Transmission dynamics of lyssavirus in Myotis myotis: mechanistic modelling study based on longitudinal seroprevalence data.

We investigated the transmission dynamics of lyssavirus in Myotis myotis and Myotis blythii, using serological, virological, demographic and ecological data collected between 2015 and 2022 from two maternity colonies in northern Italian churches. Despite no lyssavirus detection in 556 bats sampled over 11 events by reverse transcription-polymerase chain reaction (RT-PCR), 36.3% of 837 bats sampled over 27 events showed neutralizing antibodies to European bat lyssavirus 1, with a significant increase in summers. By fitting sets of mechanistic models to seroprevalence data, we investigated factors that influenced lyssavirus transmission within and between years. Five models were selected as a group of final models: in one model, a proportion of exposed bats (median model estimate: 5.8%) became infectious and died while the other exposed bats recovered with immunity without becoming infectious; in the other four models, all exposed bats became infectious and recovered with immunity. The final models supported that the two colonies experienced seasonal outbreaks driven by: (i) immunity loss particularly during hibernation, (ii) density-dependent transmission, and (iii) a high transmission rate after synchronous birthing. These findings highlight the importance of understanding ecological factors, including colony size and synchronous birthing timing, and potential infection heterogeneities to enable more robust assessments of lyssavirus spillover risk.

One health surveillance strategy for coronaviruses in Italian wildlife.

The recent reinforcement of CoV surveillance in animals fuelled by the COVID-19 pandemic provided increasing evidence that mammals other than bats might hide further diversity and play critical roles in human infectious diseases. This work describes the results of a two-year survey carried out in Italy with the double objective of uncovering CoV diversity associated with wildlife and of excluding the establishment of a reservoir for SARS-CoV-2 in particularly susceptible or exposed species. The survey targeted hosts from five different orders and was harmonised across the country in terms of sample size, target tissues, and molecular test. Results showed the circulation of 8 CoV species in 13 hosts out of the 42 screened. Coronaviruses were either typical of the host species/genus or normally associated with their domestic counterpart. Two novel viruses likely belonging to a novel CoV genus were found in mustelids. All samples were negative for SARS-CoV-2, with minimum detectable prevalence ranging between 0.49% and 4.78% in the 13 species reaching our threshold sample size of 59 individuals. Considering that within-species transmission in white-tailed deer resulted in raising the prevalence from 5% to 81% within a few months, this result would exclude a sustained cycle after spillback in the tested species.

Main causes of death of free-ranging bats in Turin province (North-Western Italy): gross and histological findings and emergent virus surveillance.

BACKGROUND: Bats are recognized as reservoir species for multiple viruses. However, little is known on bats' health and mortality. Thus, this study aimed to investigate the main causes of death of bats from Turin province (North-western Italy) and to describe gross and histopathological lesions potentially associated with the presence of selected bat viruses. RESULTS: A total of 71 bats belonging to 9 different species of the families Vespertilionidae and Molossidae were necropsied and samples of the main organs were submitted to histopathological examination. Also, aliquots of the small intestine, liver, spleen, lung, and brain were collected and submitted to biomolecular investigation for the identification of Coronaviridae, Poxviridae, Reoviridae (Mammalian orthoreovirus species), Rhabdoviridae (Vaprio ledantevirus and Lyssavirus species) and Kobuvirus. The majority of bats died from traumatic lesions due to unknown trauma or predation (n = 40/71, 56.3%), followed by emaciation (n = 13/71,18.3%). The main observed gross lesions were patagium and skin lesions (n = 23/71, 32.4%), forelimbs fractures (n = 15/71, 21.1%) and gastric distension (n = 10/71,14.1%). Histologically, the main lesions consisted of lymphoplasmacytic pneumonia (n = 24/71, 33.8%), skin/patagium dermatitis (n = 23/71, 32.4%), liver steatosis and hepatitis (n = 12, 16.9%), and white pulp depletion in the spleen (n = 7/71, 9.8%). Regarding emergent bat viruses, only poxvirus (n = 2, 2.8%) and orthoreovirus (n = 12/71, 16.9%) were detected in a low percentage of bats. CONCLUSIONS: Trauma is the main lesion observed in bats collected in Turin province (North-western Italy) associated with forelimb fractures and the detected viral positivity rate seems to suggest that they did not represent a threat for human health.

Isolation of a novel Rhabdovirus from an insectivorous bat (Pipistrellus kuhlii) in Italy.

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.

First identification of mammalian orthoreovirus type 3 in diarrheic pigs in Europe.

Mammalian Orthoreoviruses 3 (MRV3) have been described in diarrheic pigs from USA and Asia. We firstly detected MRV3 in Europe (Italy) in piglets showing severe diarrhea associated with Porcine Epidemic Diarrhea. The virus was phylogenetically related to European reoviruses of human and bat origin and to US and Chinese pig MRV3.

The close genetic relationship of lineage D Betacoronavirus from Nigerian and Kenyan straw-colored fruit bats (Eidolon helvum) is consistent with the existence of a single epidemiological unit across sub-Saharan Africa.

Straw-colored fruit bats (Eidolon helvum), which have been identified as natural hosts for several zoonotic pathogens, such as lyssaviruses, henipaviruses, and ebolavirus, are associated with human settlements in Nigeria where they are commonly consumed as a delicacy. However, information on the viruses harbored by these bats is scarce. In this study, coronavirus sequences were detected using a nested RT-PCR targeting 440 bp of the RNA-dependent RNA polymerase (RdRp) in six of 79 fecal samples collected from an urban colony of E. helvum in Ibadan, Nigeria. Phylogenetic analysis revealed that all six sequences were monophyletic and clustered in lineage D of Betacoronavirus. The extension of two fragments allowed us to classify our sequences within the RdRp Group Unit defined for Kenyan Betacoronavirus from the same host species. These findings are consistent with the previous suggestion on the existence of a single epidemiological unit of E. helvum across sub-Saharan Africa. This theory, which is supported by the genetic structure of continental E. helvum, could facilitate viral mixing between different colonies across the continent.

PRELIMINARY EVALUATION OF SPIROTOME® DEVICE FOR LIVER BIOPSY IN GREEN IGUANAS (IGUANA IGUANA): A PILOT STUDY.

The aim of this study was to evaluate a large-core manual biopsy device (Spirotome(®), Medinvents, 3500 Hasselt, Belgium) for liver sampling and histologic diagnosis in green iguanas (Iguana iguana). The study included eight green iguanas, and two ultrasound-guided biopsies were collected for each lizard, for 16 biopsies in total. The procedure was carried out under general anesthesia induced by intravenous injection of propofol (10 mg/kg) maintained with a mixture of 2.0% isoflurane and 0.8-1.2 L/min oxygen after tracheal intubation. Fourteen (87.5%) of the 16 biopsies were considered diagnostic. Liver biopsy quality was assessed according to sample size and tissue preservation. In particular, mean length (16.2 ± 4.5 mm), width (2.2 ± 0.5 mm), area (34.8 ± 6.9 mm(2)), and number of portal areas (9.4 ± 3.9) of each biopsy were recorded for all green iguanas. The total available surface of the sections obtained from the biopsies and their grade of preservation enabled a satisfactory evaluation of the parenchymal architecture. One of the green iguanas in the study died the day after the procedure due to severe hemocoeloma. Risk assessment evaluation suggested that small green iguanas may not be suitable for this biopsy procedure.

MITE INFECTION IN A MASKED PALM CIVET (PAGUMA LARVATA) TREATED BY SELAMECTIN (STRONGHOLD®, PFIZER LTD.).

The masked palm civet (Paguma larvata) is a small Asian mammal (order Carnivora, family Viverridae) uncommon in Italy. Limited information is available about management and sanitary maintenance in captivity. A 4-mo-old masked palm civet presented with pruritus, itch, scratching, and disorexia. On physical examination, alopecia and crusts were detected on the ventral and lateral trunk, tail, legs, and lips. Skin scrapings and cytology revealed Notoedres spp. and bacterial infection. On histopathology, parasitic dermatitis was observed with the presence of a Sarcoptidae mite and Demodex spp. Selamectin spot-on (15 mg/kg every 2 wk, three applications) and marbofloxacin per os (2.5 mg/kg once daily for 2 wk) were administered, and the animal recovered in 1 mo. With the good response to this therapy, a notoedric mange was thought to be the main problem. This is the first report about the use of selamectin to treat a mite infection in masked palm civet.

Evaluation of liver parenchyma and perfusion using dynamic contrast-enhanced computed tomography and contrast-enhanced ultrasonography in captive green iguanas (Iguana iguana) under general anesthesia.

BACKGROUND: Contrast-enhanced diagnostic imaging techniques are considered useful in veterinary and human medicine to evaluate liver perfusion and focal hepatic lesions. Although hepatic diseases are a common occurrence in reptile medicine, there is no reference to the use of contrast-enhanced ultrasound (CEUS) and contrast-enhanced computed tomography (CECT) to evaluate the liver in lizards. Therefore, the aim of this study was to evaluate the pattern of change in echogenicity and attenuation of the liver in green iguanas (Iguana iguana) after administration of specific contrast media. RESULTS: An increase in liver echogenicity and density was evident during CEUS and CECT, respectively. In CEUS, the mean ± SD (median; range) peak enhancement was 19.9% ± 7.5 (18.3; 11.7-34.6). Time to peak enhancement was 134.0 ± 125.1 (68.4; 59.6-364.5) seconds. During CECT, first visualization of the contrast medium was at 3.6 ± 0.5 (4; 3-4) seconds in the aorta, 10.7 ± 2.2 (10.5; 7-14) seconds in the hepatic arteries, and 15 ± 4.5 (14.5; 10-24) seconds in the liver parenchyma. Time to peak was 14.1 ± 3.4 (13; 11-21) and 31 ± 9.6 (29; 23-45) seconds in the aorta and the liver parenchyma, respectively. CONCLUSION: CEUS and dynamic CECT are practical means to determine liver hemodynamics in green iguanas. Distribution of contrast medium in iguana differed from mammals. Specific reference ranges of hepatic perfusion for diagnostic evaluation of the liver in iguanas are necessary since the use of mammalian references may lead the clinician to formulate incorrect diagnostic suspicions.

Disseminated mycobacteriosis in a stranded loggerhead sea turtle (Caretta caretta).

A loggerhead sea turtle (Caretta caretta) was found stranded alive along the Adriatic coast close to Ancona, Italy, displaying obtundation, tachypnea, and increased respiratory effort. It died a few hours after admission, and a postmortem examination was immediately performed. Miliary yellowish nodules were evident in the liver, and a lower number in the heart, stomach, and gut wall. Hundreds of whitish nodules were scattered in the lungs, with the majority of the pulmonary parenchyma being replaced by the lesions. Histologically, all nodular lesions consisted of a small central area of necrosis with acid-fast bacilli surrounded by epithelioid cells, macrophages, and lymphocytes. Giant cells were found in the spleen and the liver. Kidneys, lungs, liver, spleen, brain, and skin lesions were inoculated aseptically onto general isolation media and selective isolation media for mycobacteria. The isolate showed a restriction pattern identical to Mycobacterium chelonae by polymerase chain reaction-restriction fragment length polymorphism. To the best of the authors' knowledge, this is the first description of a disseminated infection caused by a potentially pathogenic mycobacteria in a stranded, free-ranging loggerhead sea turtle. Veterinary staff and biologists who handle sea turtles with suspected mycobacterial disease should protect themselves appropriately.

Comparison of Pan-Lyssavirus RT-PCRs and Development of an Improved Protocol for Surveillance of Non-RABV Lyssaviruses.

Rabies is a zoonotic and fatal encephalitis caused by members of the Lyssavirus genus. Among them, the most relevant species is Lyssavirus rabies, which is estimated to cause 60,000 human and most mammal rabies deaths annually worldwide. Nevertheless, all lyssaviruses can invariably cause rabies, and therefore their impact on animal and public health should not be neglected. For accurate and reliable surveillance, diagnosis should rely on broad-spectrum tests able to detect all known lyssaviruses, including the most divergent ones. In the present study, we evaluated four different pan-lyssavirus protocols widely used at an international level, including two real-time RT-PCR assays (namely LN34 and JW12/N165-146), a hemi-nested RT-PCR and a one-step RT-PCR. Additionally, an improved version of the LN34 assay ((n) LN34) was developed to increase primer-template complementarity with respect to all lyssavirus species. All protocols were evaluated in silico, and their performance was compared in vitro employing 18 lyssavirus RNAs (encompassing 15 species). The (n) LN34 assay showed enhanced sensitivity in detecting most lyssavirus species, with limits of detection ranging from 10 to 100 RNA copies/µL depending on the strain, while retaining high sensitivity against Lyssavirus rabies. The development of this protocol represents a step forward towards improved surveillance of the entire Lyssavirus genus.

Discovery of a coronavirus in the Eurasian badger (Meles meles) belonging to a putative new genus.

In the aftermath of COVID-19, coronaviruses gained renewed attention by the scientific community. The study reports the identification and genetic characterization of a novel coronavirus in the European badger (Meles meles) obtained in the framework of passive surveillance implemented in Italian wildlife in response to the pandemic. Positive samples were characterized using next generation sequencing as well as genetic and phylogenetic analyses, aiming for taxonomic placement under ICTV guidelines of the viruses contained in each sample. Results obtained for six conserved domains within the polyprotein showed that the virus clustered as outgroup and shared <46% amino acid identity with other coronaviruses, supporting the assumption that it belongs to a new putative genus Epsiloncoronavirus. This finding highlights that mammals still hide diverse coronaviruses whose zoonotic and epizootic potential remains unknown.

Synanthropic and Wild Animals as Sentinels of Zoonotic Agents: A Study of Leptospira Genotypes Circulating in Northeastern Italy.

Leptospirosis is an infectious disease widely reported in veterinary practice and a worldwide zoonosis. In Northeastern Italy, different serogroups and genotypes of Leptospira have been described in ill dogs, the most commonly detected being Icterohaemorragiae (ICT) ST 17, Australis (AUS) ST 24 and ST 198, Pomona (POM) ST 117 and ST 289, and Sejroe (SEJ) ST 155. However, there is little information available on the environmental exposure to Leptospira of wild and synanthropic animals. The aim of this study was to identify the circulating genotypes in potential reservoirs to fill this gap of knowledge. Between 2015 and 2022, 681 animal carcasses collected by the Public Veterinary Service were analyzed for Leptospira with a real-time PCR-based screening test, while positive samples were genotyped by multi-locus sequence typing analysis. To carry out our study, we tested 330 hedgehogs, 105 red foxes, 108 Norway rats, 79 mice, 22 coypus, 10 bank voles, 13 grey wolves, 5 common shrews and 9 greater mouse-eared bats. Five sequence types (STs) common in dogs were also found in wild animals: ST 24, ST 198, ST 17 and ST 155 in hedgehogs, ST 17 and ST 24 in foxes, ST 17 in rats, ST 17 and ST 155 in mice, and ST 117 in a wolf. In addition, to the best of the authors' knowledge, this is the first Italian report of SEJ ST 197 in a bank vole. Furthermore, this study described a previous survey conducted in 2009 on coypus (30 animals from the province of Trento and 41 from the province of Padua), referring to a serological positivity (L. Bratislava) without any molecular detection of Leptospira. This study on Leptospira in synanthropic and wild animals highlighted the importance of increasing our epidemiological knowledge of leptospirosis and its zoonotic risks.

Host Response of Syrian Hamster to SARS-CoV-2 Infection including Differences with Humans and between Sexes.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the importance of having proper tools and models to study the pathophysiology of emerging infectious diseases to test therapeutic protocols, assess changes in viral phenotypes, and evaluate the effects of viral evolution. This study provided a comprehensive characterization of the Syrian hamster (Mesocricetus auratus) as an animal model for SARS-CoV-2 infection using different approaches (description of clinical signs, viral load, receptor profiling, and host immune response) and targeting four different organs (lungs, intestine, brain, and PBMCs). Our data showed that both male and female hamsters were susceptible to the infection and developed a disease similar to the one observed in patients with COVID-19 that included moderate to severe pulmonary lesions, inflammation, and recruitment of the immune system in the lungs and at the systemic level. However, all animals recovered within 14 days without developing the severe pathology seen in humans, and none of them died. We found faint evidence for intestinal and neurological tropism associated with the absence of lesions and a minimal host response in intestines and brains, which highlighted another crucial difference with the multiorgan impairment of severe COVID-19. When comparing male and female hamsters, we observed that males sustained higher viral RNA shedding and replication in the lungs, suffered from more severe symptoms and histopathological lesions, and triggered higher pulmonary inflammation. Overall, these data confirmed the Syrian hamster as a suitable model for mild to moderate COVID-19 and reflected sex-related differences in the response against the virus observed in humans.

Antiviral mechanisms of two broad-spectrum monoclonal antibodies for rabies prophylaxis and therapy.

Rabies is an acute and lethal encephalomyelitis caused by lyssaviruses, among which rabies virus (RABV) is the most prevalent and important for public health. Although preventable through the post-exposure administration of rabies vaccine and immunoglobulins (RIGs), the disease is almost invariably fatal since the onset of clinical signs. Two human neutralizing monoclonal antibodies (mAbs), RVC20 and RVC58, have been shown to be effective in treating symptomatic rabies. To better understand how these mAbs work, we conducted structural modeling and in vitro assays to analyze their mechanisms of action, including their ability to mediate Fc-dependent effector functions. Our results indicate that both RVC20 and RVC58 recognize and lock the RABV-G protein in its pre-fusion conformation. RVC58 was shown to neutralize more potently the extra-cellular virus, while RVC20 mainly acts by reducing viral spreading from infected cells. Importantly, RVC20 was more effective in promoting effector functions compared to RVC58 and 17C7-RAB1 mAbs, the latter of which is approved for human rabies post-exposure treatment. These results provide valuable insights into the multiple mechanisms of action of RVC20 and RVC58 mAbs, offering relevant information for the development of these mAbs as treatment for human rabies.

Isolation and genome characterization of Lloviu virus from Italian Schreibers's bats.

Lloviu cuevavirus (LLOV) was the first identified member of Filoviridae family outside the Ebola and Marburgvirus genera. A massive die-off of Schreibers's bats (Miniopterus schreibersii) in the Iberian Peninsula in 2002 led to its initial discovery. Recent studies with recombinant and wild-type LLOV isolates confirmed the zoonotic nature of the virus in vitro. We examined bat samples from Italy for the presence of LLOV in an area outside of the currently known distribution range of the virus. We detected one positive sample from 2020, sequenced the complete coding region of the viral genome and established an infectious isolate of the virus. In addition, we performed the first comprehensive evolutionary analysis of the virus, using the Spanish, Hungarian and the Italian sequences. The most important achievement of this study is the establishment of an additional infectious LLOV isolate from a bat sample using the SuBK12-08 cells, demonstrating that this cell line is highly susceptible to LLOV infection and confirming the previous observation that these bats are effective hosts of the virus in nature. This result further strengthens the role of bats as the natural hosts for zoonotic filoviruses.

Alternative Methods to Current In Vivo Procedures to Address the 3Rs Tenet in Rabies Proficiency Testing.

Canine rabies is responsible for an estimated 59,000 human deaths every year. In an attempt to reach the ZeroBy30 goal, robust disease surveillance coupled with improved diagnostics play a paramount role in ensuring reliable data and gradually attesting rabies control advancements. In this context, proficiency testing is organized to harmonize rabies diagnostic capacities. In most exercises, rabies-positive samples consist of brains collected from intracerebrally inoculated mice. This procedure causes distress and severe suffering to animals, raising important ethical concerns that can no longer be ignored. In the last decades, the 3Rs tenet (Replace, Reduce, Refine) has been successfully implemented in several scientific areas, and we strongly support its application in the framework of rabies proficiency testing. Here, we discuss cell-based technologies as innovative sustainable in vitro candidate systems to replace in vivo experiments for the production of proficiency testing samples. The application of these alternative methods can allow completely in vitro or ex vivo production of rabies proficiency testing panels, which would represent an important replacement or reduction/refinement for current in vivo procedures.

The Importance of Accurate Host Species Identification in the Framework of Rabies Surveillance, Control and Elimination.

Accurate host identification is paramount to understand disease epidemiology and to apply appropriate control measures. This is especially important for multi-host pathogens such as the rabies virus, a major and almost invariably fatal zoonosis that has mobilized unanimous engagement at an international level towards the final goal of zero human deaths due to canine rabies. Currently, diagnostic laboratories implement a standardized identification using taxonomic keys. However, this method is challenged by high and undiscovered biodiversity, decomposition of carcasses and subjective misevaluation, as has been attested to by findings from a cohort of 242 archived specimens collected across Sub-Saharan Africa and submitted for rabies diagnosis. We applied two simple and cheap methods targeting the Cytochrome b and Cytochrome c oxidase subunit I to confirm the initial classification. We therefore suggest prioritizing a standardized protocol that includes, as a first step, the implementation of taxonomic keys at a family or subfamily level, followed by the molecular characterization of the host species.

Identification of Dobrava-Belgrade Virus in Apodemus flavicollis from North-Eastern Italy during Enhanced Mortality.

Hantaviruses include several zoonotic pathogens that cause different syndromes in humans, with mortality rates ranging from 12 to 40%. Most commonly, humans get infected through the inhalation of aerosols or dust particles contaminated with virus-containing rodent excreta. Hantaviruses are specifically associated with the host species, and human cases depend on the presence and the dynamics of reservoir hosts. In this letter, we report the identification of Dobrava-Belgrade virus (DOBV) in the yellow-necked mouse (Apodemus flavicollis) from Italy. The virus was detected in the mountainous area of the province of Udine, bordering Austria and Slovenia, during an event of enhanced mortality in wild mice and voles. Despite serological evidence in rodents and humans that suggested the circulation of hantaviruses in Italy since 2000, this is the first virological confirmation of the infection. Phylogenetic analyses across the whole genome of the two detected viruses confirmed the host-specificity of DOBV sub-species and showed the highest identity with viruses identified in Slovenia and Croatia from both A. flavicollis and humans, with no signs of reassortment. These findings highlight the need for ecologists, veterinarians and medical doctors to come together in a coordinated approach in full compliance with the One Health concept.