SPATIAL AND TEMPORAL DYNAMICS OF PESTIVIRUS, CRIMEAN-CONGO HEMORRHAGIC FEVER AND HEPATITIS E VIRUS: A PHYLOGENETIC APPROACH

The general aim of the PhD project was to apply phylogenetic analysis to viral sequences obtained in different geographic areas at different times, in order to reconstruct the most probable places of origin and pathways of dispersion of infections. Viral population growth and evolution leave a measurable imprint on the genome of viruses over the course of years, months or even days and occur simultaneously with geographic dispersal (Holmes, 2008; Pybus & Rambaut, 2009). This interaction characterizes a spatial phylodynamic process that can be recovered from genomic data using phylogeographic analyses (Faria et al., 2011). The research activity has been focused on Pestivirus genus, that includes pathogens of livestock (Bovine viral diarrhea virus - BDV) and wildlife (Border disease virus - BDV), and on zoonotic emerging diseases, involving in their epidemiological cycle both livestock and wildlife (Crimean-Congo hemorrhagic fever - CCHF, Hepatitis E virus- HEV). Concerning BDV, since 2001 several outbreaks of disease have been reported in Pyrenean chamois in Spain, France and Andorra. These outbreaks have decimated several Pyrenean chamois populations, with mortalities ranging from 40% to 85%. The infection has become endemic in the Central and Eastern Pyrenees. The aim of this study was to clarify the origin and dispersion of the Pyrenean chamois BDV genetic variant by reconstructing the spatial and temporal dynamics of BDV 5\u2019 UTR sequences of Pyrenean chamois, 10 novel sequences and 41 retrieved from public databases and Sheep BDV sequences (n=44) from Spain and France were also retrieved. The phylogenetic analysis was performed using a Bayesian Markov chain Monte Carlo (MCMC) method implemented in the BEAST v.1.74 package. The chamois clade originated from sheep BDV genotype 4, generating a founder effect due to intra-species spread and spatial dispersion. The time of the most recent common ancestor estimates for the chamois clade dated back to a time span between 1974 and 1996, with a mean estimation falling in 1988. The pathway of dispersion of isolates suggests a complex exchange between neighboring Pyrenean chamois populations, still going on such as Western direction. Genetic typing of bovine viral diarrhea virus (BVDV) has distinguished BVDV-1 and BVDV-2 species and an emerging putative third species (HoBi-like virus), recently detected in southern Italy, signaling the occurrence of natural infection in Europe. Recognizing the need to update the data on BVDV genetic variability in Italy for mounting local and European alerts, a wide collection of 5\u2019 UTR sequences (n = 371) was selected to identify the frequency of genotypes and subtypes at the herd level. BVDV-1 had the highest frequency, followed by sporadic BVDV-2. No novel HoBi-like viruses were identified. Four distribution patterns of BVDV-1 subtypes were observed: highly prevalent subtypes with a wide temporal-spatial distribution (1b and 1e), low prevalent subtypes with a widespread geographic distribution (1a, 1d, 1g, 1h, and 1k) or a restricted geographic distribution (1f), and sporadic subtypes detected only in single herds (1c, 1j, and 1l). BVDV-1c, k, and l are reported for the first time in Italy. Italy is one of the countries with the highest genetic diversity of BVDV worldwide. Northern Italy ranked first for BVDV introduction, prevalence, and dispersion. Nevertheless, the presence of sporadic variants in other restricted areas suggests the risk of different routes of BVDV introduction. CCHF is a zoonosis mainly transmitted by ticks that causes sporadic cases and severe hemorrhagic fever of acute human disease with a mortality rate of 5-60% and it has recently emerged in the Balkans and eastern Mediterranean areas. In order to reconstruct the origin and pathway of the worldwide dispersion of the virus at global and regional (eastern European) level, we investigated the phylogeography of the infection by analysing 121 publicly available CCHFV S gene sequences including two recently characterised Albanian isolates. The spatial and temporal phylogeny was reconstructed using a Bayesian Markov chain Monte Carlo approach. CCHFV phylogeographic reconstruction suggests that the disease originated about one thousand years ago from a common ancestor probably located in Africa. The virus then spread to Asia in the XV century and entered Europe on at least two occasions: the first in the early 1800s and the second in the early 1900s. The most probable location for the origin of the European clade was Russia, but Turkey played a central role in spreading the virus throughout Europe. Our data suggest that the movement of wild and domestic ungulates from endemic areas probably represent the main cause of virus dissemination in Eastern Europe. Hepatitis E virus is classified into four genotypes that have different geographical and host distributions. The main cause of sporadic autochthonous type E acute hepatitis in developed countries is genotype 3, which has a worldwide distribution and widely infects pigs. The aim of this study was to make hypotheses concerning the origin and global dispersion routes of this genotype by reconstructing the spatial and temporal dynamics of 208 HEV genotype 3 ORF-2 sequences (retrieved from public databases) isolated in different geographical areas. The evolutionary rates, time of the most recent common ancestors (tMRCAs), epidemic growth and phylogeography of HEV-3 were co-estimated using a MCMC Bayesian method. On the basis of time-scaled phylogeographical reconstruction, we hypothesise that HEV-3, after originating in the early 1800s in Europe, reached Asia in the first decades of 1900, and then moved to America probably in the 1970s-1980s. Analysis of the skyline plot showed a sharp increase of the number of infections between the 1980s and 2005, suggesting the intervention of new and highly efficient routes of transmission, possibly related to changes in the pig industry

Host range of mammalian orthoreovirus type 3 widening to alpine chamois

Mammalian orthoreoviruses (MRV) type 3 have been recently identified in human and several animal hosts, highlighting the apparent lack of species barriers. Here we report the identification and genetic characterization of MRVs strains in alpine chamois, one of the most abundant wild ungulate in the Alps. Serological survey was also performed by MRV neutralization test in chamois population during five consecutive years (2008-2012). Three novel MRVs were isolated on cell culture from chamois lung tissues. No respiratory or other clinical symptoms neither lung macroscopic lesions were observed in the chamois population. MRV strains were classified as MRV-3 within the lineage III, based on S1 phylogeny, and were closely related to Italian strains identified in dog, bat and diarrheic pig. The full genome sequence was obtained by next-generation sequencing and phylogenetic analyses showed that other segments were more similar to MRVs of different geographic locations, serotypes and hosts, including human, highlighting genome reassortment and lack of host specific barriers. By using serum neutralization test, a high prevalence of MRV-3 antibodies was observed in chamois population throughout the monitored period, showing an endemic level of infection and suggesting a self-maintenance of MRV and/or a continuous spill-over of infection from other animal species

BVDV permissiveness and lack of expression of co-stimulatory molecules on PBMCs from calves pre-infected with BVDV

Bovine viral diarrhea virus (BVDV) has been detected in peripheral blood mononuclear cells (PBMCs) of immunocompetent animals, not being clear whether the development of a specific humoral immune response can prevent BVDV infection. The aim of this study was to evaluate the ability of non-cytopathic BVDV to replicate and produce infectious virus in PBMCs from calves pre-infected with BVDV and to elucidate the immunomodulatory effect of BVDV on these cells in an in vitro model. Quantification of virus was by quantitative PCR, while its replicative capacity and shedding into the extracellular environment was evaluated by viral titration. Apoptosis was assessed by flow cytometry analysis of annexin V and propidium iodide, and by expression of caspase-3/7. Flow cytometry was used to analyze the expression of CD14/CD11b/CD80, CD4/CD8/CD25, MHC-I/MHC-II and B-B2 markers. Our results showed that PBMCs from cattle naturally infected with BVDV were more susceptible to in vitro BVDV infection and showed a more severe apoptosis response than those from na\uefve animals. Non-cytopathic BVDV in vitro infection also resulted in a lack of effect in the expression of antigen presentation surface markers. All these findings could be related to the immunosuppressive capacity of BVDV and the susceptibility of cattle to this infection

Are tree squirrels involved in the circulation of flaviviruses in Italy?

West Nile virus (WNV), Usutu virus (USUV) and tick-borne encephalitis virus (TBEV) are emerging zoonotic flaviviruses (family Flaviviridae), which have circulated in Europe in the past decade. A cross-sectional study was conducted to assess exposure to these antigenically related flaviviruses in eastern grey squirrels (Sciurus carolinensis) in Italy. Seventeen out of 158 (10.8%; CI95%: 5.9-15.6) squirrels' sera tested through bELISA had antibodies against flaviviruses. Specific neutralizing antibodies to WNV, USUV and TBEV were detected by virus neutralization tests. Our results indicate that tree squirrels are exposed to Culex and tick-borne zoonotic flaviviruses in Italy. Moreover, this study shows for the first time USUV and TBEV exposure in grey squirrels, broadening the host range reported for these viruses. Even though further studies are needed to define the real role of tree squirrels in the epidemiology of flaviviruses in Europe, this study highlights that serology could be an effective approach for future investigations aimed at broadening our knowledge about the species exposed to these zoonotic infections

Renal cell carcinoma incidence rates and trends in young adults aged 20-39 years

Background: The burden of renal cell carcinoma (RCC) in young adults received marginal attention. We assessed contemporary gender, race and stage-specific incidence and trends of RCC among young adults (20-39 years-old) in the United States.Methods: Within Surveillance, Epidemiology, and End Results database (2000-2016), patients aged 20-39 years with histologically confirmed RCC were included. Age-standardized incidence rates (ASR per 100,000 person-years) were estimated. Temporal trends were calculated through joinpoint regression analyses to describe the average annual percent change (AAPC).Results: From 2000-2016, 7767 new RCC cases were recorded (ASR 0.6, AAPC + 5.0 %, p < 0.001). ASRs were higher in males than in females (0.7 and 0.5, respectively) and increased significantly in both genders (AAPC + 5.0 % and + 4.7 % both p < 0.001, respectively). Non-Hispanic American Indian/Alaska Native had the highest incidence (ASR 1.0) vs. non-Hispanic Asian or Pacific Islander the lowest (ASR 0.3). ASRs significantly increased in all ethnic groups. T1aNOMO and T1bNOMO stages showed the highest incidence and increase (ASR 0.3, AAPC + 5.9 %, p < 0.001 and ASR 0.1, AAPC + 5.7 %, p < 0.001, respectively). Also regional and distant stages increased (AAPC + 3.7 %, p = 0.001 and AAPC + 1.5 %, p = 0.06). The most frequent tumor characteristics were G2 (44.4 %, ASR 0.3, AAPC + 6.3 %, p < 0.001) and G1 (13.1 %, ASR 0.1, AAPC + 1.1 %, p = 0.2), as well as clear cell histology (54.8 %, ASR 0.3, AAPC + 7.6 %, p < 0.001).Conclusions: RCC in young adults is rare, but increasing. This is mainly due to T1aN0M0 tumors. Nonetheless, also regional diseases are significantly increasing. Differences between ethnic groups exist and may warrant further research

Detection and genetic characterization of domestic cat hepadnavirus in cats with cavitary effusions

: After the identification of the novel domestic cat hepadnavirus (DCH) in 2018, its potential pathogenetic role in feline hepatic diseases has been suggested. Following the detection of DCH in a cat's serum and peritoneal effusion, the aim of this study was to retrospectively investigate the presence of DCH in cats with and without cavitary effusions along with DCH presence in effusions. Stored serum and effusion samples from cats with and without effusions admitted to the Veterinary Teaching Hospital of Lodi (Italy) in 2020-2022 were included based on results of hematobiochemical parameters. Effusions were classified based on cytological and physicochemical findings. The likelihood of liver damage was estimated based on clinical and laboratory findings. Samples were tested for DCH presence by quantitative PCR (qPCR). Positive samples were subjected to whole genome sequencing and phylogenetic analysis. DCH was detected in both serum and peritoneal effusion samples of 2/72 (2.8%) enrolled cats, included in the group with effusions (2/33; 6.1%), with one cat showing inflammatory and the other non-inflammatory effusion. Both DCH-positive cats belonged to the group with a likelihood of liver damage (2/22, 9.1%). Phylogeny showed that the DCH sequences from this study clustered with the prototypic Australian strain but were not included in the clade with other Italian DCH sequences. Results suggest the circulation of different DCH variants in Italy and show the presence of DCH in effusion samples from DCH-positive cats, mirroring the presence of HBV in body fluids from HBV-infected humans. Further studies are still recommended to define the pathogenic role of DCH in cats

Metabolic syndrome predicts worse perioperative outcomes in patients treated with partial nephrectomy for renal cell carcinoma

OBJECTIVE: To test the association between metabolic syndrome (MetS) and its components (high blood pressure, body mass index [BMI] 65 30, altered fasting glucose, low high-density lipoprotein cholesterol and high triglycerides) on perioperative outcomes after partial nephrectomy (PN). METHODS: Within the National Inpatient Sample database (2000-2015) we identified all PN patients. First, temporal trends of MetS were reported. Second, the effect of MetS components was tested in multivariable logistic regression models predicting overall and specific perioperative complications. Third, we tested for dose-response from the concomitant effect of multiple MetS components. All models were weighted and adjusted for clustering, as well as all available patient and hospital characteristics. RESULTS: Of 25,875 patients: (1) 59.3% had high blood pressure, (2) 14.7% had BMI 65 30, (3) 21.7% had altered fasting glucose, (4) 20.2% had high triglycerides, and (5) <0.01% had low high-density lipoprotein cholesterol. One vs 2 vs 3 vs 4 MetS components were recorded in 34.9% vs 22.9% vs 8.9% vs 2.2% patients. Of all, 11.1% exhibited 65 3 components and qualified for MetS. The rates of MetS increased over time (estimated annual percentage changes: +12.0%;P <.001). The 4 tested MetS components (high blood pressure, BMI 65 30, altered fasting glucose, and high triglycerides) achieved independent predictor status in multivariable models predicting overall, cardiac, miscellaneous medical, vascular, and respiratory complications, as well as transfusions. Moreover, a statistically significant dose-response was confirmed for the same endpoints. CONCLUSION: MetS and its components consistently and strongly predict perioperative complications after PN. Moreover, the strength of the effect was directly proportional to the number of MetS components exhibited by each individual patient, even if formal MetS diagnosis of 65 3 components has not been m

Highlighting priority areas for bovine viral diarrhea control in Italy : a phylogeographic approach

The prevalence and genetic diversity of bovine viral diarrhea virus (BVDV) in a geographic area are largely influenced by live animal trade and management practices. Despite control and eradication programs currently underway in several European countries, the risk of BVDV spread within and among countries is still present. BVDV-1 is the predominant type circulating in European cattle population. In this study, a phylogeographic analysis was applied to the BVDV-1 highest prevalent subtypes in Italy to reconstruct the origin and spatial-temporal distribution and to trace main viral flows between different locations to highlight priority areas for BVDV control. A comprehensive dataset of BVDV-1b (n\u202f=\u202f173) and 1e (n\u202f=\u202f172) 5' UTR sequences was analysed, including both novel and published sequences from Italy and from European countries bordering and/or with commercial cattle flows with Italy. A common phylogeographic pattern was observed for BVDV-1b and 1e subtypes: interspersion from multiple Italian areas and European countries was widespread until the end of the last century, whereas significant local clusters were observed starting from 2000. These findings support a continuous viral flow among different areas over long time scales with no evidence of significant geographical structure, while local transmission networks are limited to more recent years. Northern Italy has been confirmed as the area of origin of the main clades of both BVDV subtypes at national level, acting both as a crucial area for introduction and a maintenance source for other areas. Piedmont, Central and Southern Italian regions contributed to limited geographical distribution and local BVDV-1b and 1e persistence. On the whole, priority control measures for BVDV-1b and 1e in Italy should be focused on: i) implementation of BVDV systematic control in all Northern Italian regions to break the viral flow from larger to smaller animal populations; and ii) breaking the dynamics of infections in regions with self-maintenance of BVDV by voluntary control programs

PTX3 is up-regulated in epithelial mammary cells during S. aureus intramammary infection in goat

Pentraxins are a superfamily of conserved molecules with immune functions such as complement activation and opsonization. PTX3 is the prototypic long pentraxin and is produced by different cell populations after pro-inflammatory stimuli. Different studies have demonstrated the up-regulation of PTX3 during ruminant mastitis, but its role is still unknown. The aim of this study was to elucidate the role of PTX3 in the immune response to S. aureus intra-mammary infection (IMI). Given that no data are available on PTX3 expression in goat tissues, we first studied its pattern of expression  in goat normal tissues. Then we investigated the role of PTX3 during mammary infection, comparing its expression in healthy and infected blood, milk and tissues. Six healthy goats were infused with PBS in the right udder and with S. aureus in the left udder. Mammary biopsies from udders were collected 30h post infection, formalin fixed and routinely processed for microscopic evaluation or immediately stored in RNAlater. Tissue samples were collected at the slaughterhouse from healthy goats and were immediately stored in RNAlater. Blood and milk were collected from healthy and infected goats; cells from blood and milk were isolated and processed for RNA extraction or for cytospins; milk fat globules were obtained through milk centrifugation and immediately processed for RNA extraction. Total RNA from different organs, blood or milk cells, milk fat globules and mammary tissues was extracted and used as template in qPCR for PTX3. PTX3 expression was investigated by immunohistochemistry on formalin fixed paraffin embedded mammary tissue samples and on cytospins of isolated goat blood and milk cells. PTX3 mRNA was expressed with very high levels in bone marrow, mammary gland, aorta, pancreas, skin and lung. Given the high expression in the mammary gland, we investigated which cell population expressed PTX3. PTX3 was mainly expressed in the apical cytoplasmic portion of mammary gland epithelial cells, and in macrophages. During S. aureus infection PTX3 was up-regulated by epithelial cells. Macrophages and mammary secretum didn’t show PTX3 modulation, but PMNs recruited during infection were variably intensely positive. PTX3 mRNA expression was low in healthy organs and tissues of goats as has been reported indeed the molecules commonly induced after pro-inflammatory stimulation. As expected, PTX3 was constitutively expressed in bone marrow, rich in PMNs and monocytes, in aorta covered by endothelium and in the skin. PTX3 was up-regulated in epithelial mammary cells and in milk cells after S. aureus infection, demonstrating that it represents a first line of immune defense in goat udder. No modulation was observed in macrophages, in the secretum and in the ductal epithelial cells. Further experiments are needed to elucidate the role of PTX3 in the pathogenesis of S. aureus infection