuhlii/Italy/5515-2/2012. ##Assembly-Data-START## Assembly Method :: Lasergene v. 12 Sequencing Technology :: IonTorrent ##Assembly-Data-END##
Publication Preview
GenBank Accession Numbers KU194667-KU194675 represent sequences from 9 segments of Mammalian orthoreovirus 3 T3/Pipistrellus_kuhlii/Italy/5515-3/2012. ##Assembly-Data-START## Assembly Method :: Lasergene v. 12 Sequencing Technology :: Illumina ##Assembly-Data-END##
Davide Lelli
added 2 research items
Background: Rhabdoviridae is one of the most ecologically diverse families of RNA viruses which can infect a wide range of vertebrates and invertebrates. Bats, among mammals, are pointed to harbor a significantly higher proportion of unknown or emerging viruses with zoonotic potential. Herein, we report the isolation of a novel rhabdovirus, detected in the framework of a virological survey on bats implemented in North Italy. Methods: Virus isolation and identification were performed on samples of 635 bats by using cell cultures, negative staining electron microscopy and PCRs for different viruses. NGS was commonly performed on cell culture supernatants showing cytopathic effect or in case of samples resulted positive by at least one of the PCRs included in the diagnostic protocol. Results: A rhabdovirus was isolated from different organs of a Pipistrellus kuhlii. Virus identification was obtained by electron microscopy and NGS sequencing. The complete genome size was 11,774 nt comprised 5 genes, encoding the canonical rhabdovirus structural proteins, and an additional transcriptional unit (U1) encoding a hypothetical small protein (157aa) (3'-N-P-M-G-U1-L-5'). The genome organization and phylogenetic analysis suggest that the new virus, named Vaprio virus (VAPV), belongs to the recently established genus Ledantevirus (subgroup B) and it is highly divergent to its closest known relative, Le Dantec virus (LDV) (human, 1965 Senegal). A specific RT-PCR amplifying a 350 bp fragment of the ORF 6 gene, encoding for L protein, was developed and used to test retrospectively a subset of 76 bats coming from the same area and period, revealing two more VAPV positive bats. Conclusions: VAPV is a novel isolate of chiropteran rhabdovirus. Genome organization and phylogenetic analyses demonstrated that VAPV should be considered a novel species within the genus Ledantevirus for which viral ecology and disease associations should be investigated.
Publication Preview
GenBank Accession Numbers KU194659-KU194666 represent sequences from 8 segments of Mammalian orthoreovirus 3 T3/Pipistrellus_kuhlii/Italy/5515-2/2012. ##Assembly-Data-START## Assembly Method :: Lasergene v. 12 Sequencing Technology :: IonTorrent ##Assembly-Data-END##
Davide Lelli
added a project goal
Bats are universally recognised as main reservoir for emerging and potentially zoonotic viruses. Poor eco-epidemiological data are available on the circulation of the most important zoonotic viruses in the Italian bat population. We will undertake passive and active surveillance in Italian bats mainly targeted on the detection of coronaviruses (CoVs), lyssaviruses (LYSVs) and orthoreoviruses (MRVs). The occurrence of selected emerging viral infections relevant for either bat conservation or public health will also be investigated. This project aims to cover both human health and biological conservation issues by enhancing survey for major viral infections in bats.