Martina Castellan

The Lyssavirus genus includes seventeen viral species able to cause rabies, an acute and almost invariably fatal encephalomyelitis of mammals. Rabies virus (RABV), which represents the type species of the genus, is a multi-host pathogen that over the years has undergone multiple events of host-switching, thus occupying several geographical and ecological niches. In contrast, non-RABV lyssaviruses are mainly confined within a single natural host with rare spillover events. In this scenario, unveiling the mechanisms underlying the host immune response against a virus is crucial to understand the dynamics of infection and to predict the probability of colonization/adaptation to a new target species. Presently, the host response to lyssaviruses has only been partially explored, with the majority of data extrapolated from RABV infection. West Caucasian bat virus (WCBV), a divergent lyssavirus, has recently been associated with a spillover event to a domestic cat, raising concern about the risks to public health due to the circulation of the virus in its natural host. Through this study we have investigated the immune response determined by the WCBV versus two widely known lyssaviruses. We selected the Syrian hamster as representative of an accidental host, and chose the intramuscular route in order to mimic the natural infection. In hamsters, WCBV was highly pathogenic, determining 100% lethality and mild encephalitis. In comparison with Duvenhage virus (DUVV) and RABV, we found that WCBV displayed an intermediate ability to promote cellular antiviral response, produce pro-inflammatory cytokines, and recruit and activate lymphocytes in the hamsters’ central nervous system. IMPORTANCE: Although all lyssaviruses cause fatal encephalomyelitis in mammals, they display a different host tropism and pathogenicity, with the ecology of Rabies virus (RABV) continually evolving and adapting to new host species. In 2020, West Caucasian bat virus (WCBV) was identified as the causative agent of rabies in a domestic cat in Italy. This event raised concerns about its public health consequences, due to the absence of biologicals against the infection. Our study investigates the host immune response triggered by WCBV in comparison with a pathogenic strain of RABV and the low pathogenic Duvenhage lyssavirus (DUVV), as a proxy to understand the mechanisms leading to lyssavirus spillover and pathogenicity. We overall confirm that previous evidence indicating an inverse relationship between lyssavirus pathogenicity and immune response is applicable for WCBV as well. Importantly, this work represents the first transcriptomic analysis of the WCBV interaction in the central nervous system with an accidental host.

The Lyssavirus genus includes seventeen neurotropic viral species which are able to cause rabies, an acute and almost invariably fatal encephalomyelitis of mammals. Rabies virus (RABV), the genus prototype, is a multi-host pathogen that undergoes multiple events of host-switching, thus occupying several geographical and ecological niches. In contrast, non-RABV lyssaviruses are mainly confined within a single natural host with rare spillover events never followed by adaptation to new accidental host species. In this scenario, unveiling the mechanisms underlying the host immune response against a virus is crucial to understand the dynamics of infection but also to predict the probability of colonization and adaptation to a new target species. Presently, the host response to lyssaviruses has only been partially explored, with the majority of data inferred from RABV infection, under the assumption that all members of the genus exhibit a similar behavior. Through our study we have investigated the immune response determined by the West Caucasian bat virus (WCBV). Indeed, WCBV has been recently associated with a spillover event to a domestic cat, raising concern about the risks for public health due to the circulation of the virus in its natural host. We selected the Syrian hamster as an animal model representative for an accidental host, and chose the intramuscular route in order to mimic the natural route of infection. In hamsters, WCBV was highly pathogenic, determining 100% lethality and mild encephalitis. In comparison with Duvenhage virus (DUVV) and RABV, we found that WCBV displayed an intermediate ability to promote cellular antiviral response, produce pro-inflammatory cytokines, recruit and activate lymphocytes in the hamsters’ central nervous system. Author Summary: Viruses belonging to the genus Lyssavirus cause rabies, a zoonotic and almost invariably fatal encephalomyelitis. However, not all lyssaviruses behave in the same way, with Rabies virus (RABV) being a multi-host pathogen and non-RABV ones remaining mostly confined to their natural hosts. Host-pathogen interaction might help to elucidate the mechanisms behind the differences observed among lyssaviruses in terms of disease dynamics and ability to cross the species barrier, adapting to novel hosts. In our study, we have investigated the host immune response triggered by the WCBV strain spilled-over from its host (the bent-winged bat) to a domestic cat in 2020. Our study offers a key to understand and generalize the mechanisms underlying lyssavirus spillover and pathogenicity in accidental hosts. More generally, our work confirms and marshals previous, fragmentary evidence indicating a strong and inverse relationship between lyssavirus pathogenicity and immune response induction.

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.

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 phenotype and evaluate the effect of viral evolution. This study provides 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 are susceptible to the infection and develop a disease similar to the one observed in patients with COVID-19, including moderate to severe pulmonary lesions, inflammation and recruitment of the immune system in lungs and at 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, highlighting another crucial difference with the multi-organ impairment of severe COVID-19. When comparing male and female hamsters, it was 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 confirm the Syrian hamster as a suitable model for mildmoderate COVID-19 and reflect sex-related differences in the response against the virus observed in humans.

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.

Tick borne encephalitis virus (TBEV) is the causative agent of Tick borne encephalitis in humans, a severe zoonosis occurring in the Paleartic region mainly transmitted through ticks belonging to the genus Ixodes. In Italy, TBEV is restricted to few foci in the north-eastern part of the country. This report describes for the first time a case of clinical TBE in a roe deer, occurred in the Belluno province, Veneto region, an area highly endemic for the presence of the virus. The affected roe deer showed ataxia, staggering movements, muscle tremors and persistent teeth grinding causing hypersalivation. At necropsy, the macroscopic picture was inconclusive. RNA of TBEV was detected by real-time RT-PCR. Phylogenetic analysis revealed a close relationship to TBEV of the European subtype, and 100% similarity with a virus from the bordering Trento Province. The histological examination of the midbrain confirmed the viral etiology and specific immunofluorescence indicated the presence of a Flavivirus infection and characterized the pattern of infection in the neurons. This report underlines for the first time the occurrence of clinical encephalitic manifestations due to TBEV in a roe deer, thussuggesting to include this pathogen in the frame of differential diagnosis in this species.

Tick-borne encephalitis virus (TBEV) is the causative agent of tick-borne encephalitis (TBE), a severe zoonosis occurring in the Palearctic region mainly transmitted through Ixodes ticks. In Italy, TBEV is restricted to the north-eastern part of the country. This report describes for the first time a case of clinical TBE in a roe deer (Capreolus capreolus L.). The case occurred in the Belluno province, Veneto region, an area endemic for TBEV. The affected roe deer showed ataxia, staggering movements, muscle tremors, wide-base stance of the front limbs, repetitive movements of the head, persistent teeth grinding, hypersalivation and prolonged recumbency. An autopsy revealed no significant lesions to explain the neurological signs. TBEV RNA was detected in the brain by real-time RT-PCR, and the nearly complete viral genome (10,897 nucleotides) was sequenced. Phylogenetic analysis of the gene encoding the envelope protein revealed a close relationship to TBEV of the European subtype, and 100% similarity with a partial sequence (520 nucleotides) of a TBEV found in ticks in the bordering Trento province. The histological examination of the midbrain revealed lymphohistiocytic encephalitis, satellitosis and microgliosis, consistent with a viral etiology. Other viral etiologies were ruled out by metagenomic analysis of the brain. This report underlines, for the first time, the occurrence of clinical encephalitic manifestations due to TBEV in a roe deer, suggesting that this pathogen should be included in the frame of differential diagnoses in roe deer with neurologic disease.

Glioblastoma (GBM) is a devastating human malignancy. GBM stem-like cells (GSCs) drive tumor initiation and progression. Yet the molecular determinants defining GSCs in their native state in patients remain poorly understood. Here we used single-cell datasets and identified GSCs at the apex of the differentiation hierarchy of GBM. By reconstructing the GSCs’ regulatory network, we identified the YAP/TAZ coactivators as master regulators of this cell state, irrespectively of GBM subtypes. YAP/TAZ are required to install GSC properties in primary cells downstream of multiple oncogenic lesions and are required for tumor initiation and maintenance in vivo in different mouse and human GBM models. YAP/TAZ act as main roadblock of GSC differentiation, and their inhibition irreversibly locks differentiated GBM cells into a nontumorigenic state, preventing plasticity and regeneration of GSC-like cells. Thus, GSC identity is linked to a key molecular hub integrating genetics and microenvironmental inputs within the multifaceted biology of GBM.

How the behaviour of somatic stem cells (SCs) is influenced by mechanical signals remains a black-box in cell biology. Here we show that YAP/TAZ regulation by cell shape and rigidity of the extracellular matrix (ECM) dictates a pivotal SC decision: to remain undifferentiated and grow, or to activate a terminal differentiation programme. Notably, mechano-activation of YAP/TAZ promotes epidermal stemness by inhibition of Notch signalling, a key factor for epidermal differentiation. Conversely, YAP/TAZ inhibition by low mechanical forces induces Notch signalling and loss of SC traits. As such, mechano-dependent regulation of YAP/TAZ reflects into mechano-dependent regulation of Notch signalling. Mechanistically, at least in part, this is mediated by YAP/TAZ binding to distant enhancers activating the expression of Delta-like ligands, serving as ‘in cis’ inhibitors of Notch. Thus YAP/TAZ mechanotransduction integrates with cell–cell communication pathways for fine-grained orchestration of SC decisions.