Efficient isolation on Vero.DogSLAMtag cells and full genome characterization of Dolphin Morbillivirus (DMV) by next generation sequencing

The Dolphin Morbillivirus (DMV) genome from the frst Mediterranean epidemic (1990-\u201992) is the only cetacean Morbillivirus that has been completely sequenced. Here, we report the frst application of next generation sequencing (NGS) to morbillivirus infection of aquatic mammals. A viral isolate, representative of the 2006-\u201908 Mediterranean epidemic (DMV_IZSPLV_2008), efciently grew on Vero.DogSLAMtag cells and was submitted to whole genome characterization by NGS. The fnal genome length was 15,673 nucleotides, covering 99.82% of the DMV reference genome. Comparison of DMV_IZSPLV_2008 and 1990-\u201992 DMV strain sequences revealed 157 nucleotide mutations and 47 amino acid changes. The sequence similarity was 98.7% at the full genome level. Whole-genome phylogeny suggested that the DMV strain circulating during the 2006-\u201908 epidemics emerged from the 1990-\u201992 DMV strain. Viral isolation is considered the \u201cgold standard\u201d for morbillivirus diagnostics but efcient propagation of infectious virus is difcult to achieve. The successful cell replication of this strain allowed performing NGS directly from the viral RNA, without prior PCR amplifcation. We therefore provide to the scientifc community a second DMV genome, representative of another major outbreak. Interestingly, genome comparison revealed that the neglected L gene encompasses 74% of the genetic diversity and might serve as \u201chypervariable\u201d target for strain characterization

Development and validation of an indirect ELISA as a confirmatory test for surveillance of infectious bovine rhinotracheitis in vaccinated herds

BACKGROUND: Bovine herpesvirus 1 (BoHV1) is a member of the viral subfamily of Alphaherpesvirinae that infects various species, including cattle, sheep, and goats. The virus causes infectious bovine rhinotracheitis (IBR), which is included in a European list of diseases that may require control and eradication programs. The lack of confirmatory tests affects the validity of diagnostic tools, especially those used for vaccinated herds. In this study, we report the development and validation of an indirect enzyme-linked immunosorbent assay (ELISA) based on BoHV1 glycoprotein E, which was expressed as a secreted recombinant antigen in a mammalian cell system. The performance of the new rec-gE ELISA was compared with that of commercially available indirect and/or blocking ELISAs. RESULTS: The sample set included blood sera from animals from IBR-positive farms, IBR-free farms, and marker-vaccinated farms. The indirect ELISA proposed in this study is based on antibody reactivity against BoHV1 gE, and showed high sensitivity and specificity (98.41 and 99.76 %, respectively). CONCLUSIONS: The ELISA performed well, in terms of both its diagnostic sensitivity and specificity, and as a confirmatory methodology, and therefore should improve the diagnostic protocols used for IBR surveillance

Isolation and molecular characterisation of Halicephalobus gingivalis in the brain of a horse in Piedmont, Italy

Background: A fatal case of meningoencephalitis was reported in a 13-year-old Koninklijk Warmbloed Paard Nederland stallion, suspected of West Nile virus (WNV) infection, in the Piedmont region of Italy. Clinical signs included right head tilt and circling, depression alternated with excitability, fever and lateral strabismus. Combined treatment consisting of dimethylsulfoxide, dexamethasone, sulphonamides and sedative was administered, but because of the poor conditions the horse was euthanatized and submitted for necropsy. Results: At post-mortem examination no skin lesions were observed, all organs appeared normal on gross evaluation and only head and blood samples were further investigated. Neuropathological findings consisted of granulomatous meningoencephalitis and larvae and adult females of Halicephalobus gingivalis were isolated and identified from the digested brain. Frozen brain was submitted to PCR amplification and 220 bp multiple sequence alignment was analysed by Bayesian phylogenetic analysis. Conclusions: Phylogenetic inference revealed that the isolate belongs to H. gingivalis Lineage 3. WN surveillance can help to deepen our knowledge of horse neurological disorders investigating their causes and incidence. Moreover, it can help to understand the geographic distribution of the H. gingivalis, to unravel epidemiological information, and to estimate risk for humans

Mosquito-Borne Diseases and ‘One Health’: The Northwestern Italian Experience

In Italy, the surveillance of Mosquito-Borne Diseases (MBDs) is regulated by two national preparedness plans: (1) for West Nile and Usutu viruses, integrating human and veterinary surveillance in order to early detect viruses circulation and to quickly apply control measures aimed at reducing the risk of transmission through blood and blood components and (2) for Arbovirosis transmitted by Aedes mosquitoes, mainly Chikungunya, Dengue and Zika viruses, based on surveillance of both imported and autochthonous human cases. This chapter reports the results of the application of these National Plans in Northwestern Italy and their impact for human health. In detail, we present the coordinated activities enforced in Piemonte and Liguria Regions, as a good example of the ‘One Health approach’ to control MBDs and prevent human transmission

Specific capture and whole‑genome phylogeography of Dolphin morbillivirus

Dolphin morbillivirus (DMV) is considered an emerging threat having caused several epidemics worldwide. Only few DMV genomes are publicly available. Here, we report the use of target enrichment directly from cetacean tissues to obtain novel DMV genome sequences, with sequence comparison and phylodynamic analysis. RNA from 15 tissue samples of cetaceans stranded along the Italian and French coasts (2008–2017) was purified and processed using custom probes (by bait hybridization) for target enrichment and sequenced on Illumina MiSeq. Data were mapped against the reference genome, and the novel sequences were aligned to the available genome sequences. The alignment was then used for phylogenetic and phylogeographic analysis using MrBayes and BEAST. We herein report that target enrichment by specific capture may be a successful strategy for whole-genome sequencing of DMV directly from field samples. By this strategy, 14 complete and one partially complete genomes were obtained, with reads mapping to the virus up to 98% and coverage up to 7800X. The phylogenetic tree well discriminated the Mediterranean and the NE-Atlantic strains, circulating in the Mediterranean Sea and causing two different epidemics (2008–2015 and 2014–2017, respectively), with a limited time overlap of the two strains, sharing a common ancestor approximately in 1998

A Qualitative PCR Assay for the Discrimination of Bubaline Herpesvirus 1, Bovine Herpesvirus 1 and Bovine Herpesvirus 5

Bubaline herpesvirus 1 (BuHV-1), Bovine herpesvirus 1 (BoHV-1) and Bovine herpesvirus 5 (BoHV-5) are classified in the genus Varicellovirus, subfamily Alphaherpesvirinae. BoHV-1 is the causative agent of infectious bovine rhinotracheitis, BoHV-5 induces moderate disease in adult cattle while BuHV-1 has instead been associated with a decline in livestock production of water buffaloes. The aim of this study was to develop a qualitative PCR assay that allows the discrimination of BuHV-1, BoHV-1 and BoHV-5. The alignment of homologous genes identified specific nucleotide sequences of BuHV- 1, BoHV-1 and BoHV-5. The design of the primers and the optimization of the PCR assay were focused on the target sequences located on the portions of gD, gE and gG genes. This assay involved the use of three different PCR end-points: the PCR of a portion of the gD gene identified only the presence of BoHV-1; the PCR of a portion of the gE gene confirmed the presence of both BoHV-5 and BuHV-1; the PCR of a portion of the gG gene discriminated between BoHV-5 and BuHV-1, as the amplification product was observed only for BoHV-5. This qualitative PCR assay allowed the differentiation of BoHV-1 and BoHV-5 infections both in cattle and water buffaloes and heterologous BuHV-1 infections in bovine