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Human rabies cases today are predominantly associated with infection from rabid domestic dogs. Unlike dogs, a common global reservoir species that perpetuates rabies viruses (RABV) within their populations, domestic cats are much less frequently reported or vaccinated. Epidemiologically, cats are important vectors of lyssaviruses but are not viral reservoirs. Typically, cats are incidental hosts only, infected with the predominant lyssavirus in their geographic locale. Human cases associated with rabid cats have occurred in Africa, Asia, Europe and throughout the Americas. As adept, solitary hunters, wild and domestic felids are at risk of lyssavirus infection based upon interactions with infected prey, such as bats, or from transmission by other mesocarnivores, such as rabid dogs, foxes, jackals, raccoons, and skunks. Current veterinary vaccines provide safe and effective immunity in cats against phylogroup I lyssaviruses, such as RABV, but not against divergent lyssaviruses in phylogroups II-IV. With the focus upon the global elimination of canine rabies, the emergence of rabies in cats represents a concerning trend. Clearly, education about the occurrence of rabies in cats needs to be improved, as well as the routine vaccination of cats to reduce the associated risks to public health, agriculture, and conservation biology from a One Health perspective.

Abstract 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.

Abstract 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.

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.

Abstract Rabies is a neglected disease caused by a neurotropic Lyssavirus, transmitted to humans predominantly by the bite of infected dogs. Rabies is preventable with vaccines or proper post‐exposure prophylaxis (PEP), but it still causes about 60,000 deaths every year. No cure exists after the onset of clinical signs, and the case‐fatality rate approaches 100% even with advanced supportive care. Here, we report that a combination of two potent neutralizing human monoclonal antibodies directed against the viral envelope glycoprotein cures symptomatic rabid mice. Treatment efficacy requires the concomitant administration of antibodies in the periphery and in the central nervous system through intracerebroventricular infusion. After such treatment, recovered mice presented good clinical condition, viral loads were undetectable, and the brain inflammatory profile was almost normal. Our findings provide the unprecedented proof of concept of an antibody‐based therapeutic approach for symptomatic rabies.

ABSTRACTA second case of a novel rabies variant described once in a capuchin monkey from Mato Grosso, Brazil, was discovered in a rabid wild kinkajou from the same region, indicating a public health risk following exposure to either of the two animals.

Rabies has been listed as a priority zoonotic disease in many African countries and the countdown to reach the goal of eliminating dog-mediated human rabies deaths by 2030 means that disease control measures need to be applied fast. In this context, an essential pillar of any national plan to control rabies is the implementation of reliable diagnostic techniques to ensure the success of field surveillance systems. Although many African countries have received international support for the control of rabies–some countries, like Benin, have not received a similar level of support. Indeed, until 2018, Benin was not able to diagnose rabies and rabies diagnosis in animals as well as humans relied solely on observed clinical symptoms. Although the Central Veterinary Laboratory (CVL) of Parakou had the equipment to implement two recommended tests, the lack of specific reagents and skills prevented the implementation of a rabies diagnostic service. Here we present the joint efforts of the national authorities in Benin, intergovernmental agencies, and non-governmental organizations to assess the strengths and weaknesses of the government's rabies control efforts. We have applied the Stepwise Approach toward Rabies Elimination (SARE) analysis, implemented rabies diagnostic capacities at the CVL of Parakou, characterized strains of rabies virus circulating in Benin, and finally integrated an inter-laboratory comparison program.

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.

The illegal trade of animals poses several health issues to the global community, among which are the underestimated risk for spillover infection and the potential for an epizootic in both wildlife and domestic naïve populations. We herein describe the genetic and antigenic characterization of viruses of the specie Carnivore protoparvovirus 1 detected at high prevalence in puppies illegally introduced in North Eastern Italy and compared them with those circulating in wild carnivores from the same area. We found evidence of a wide diversity of canine parvoviruses (CPV-2) belonging to different antigenic types in illegally imported pups. In wildlife, we found a high circulation of feline parvovirus (FPV) in golden jackals and badgers, whereas CPV-2 was observed in one wolf only. Although supporting a possible spillover event, the low representation of wolf samples in the present study prevented us from inferring the origin, prevalence and viral diversity of the viruses circulating in this species. Therefore, we suggest performing more thorough investigations before excluding endemic CPV-2 circulation in this species.

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.

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.

Illegal animal trade (pet, wildlife, animal products, etc.) is an example of transnational organized crime (T.O.C.) that generates a large business with huge profit margins. This criminal activity causes several negative effects on human health (zoonoses), animal health and welfare, market protection, consumer fraud and may be used as tool of agro/bio-terrorism. Illegal animal trade can facilitate the spread of zoonoses that are defined as diseases and infections that are transmitted by vertebrate animals to man. Humans are affected by more than 1,700 known pathogens: 60% of existing human infectious diseases are zoonotic and at least 75% of emerging infectious diseases of humans have an animal origin and 72% of zoonoses originate from wildlife or exotic animals. The Bio-Crime Project was developed in 2017 by Friuli Venezia Giulia Region (Italy) and Land Carinthia (Austria) together with other public institutions to combat illegal animal trade and to reduce the risk of disease transmission from animals to humans. Project partners agreed that a multi-agency approach was required to tackle the illegal animal trade that was high value, easy to undertake and transnational crime. The Bio-crime model of cross-border cooperation introduces the novel approach of replicating the cooperative framework given by the triad of Veterinary Public Health, Justice and Law Enforcements/Customs across borders using the International Police and Custom Cooperation Centres (IPCCCs) as a connection link among public entities of the neighbor countries. This model has been recognized as a best practice at European level because it can be easily replicated and scaled up without any supplementary cost for Member States.

To achieve the goal of eliminating dog-mediated human rabies deaths by 2030, many African countries have agreed to list rabies as a priority zoonotic disease and to undertake both short and long-term control programs. Within this context, reliable local diagnosis is essential for the success of field surveillance systems. However, a harmonized, sustainable and supportive diagnostic offer has yet to be achieved in the continent. We herewith describe the organization and outcome of a proficiency test (PT) for the post-mortem diagnosis of rabies in animals, involving thirteen veterinary laboratories and one public health laboratory in Africa. Participants were invited to assess both the performance of the Direct Fluorescent Antibody (DFA) test and of a conventional RT-PCR. From the submitted results, while thirteen laboratories proved to be able to test the samples through DFA test, eleven performed the RT-PCR method; ten applied both techniques. Of note, the number of laboratories able to apply rabies RT-PCR had increased from four to ten after the exercise. Importantly, results showed a higher proficiency in applying the molecular test compared to the DFA test (concordance, sensitivity and specificity: 98.2%, 96.97% and 100% for RT-PCR; 87.69%, 89.23% and 86.15% for DFA test), indicating the feasibility of molecular methods to diagnose animal pathogens in Africa. Another positive outcome of this approach was that negative and positive controls were made available for further in-house validation of new techniques; in addition, a detailed questionnaire was provided to collect useful and relevant information on the diagnostic procedures and biosafety measures applied at laboratory level.

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.

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.

Abstract 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.

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.

The illegal trade of companion animals in the European Union poses several legal, ethical and health issues to the entire community. In the framework of the Biocrime Interreg project between Italy and Austria, we surveyed puppies and kittens confiscated at the borders to identify the most frequent pathogens associated with (i) the risk of spread within the shelter, (ii) the development of fatal disease and (iii) the zoonotic potential. From January 2018 to December 2020, we examined a total of 613 puppies and 62 kittens coming from 44 requisitions. Feces, skin specimens and blood sera from confiscated animals were tested to verify the presence of major infections and to assess the rabies post-vaccination immunity. Out of the total of individuals under investigation, necropsies and laboratory investigations were also performed on 79 puppies and three kittens that had died during the observation period. Results indicated a high prevalence of Canine Parvovirus (CPV) and Giardia spp. infections, CPV as the most likely cause of fatal gastroenteritis in puppies and Salmonella and Microsporum canis as major zoonotic pathogens. Conversely, both extended spectrum beta lactamases Escherichia coli and methicillin resistant Staphylococcus pseudintermedius strains as rare findings. Results highlighted that illegal animal trade could expose the human population to potential zoonotic risk and naïve animal population to potentially disrupting epidemic waves, both of these issues being largely underestimated when buying companion animals.

Information on the population dynamics of a reservoir species have been increasingly adopted to understand and eventually predict the dispersal patterns of infectious diseases throughout an area. Although potentially relevant, to date there are no studies which have investigated the genetic structure of the red fox population in relation to infectious disease dynamics. Therefore, we genetically and spatially characterised the red fox population in the area stretching between the Eastern and Dinaric Alps, which has been affected by both distemper and rabies at different time intervals. Red foxes collected from north-eastern Italy, Austria, Slovenia and Croatia between 2006-2012, were studied using a set of 21 microsatellite markers. We confirmed a weak genetic differentiation within the fox population using Bayesian clustering analyses, and we were able to differentiate the fox population into geographically segregated groups. Our finding might be due to the presence of geographical barriers that have likely influenced the distribution of the fox population, limiting in turn gene flow and spread of infectious diseases. Focusing on the Italian red fox population, we observed interesting variations in the prevalence of both diseases among distinct fox clusters, with the previously identified Italy 1 and Italy 2 rabies as well as distemper viruses preferentially affecting different sub-groups identified in the study. Knowledge of the regional-scale population structure can improve understanding of the epidemiology and spread of diseases. Our study paves the way for an integrated approach for disease control coupling pathogen, host and environmental data to inform targeted control programs in the future.

Abstract Currently available rabies post‐exposure prophylaxis (PEP) for use in humans includes equine or human rabies immunoglobulins (RIG). The replacement of RIG with an equally or more potent and safer product is strongly encouraged due to the high costs and limited availability of existing RIG. In this study, we identified two broadly neutralizing human monoclonal antibodies that represent a valid and affordable alternative to RIG in rabies PEP. Memory B cells from four selected vaccinated donors were immortalized and monoclonal antibodies were tested for neutralizing activity and epitope specificity. Two antibodies, identified as RVC20 and RVC58 (binding to antigenic site I and III, respectively), were selected for their potency and broad‐spectrum reactivity. In vitro, RVC20 and RVC58 were able to neutralize all 35 rabies virus (RABV) and 25 non‐RABV lyssaviruses. They showed higher potency and breath compared to antibodies under clinical development (namely CR57, CR4098, and RAB1) and commercially available human RIG. In vivo, the RVC20–RVC58 cocktail protected Syrian hamsters from a lethal RABV challenge and did not affect the endogenous hamster post‐vaccination antibody response.

Bats are often claimed to be a major source for future viral epidemics, as they are associated with several viruses with zoonotic potential. Here we describe the presence and biodiversity of bats associated with intensive pig farms devoted to the production of heavy pigs in northern Italy. Since chiropters or signs of their presence were not found within animal shelters in our study area, we suggest that fecal viruses with high environmental resistance have the highest likelihood for spillover through indirect transmission. In turn, we investigated the circulation of mammalian orthoreoviruses (MRVs), coronaviruses (CoVs) and astroviruses (AstVs) in pigs and bats sharing the same environment. Results of our preliminary study did not show any bat virus in pigs suggesting that spillover from these animals is rare. However, several AstVs, CoVs and MRVs circulated undetected in pigs. Among those, one MRV was a reassortant strain carrying viral genes likely acquired from bats. On the other hand, we found a swine AstV and a MRV strain carrying swine genes in bat guano, indicating that viral exchange at the bat–pig interface might occur more frequently from pigs to bats rather than the other way around. Considering the indoor farming system as the most common system in the European Union (EU), preventive measures should focus on biosecurity rather than displacement of bats, which are protected throughout the EU and provide critical ecosystem services for rural settings.

Mammalian Orthoreoviruses (MRV) are segmented dsRNA viruses in the family Reoviridae. MRVs infect mammals and cause asymptomatic respiratory, gastro-enteric and, rarely, encephalic infections. MRVs are divided into at least three serotypes: MRV1, MRV2 and MRV3. In Europe, swine MRV (swMRV) was first isolated in Austria in 1998 and subsequently reported more than fifteen years later in Italy. In the present study, we characterized two novel reassortant swMRVs identified in one same Italian farm over two years. The two viruses shared the same genetic backbone but showed evidence of reassortment in the S1, S4, M2 segments and were therefore classified into two serotypes: MRV3 in 2016 and MRV2 in 2018. A genetic relation to pig, bat and human MRVs and other unknown sources was identified. A considerable genetic diversity was observed in the Italian MRV3 and MRV2 compared to other available swMRVs. The S1 protein presented unique amino acid signatures in both swMRVs, with unexpected frequencies for MRV2. The remaining genes formed distinct and novel genetic groups that revealed a geographically related evolution of swMRVs in Italy. This is the first report of the complete molecular characterization of novel reassortant swMRVs in Italy and Europe, which suggests a greater genetic diversity of swMRVs never identified before.

As a neglected zoonotic disease, rabies causes approximately 5.9 × 104 human deaths annually, primarily affecting low- and middle-income countries in Asia and Africa. In those regions, insufficient surveillance is hampering adequate medical intervention and is driving the vicious cycle of neglect. Where resources to provide laboratory disease confirmation are limited, there is a need for user-friendly and low-cost reliable diagnostic tools that do not rely on specialized laboratory facilities. Lateral flow devices (LFD) offer an alternative to conventional diagnostic methods and may strengthen control efforts in low-resource settings. Five different commercially available LFDs were compared in a multi-centered study with respect to their diagnostic sensitivity and their agreement with standard rabies diagnostic techniques. Our evaluation was conducted by several international reference laboratories using a broad panel of samples. The overall sensitivities ranged from 0% up to 62%, depending on the LFD manufacturer, with substantial variation between the different laboratories. Samples with high antigen content and high relative viral load tended to test positive more often in the Anigen/Bionote test, the latter being the one with the best performance. Still, the overall unsatisfactory findings corroborate a previous study and indicate a persistent lack of appropriate test validation and quality control. At present, the tested kits are not suitable for in-field use for rabies diagnosis, especially not for suspect animals where human contact has been identified, as an incorrect negative diagnosis may result in human casualties. This study points out the discrepancy between the enormous need for such a diagnostic tool on the one hand, and on the other hand, a number of already existing tests that are not yet ready for use.

Rabies is a neglected zoonotic disease that causes an estimated 60,000 human deaths annually. The main burden lies on developing countries in Asia and Africa, where surveillance and disease detection is hampered by absence of adequate laboratory facilities and/or the difficulties of submitting samples from remote areas to laboratories. Under these conditions, easy-to-use tests such as immunochromatographic assays, i.e. lateral flow devices (LFD), may increase surveillance and improve control efforts. Several LFDs for rabies diagnosis are available but, except for one, there are no data regarding their performance. Therefore, we compared six commercially available LFDs for diagnostic and analytical sensitivity, as well as their specificity and their diagnostic agreement with standard rabies diagnostic techniques using different sample sets, including experimentally infected animals and several sets of field samples. Using field samples the sensitivities ranged between 0% up to 100% depending on the LFD and the samples, while for experimentally infected animals the maximum sensitivity was 32%. Positive results in LFD could be further validated using RT-qPCR and sequencing. In summary, in our study none of the tests investigated proved to be satisfactory, although the results somewhat contradict previous studies, indicating batch to batch variation. The high number of false negative results reiterates the necessity to perform a proper test validation before being marketed and used in the field. In this respect, marketing authorization and batch release control could secure a sufficient quality for these alternative tests, which could then fulfil their potential.

Genetic characterization of a selection of influenza virus (H5N1) samples, circulating in 8 Nigerian states over a 39-day period in early 2007, indicates that a new reassortant strain is present in 7 of the 8 states. Our study reports an entirely different influenza virus (H5N1) reassortant becoming predominant and widespread in poultry.

Since 2006 the red fox (Vulpes vulpes) population in north-eastern Italy has experienced an epidemic of canine distemper virus (CDV). Additionally, in 2008, after a thirteen-year absence from Italy, fox rabies was re-introduced in the Udine province at the national border with Slovenia. Disease intervention strategies are being developed and implemented to control rabies in this area and minimise risk to human health. Here we present empirical data and the epidemiological picture relating to these epidemics in the period 2006-2010. Of important significance for epidemiological studies of wild animals, basic mathematical models are developed to exploit information collected from the surveillance program on dead and/or living animals in order to assess the incidence of infection. These models are also used to estimate the rate of transmission of both diseases and the rate of vaccination, while correcting for a bias in early collection of CDV samples. We found that the rate of rabies transmission was roughly twice that of CDV, with an estimated effective contact between infected and susceptible fox leading to a new infection occurring once every 3 days for rabies, and once a week for CDV. We also inferred that during the early stage of the CDV epidemic, a bias in the monitoring protocol resulted in a positive sample being almost 10 times more likely to be collected than a negative sample. We estimated the rate of intake of oral vaccine at 0.006 per day, allowing us to estimate that roughly 68% of the foxes would be immunised. This was confirmed by field observations. Finally we discuss the implications for the eco-epidemiological dynamics of both epidemics in relation to control measures.