Identification and Characterisation of a Myxoma Virus Detected in the Italian Hare (Lepus corsicanus).

Myxoma virus (MYXV) is a Leporipoxvirus (genus) belonging to the family Poxviridae; it is characterised by a genome of approximately 161 kb dsDNA encoding for several proteins that play an essential role in both host spectrum determination and immunomodulation. The healthy reservoir of the virus is Sylvilagus spp. At the same time, in wild and domestic European rabbits (Oryctolagus cuniculus), MYXV is the etiologic agent of myxomatosis, a disease with an extremely high mortality rate. In 2014, an interspecies jump of MYXV was reported in Lepus europaeus in the UK. In 2018, myxomatosis induced by a new recombinant strain called MYXV-To was identified during a large outbreak in Iberian hares (Lepus granatensis) in Spain. Here, we describe the case of myxomatosis in another hare species: an adult male Italian hare (Lepus corsicanus) found dead in 2018 in Sicily with lesions suggestive of myxomatosis and treponema infection. Laboratory tests, e.g., end-point PCR and negative staining electron microscopy, confirmed the presence of both pathogens. MYXV was then isolated from tissue samples in permissive cells and sequenced using NGS technology. Main genomic differences concerning known MYXV strains are discussed.

Paslahepevirus balayani (Hepatitis E Virus) in Italian Nonungulate Wildlife: Molecular Detection and Characterization of an Isolate from a Crested Porcupine (Hystrix cristata).

Paslahepevirus balayani (hepatitis E virus [HEV]) is the causative agent of hepatitis E, a worldwide zoonosis involving a wide range of hosts among domestic and wild animals. This species is characterized by a great genomic heterogeneity and includes eight genotypes, HEV-1 to HEV-8. The HEV-3 genotype is one of the most common types circulating in Italy in humans and Suidae. Although domestic and wild Sus scrofa and deer (Cervidae) are recognized as the main reservoirs of HEV, several other wild species are potential carriers. A total of 228 liver samples were collected from nonungulate wild animals, found dead, in the framework of the regional passive surveillance program in Umbria and Marche regions (central Italy) during 2018-20. These were tested using real-time reverse-transcriptase PCR (RT-PCR) for detection of RNA of HEV-1 to HEV-4 and confirmed by nested RT-PCR assay. One of the 11 samples collected from crested porcupines (Hystrix cristata) tested positive for the presence of HEV RNA; all other samples were negative. Sequence analysis based on the full-length genome revealed that this isolate, 49434/UM/2018 (accession no. OL658617), belongs to the HEV-3e subtype. These findings suggest a potential role of crested porcupines as a carrier of HEV infection.

Comparative analysis of two genomes of Chlamydia pecorum isolates from an Alpine chamois and a water buffalo.

BACKGROUND: To date, whole genome sequencing has been performed mainly for isolates of Chlamydia trachomatis, C. pneumoniae, C. psittaci and C. abortus, but only a few isolates of C. pecorum have been entirely sequenced and this makes it difficult to understand its diversity and population structure. In this study the genome of two C. pecorum strains isolated from the lung of an Alpine chamois affected with pneumonia (isolate PV7855) and the brain of a water buffalo affected with meningoencephalomyelitis (isolate PV6959), were completely sequenced with MiSeq system (Illumina) and analyzed in their most polymorphic regions. RESULTS: The genome length and GC content of the two isolates were found to be consistent with other C. pecorum isolates and the gene content of polymorphic membrane proteins and plasticity zone was found to be very similar. Some differences were observed in the phospholipase genes for both isolates and in the number of genes in the plasticity zone, such as the presence of some hypothetical proteins in PV6959, not present in any other genomes analyzed in this study. Interestingly, PV6959 possesses an extra pmp and has an incomplete tryptophan biosynthesis operon. Plasmids were detected in both isolates. CONCLUSIONS: Genome sequencing of the two C. pecorum strains did not reveal differences in length and GC content despite the origin from different animal species with different clinical disease. In the plasticity zone, the differences in the genes pattern might be related to the onset of specific symptoms or infection of specific hosts. The absence of a tryptophan biosynthesis pathway in PV6959 may suggest a strict relationship between C. pecorum and its host.

Genomic Epidemiology and Heterogeneity of SRLV in Italy from 1998 to 2019.

Small ruminant lentiviruses (SRLV) are viruses that retro-transcribe RNA to DNA and show high rates of genetic variability. SRLV affect animals with strains specific for each host species (sheep or goats), resulting in a series of clinical manifestations depending on the virulence of the strain, the host's genetic background and farm production system. The aim of this work was to present an up-to-date overview of the genomic epidemiology and genetic diversity of SRLV in Italy over time (1998-2019). In this study, we investigated 219 SRLV samples collected from 17 different Italian regions in 178 geographically distinct herds by CEREL. Our genetic study was based on partial sequencing of the gag-pol gene (800 bp) and phylogenetic analysis. We identified new subtypes with high heterogeneity, new clusters and recombinant forms. The genetic diversity of Italian SRLV strains may have diagnostic and immunological implications that affect the performance of diagnostic tools. Therefore, it is extremely important to increase the control of genomic variants to improve the control measures.

Hepatitis E virus (HEV) genotype 3 diversity: Identification of a novel HEV subtype in wild boar in Central Italy.

In the last decade in Europe, the number of autochthonous cases of hepatitis E has significantly increased. Most of the cases arise from foodborne infections caused by the zoonotic hepatitis E virus (HEV) genotypes HEV-3 and HEV-4. Several human cases have been linked to consumption of raw or undercooked animal products of both pork (liver sausages) and wild boar meat. In this study, the occurrence of HEV infection was investigated in 611 livers and 88 paired lungs from wild boars collected during the hunting seasons of 2016-2020 in the Umbria-Marche Apennines (Central Italy). Using real-time reverse transcription polymerase chain reaction, 15 liver samples (2.45%) and one lung sample were found to be positive for HEV RNA. The phylogenetic tree built on the partial ORF2 gene revealed that the detected HEV strains belonged to HEV-3f (n = 5), HEV-3e (n = 1) and HEV-3c (n = 1) subtypes. Interestingly, 8 strains were genetically placed in a different cluster, further away from all other subtypes. To corroborate this finding, four complete genomes were obtained by next generation sequencing. The full genome of the HEV strains clustered together with another wild boar strain previously detected in Southern Italy in 2015 but the strains were divergent from all the HEV-3 strains classified in any subtype defined so far. Thus, these strains represent a novel subtype that might have originated in Italy, which we have tentatively named HEV-3n.

Near-complete nucleotide sequence of a border disease virus genotype BDV-7 isolate from Italy.

To gain further insight into the genomic features of border disease virus (BDV), we determined the nearly complete genome sequence of isolate TO/121/04 from an aborted ovine fetus. Its genome contains a single open reading frame (ORF), which comprises 11,681 nucleotides encoding a polyprotein of 3893 amino acids. Phylogenetic analysis of the near full-length genome sequence showed that the BDV isolate differed significantly from all ovine pestiviruses identified so far, thus re-affirming the presence in Italy of this novel genetic group, termed BDV-7.

Cross-Reactivity Antibody Response after Vaccination with Modified Live and Killed Bovine Viral Diarrhoea Virus (BVD) Vaccines.

Pestivirus A or bovine viral diarrhoea virus (BVDV) type 1 is responsible for cosmopolitan diseases affecting cattle and other ruminants, presenting a wide range of clinical manifestations, with relevant impact on zootechnic production. The objective of the present study was to verify whether animals immunised with four commercial vaccines also developed a protective humoral immunity against other viral subgenotypes than those contained in each vaccine. Four groups of 25 bovines each were formed and vaccinated according to the manufacturer's instructions of the commercial vaccines. On sera collected 28 days after the last vaccination, virus neutralisation tests (VNT) were performed using homologous and heterologous viruses and enzyme-linked immunosorbent assay (ELISA) methods. Finally, the VNT results were comparatively evaluated through a statistical analysis. Serological results highlighted that, although with a different degree of efficiency, the four vaccines resulted in not developing a solid antibody-mediated cross-immunity against all the strains used.

Phylogenetic analysis of small ruminant lentiviruses in Germany and Iran suggests their expansion with domestic sheep.

Small ruminant lentiviruses (SRLVs) are found in sheep in Germany and Iran. SRLVs have been classified into four genotypes: A-C and E. Genotype A has been subdivided into 20 subtypes. Previous studies suggested that, first, the ancestors of genotype A are those SRLVs found in Turkey, second, the evolution of SRLVs is related to the domestication process, and, third, SRLV infection was first observed in sheep in Iceland and the source of that infection was a flock imported from Germany. This study generated, for the first time, partial SRLV sequence data from German and Iranian sheep, enhancing our knowledge of the genetic and evolutionary relationships of SRLVs, and their associations with the domestication process. Based on 54 SRLV sequences from German and Iranian sheep, our results reveal: (1) SRLV subtypes A4, A5, A11, A16 and A21 (new) are found in German sheep and A22 (new) in Iranian sheep. (2) Genotype A has potentially an additional ancestor (A22), found in Iran, Lebanon and Jordan. (3) Subtype A22 is likely an old version of SRLVs. (4) The transmission routes of some SRLVs are compatible with domestication pathways. (5) This study found no evidence of Icelandic subtype A1 in German sheep.

Serological relationship between a novel ovine pestivirus and classical swine fever virus.

The genus Pestivirus comprises globally distributed members of the family Flaviviridae, which cause severe losses in livestock. The most common species of the genus are bovine viral diarrhoea virus type 1 (BVDV-1) and type 2 (BVDV-2), classical swine fever virus (CSFV) and border disease virus (BDV). Recently, a novel ovine pestivirus was repeatedly detected in aborted lamb foetuses on a farm located in the Brescia Province (Italy). Complete genome characterization of this isolate showed that it was highly divergent from known pestivirus species and that it was genetically closely related to CSFV. The aim of this study was to determine the serological relatedness between the identified novel pestivirus and BVDV, BDV and CSFV selected strains for which homologous serum was available, by antigenic characterization performed using cross-neutralization assays. The serological relatedness was expressed as the coefficient of antigenic similarity (R). Both field and specific antisera raised against the ovine pestivirus neutralized the CSFV reference strain Diepholz with titres significantly higher than those specific for the BDV and BVDV strains. Furthermore, the calculated R values clearly indicated that the novel ovine pestivirus is antigenically more related to CSFV than to ruminant pestiviruses, in agreement with the results of the genomic analysis. This would have severe consequences on CSFV serology in the event of a switch to porcine hosts with implications for CSFV surveillance and porcine health management.

Molecular characterization of Italian bovine leukemia virus isolates reveals the presence of distinct phylogenetic clusters.

Disease caused by bovine leukemia virus (BLV) results in significant economic losses to the livestock industry. To date, there is only one report describing the strains found in Italy. BLV strains (n = 24), collected between 2012 and 2016 from four different Italian regions, were genetically analyzed by direct sequencing of a portion of the BLV env gene, and the sequences were compared with those in the GenBank database. The Italian BLVs clustered into genotypes G2, G4, G6, G7, and G8, revealing a high level of BLV genetic heterogeneity in Italy. This study provides a basis for further investigations into the evolutionary relationship between BLV strains.

Complete genome sequences of a cytophatic/noncytophatic pair of bovine viral diarrhea virus subtype 1a viruses.

The complete genome sequences of both biotypes of a pair of bovine viral diarrhea viruses isolated from a bovid affected by mucosal disease were determined by next generation sequencing. The cytopathic virus possessed a 423-base insertion derived from bovine poly ubiquitin in the NS2/3 coding region and one nucleotide change. Both biotypes showed an additional glycosylation site in their N-terminus.

Complete nucleotide sequence of a novel bovine viral diarrhea virus subtype 1 isolate from Italy.

To gain further insight into the genomic features of bovine viral diarrhea virus 1 (BVDV-1) subtypes, we sequenced the complete genome of the BVDV-1 isolate VE/245/12. This is an uncommon subtype that was isolated from a persistently infected animal. Here, we report the complete genome sequence, consisting of 12,295 nucleotides (nt) with an open reading frame of 11,694 nt encoding 3,898 amino acids. Phylogenetic analysis of the full-length genome, 5'-UTR, and Npro region confirmed that the BVDV-1 isolate differed significantly from all of the bovine pestiviruses identified so far, providing evidence for the presence of a distinct novel genetic group.

Complete Genome Sequence of a Bovine Viral Diarrhea Virus Subgenotype 1g Strain Isolated in Italy.

We report here the full-length sequence of the bovine viral diarrhea virus (BVDV) strain UM/103/04, isolated from a persistently infected cow in Italy. It belongs to the subgenotype 1g, which is described as a sporadic subgenotype in Italy. This is the first report of a complete genome sequence of BVDV-1g.

Complete Genome Sequence of a Bovine Viral Diarrhea Virus Subgenotype 1h Strain Isolated in Italy.

We sequenced the complete genome of bovine viral diarrhea virus (BVDV) strain UM/126/07. It belongs to subgenotype 1h. The complete genome is composed of 12,196 nucleotides organized as one open reading frame encoding 3,898 amino acids. This is the first report of a full-length sequence of BVDV-1h.

Genetic characterization of border disease virus (BDV) isolates from small ruminants in Italy.

Border disease virus (BDV) belongs to the Pestivirus genus in the family Flaviviridae. Genetic analyses of pestiviruses that have been isolated from sheep and goat have led to the proposal that BDV isolates can be phylogenetically segregated into at least seven clusters, subtypes BDV-1 to BDV-7. In order to investigate the genetic heterogeneity of small ruminant pestivirus isolates in Italy, a selection of 5'-UTR sequences from isolates that were collected from clinical specimens between 2002 and 2014 was analysed. Phylogenetic reconstructions indicated that the BDV-positive samples clustered within the BDV-1, BDV-3, BDV-5, and BDV-7 groups. These results suggested high genetic diversity within the Italian BDV field isolates. The phylogenetic analysis indicated the first evidence of BDV-1 and BDV-5 circulation in Italy. The marked diversity of the pestivirus isolates might reflect the sheep trade with foreign countries.

Increased genetic diversity of BVDV-1: recent findings and implications thereof.

Sequence-based genotyping was recently used to distinguish between the BVDV-1 and BVDV-2 species of the bovine viral diarrhoea virus (BVDV). Quite recently, a new putative species, BVDV-3, was also detected. The phylogenetic analysis of the 5'-untranslated region (UTR) and Npro region has revealed at least 17 distinct subtypes for BVDV-1 to date. The aim of this study was to further investigate the genetic heterogeneity of BVDV-1 in Italy, by analysing 173 virus sequences from isolates collected over an 18-year period (1997-2014). Viral RNA was extracted from the original biological samples identified as BVDV-1-positive. Reverse transcription (RT) and polymerase chain reaction (PCR) assays targeting a 288-base pair (bp) region of the 5'-UTR and a 428-bp region encoding the autoprotease Npro were performed, and the RT-PCR products were sequenced. The phylogenetic analysis of the 5'-UTR and Npro sequences re-confirmed the circulation of ten out of eleven subtypes previously discovered in Italy. Interestingly, four isolates differed significantly from all of the bovine pestiviruses identified so far, thereby providing evidence for the circulation of three novel subtypes that have not been documented so far. The growing number of reports on BVDV-1 heterogeneity, including the recent findings reported herein, raises concern related to the emergence and spread of new BVDV variants, with possible implications for animal health and disease control. This global issue needs to be addressed with the highest priority.

Activated autologous T cells exert an anti-B-cell chronic lymphatic leukemia effect in vitro and in vivo.

BACKGROUND AIMS: The impact of chronic lymphatic leukemia (CLL) tumor burden on the autologous immune system has already been demonstrated. This study attempted to elucidate the molecular mechanisms underlying T-cell immunologic deficiencies in CLL. METHODS: Freshly isolated CD3(+) T cells from patients with a diagnosis of CLL and healthy donors were analyzed by gene expression profiling. Activated T cells from 20 patients with CLL were tested in vitro for cytotoxicity against mutated and unmutated autologous B cells and DAUDI, K562 and P815 cell lines. To investigate T-cell mediated cytotoxicity in vivo, we co-transplanted OKT3-activated T lymphocytes and autologous B-cell CLL (B-CLL) cells into NOD/SCID mice. RESULTS: Gene expression profiles of peripheral blood T cells from B-CLL patients showed 25 down-regulated, and 31 up-regulated, genes that were mainly involved in cell differentiation, proliferation, survival, apoptosis, cytoskeleton formation, vesicle trafficking and T-cell activation. After culture, the T-cell count remained unchanged, CD8 cells expanded more than CD4 and a cytotoxicity index >30% was present in 5/20 patients. Cytotoxicity against B autologous leukemic cells did not correlate with B-cell mutational status. Only activated T cells exerting cytotoxicity against autologous leukemic B cells prevented CLL in a human-mouse chimera. CONCLUSIONS: This study indicates that patients with CLL are affected by a partial immunologic defect that might be somewhat susceptible to repair. This study identifies the molecular pathways underlying T-cell deficiencies in CLL and shows that cytotoxic T-cell functions against autologous B-CLL can be rebuilt at least in part in vitro and in vivo.