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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.

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.

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.

Coronaviruses (CoVs) are widespread and highly diversified in wildlife and domestic mammals and can emerge as zoonotic or epizootic pathogens and consequently host shift from these reservoirs, highlighting the importance of veterinary surveillance. All genera can be found in mammals, with α and β showing the highest frequency and diversification. The aims of this study were to review the literature for features of CoV surveillance in animals, to test widely used molecular protocols, and to identify the most effective one in terms of spectrum and sensitivity. We combined a literature review with analyses in silico and in vitro using viral strains and archive field samples. We found that most protocols defined as pan-coronavirus are strongly biased towards α- and β-CoVs and show medium-low sensitivity. The best results were observed using our new protocol, showing LoD 100 PFU/mL for SARS-CoV-2, 50 TCID50/mL for CaCoV, 0.39 TCID50/mL for BoCoV, and 9 ± 1 log2 ×10−5 HA for IBV. The protocol successfully confirmed the positivity for a broad range of CoVs in 30/30 field samples. Our study points out that pan-CoV surveillance in mammals could be strongly improved in sensitivity and spectrum and propose the application of a new RT-PCR assay, which is able to detect CoVs from all four genera, with an optimal sensitivity for α-, β-, and γ-.

In June 2020, a cat from Arezzo (Italy) that died from a neurological disease was diagnosed with West Caucasian Bat Lyssavirus (WCBV). The virus retained high identity across the whole-genome with the reference isolate found in 2002 from a Russian bent-winged bat. We applied control measures recommended by national regulations, investigated a possible interface between cats and bats using visual inspections, bioacoustics analyses and camera trapping and performed active and passive surveillance in bats to trace the source of infection. People that were exposed to the cat received full post-exposure prophylaxis while animals underwent six months of quarantine. One year later, they are all healthy. In a tunnel located near the cat’s house, we identified a group of bent-winged bats that showed virus-neutralizing antibodies to WCBV across four sampling occasions, but no virus in salivary swabs. Carcasses from other bat species were all negative. This description of WCBV in a non-flying mammal confirms that this virus can cause clinical rabies in the absence of preventive and therapeutic measures, and highlights the lack of international guidelines against divergent lyssaviruses. We detected bent-winged bats as the most probable source of infection, testifying the encroachment between these bats and pets/human in urban areas and confirming free-ranging cats as potential hazard for public health and conservation.

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.