Intranasal type I interferon treatment is beneficial only when administered before clinical signs onset in the SARS-CoV-2 hamster model.

Impaired type I interferons (IFNs) production or signaling have been associated with severe COVID-19, further promoting the evaluation of recombinant type I IFNs as therapeutics against SARS-CoV-2 infection. In the Syrian hamster model, we show that intranasal administration of IFN-α starting one day pre-infection or one day post-infection limited weight loss and decreased viral lung titers. By contrast, intranasal administration of IFN-α starting at the onset of symptoms three days post-infection had no impact on the clinical course of SARS-CoV-2 infection. Our results provide evidence that early type I IFN treatment is beneficial, while late interventions are ineffective, although not associated with signs of enhanced disease.

Hamster and ferret experimental infection with intranasal low dose of a single strain of SARS-CoV-2.

Understanding the pathogenesis of the SARS-CoV-2 infection is key to developing preventive and therapeutic strategies against COVID-19, in the case of severe illness but also when the disease is mild. The use of appropriate experimental animal models remains central in the in vivo exploration of the physiopathology of infection and antiviral strategies. This study describes SARS-CoV-2 intranasal infection in ferrets and hamsters with low doses of low-passage SARS-CoV-2 clinical French isolate UCN19, describing infection levels, excretion, immune responses and pathological patterns in both animal species. Individual infection with 103 p.f.u. SARS-CoV-2 induced a more severe disease in hamsters than in ferrets. Viral RNA was detected in the lungs of hamsters but not of ferrets and in the brain (olfactory bulb and/or medulla oblongata) of both species. Overall, the clinical disease remained mild, with serological responses detected from 7 days and 10 days post-inoculation in hamsters and ferrets respectively. The virus became undetectable and pathology resolved within 14 days. The kinetics and levels of infection can be used in ferrets and hamsters as experimental models for understanding the pathogenicity of SARS-CoV-2, and testing the protective effect of drugs.

Massive transient damage of the olfactory epithelium associated with infection of sustentacular cells by SARS-CoV-2 in golden Syrian hamsters.

Anosmia is one of the most prevalent symptoms of SARS-CoV-2 infection during the COVID-19 pandemic. However, the cellular mechanism behind the sudden loss of smell has not yet been investigated. The initial step of odour detection takes place in the pseudostratified olfactory epithelium (OE) mainly composed of olfactory sensory neurons surrounded by supporting cells known as sustentacular cells. The olfactory neurons project their axons to the olfactory bulb in the central nervous system offering a potential pathway for pathogens to enter the central nervous system by bypassing the blood brain barrier. In the present study, we explored the impact of SARS-CoV-2 infection on the olfactory system in golden Syrian hamsters. We observed massive damage of the OE as early as 2 days post nasal instillation of SARS-CoV-2, resulting in a major loss of cilia necessary for odour detection. These damages were associated with infection of a large proportion of sustentacular cells but not of olfactory neurons, and we did not detect any presence of the virus in the olfactory bulbs. We observed massive infiltration of immune cells in the OE and lamina propria of infected animals, which may contribute to the desquamation of the OE. The OE was partially restored 14 days post infection. Anosmia observed in COVID-19 patient is therefore likely to be linked to a massive and fast desquamation of the OE following sustentacular cells infection with SARS-CoV-2 and subsequent recruitment of immune cells in the OE and lamina propria.

Detection of rabies antibodies in wild boars in north-east Romania by a rabies ELISA test.

BACKGROUND: In the last few decades, Romania has been considered one of the European countries most affected by animal rabies, but a combination of oral rabies vaccination (ORV) campaigns in foxes alongside mandatory vaccination of pets has substantially decreased the number of rabies cases in recent years. The objective of this study was to detect rabies antibodies in wild boar serum and thoracic fluid samples collected during the hunting season after ORV campaigns in north-eastern Romania in order to identify if wild boars are substantial competitors to foxes for ORV baits. RESULTS: When the 312 wild boar samples were tested by ELISA (BioPro ELISA, Czech Republic), 42.31% (132/312) demonstrated rabies antibodies. In order to compare these wild boar results in terms of the percentage of immunisation, fox samples were also included in the study, and in this case only 28.40% (98/345) demonstrated rabies antibodies by ELISA. To check the diagnostic sensitivity and specificity of this ELISA, those samples with a sufficient volume from both species that had tested either negative or positive with an initial ELISA were then tested with the Fluorescent Antibody Virus Neutralisation (FAVN) assay. The overall concordance between the BioPro ELISA and FAVN test was 74.26% (75/101) in wild boar samples and 65.66% (65/99) in fox samples, 140 out of 200 samples being correlated with the two methods, although no significant statistical difference (p = 0.218) between the two species was registered. We found a good agreement by both tests for the ELISA-positive samples (91.30%), however the situation was different for the ELISA-negative samples, where a low agreement was demonstrated (41.18%). CONCLUSIONS: This study reports for the first time the presence of rabies antibodies in wild boar samples collected during the hunting season in Romania after ORV campaigns in rabies endemic areas. It is also the first study to demonstrate that ELISA BioPro can be used on wild boar samples with satisfactory results compared to the FAVN test for this species.

Molecular and serological survey of lyssaviruses in Croatian bat populations.

BACKGROUND: Rabies is the only known zoonotic disease of bat origin in Europe. The disease is caused by species belonging to the genus Lyssavirus. Five Lyssavirus species, i.e., European bat lyssavirus (EBLV)-1, EBLV-2, Bokeloh bat lyssavirus, Lleida bat lyssavirus, and West Caucasian bat virus, have been identified in European bats. More recently, a proposed sixth species, Kotalahti bat lyssavirus, was detected. Thus, in this study, active surveillance was initiated in order to obtain insights into the prevalence of lyssaviruses in Croatian bat populations and to improve our understanding of the public health threat of infected bats. RESULTS: In total, 455 bats were caught throughout Continental and Mediterranean Croatia. Antibodies were found in 20 of 350 bats (5.71%, 95% confidence interval 3.73-8.66). The majority of seropositive bats were found in Trbusnjak cave (Continental Croatia, Eastern part), and most seropositive bats belonged to Myotis myotis (13/20). All oropharyngeal swabs were negative for the presence of Lyssavirus. CONCLUSIONS: The presence of lyssaviruses in bat populations was confirmed for the first time in Croatia and Southeastern Europe. The results of this study suggest the need for further comprehensive analyses of lyssaviruses in bats in this part of Europe.

Potentially Zoonotic Bartonella in Bats from France and Spain.

We detected Bartonella in 11 of 109 insectivorous bats from France and 1 of 26 bats from Spain. These genetic variants are closely related to bat-associated Bartonella described in Finland and the United Kingdom and to B. mayotimonensis, the agent of a human endocarditis case in the United States.

Bat rabies surveillance in France: first report of unusual mortality among serotine bats.

BACKGROUND: Rabies is a fatal viral encephalitic disease that is caused by lyssaviruses which can affect all mammals, including human and bats. In Europe, bat rabies cases are attributed to five different lyssavirus species, the majority of rabid bats being attributed to European bat 1 lyssavirus (EBLV-1), circulating mainly in serotine bats (Eptesicus serotinus). In France, rabies in bats is under surveillance since 1989, with 77 positive cases reported between 1989 and 2016. CASE PRESENTATION: In the frame of the bat rabies surveillance, an unusual mortality of serotine bats was reported in 2009 in a village in North-East France. Six juvenile bats from an E. serotinus maternity colony counting ~200 individuals were found to be infected with EBLV-1. The active surveillance of the colony by capture sessions of bats from July to September 2009 showed a high detection rate of neutralising EBLV-1 antibodies (≈ 50%) in the colony. Moreover, one out of 111 animals tested was found to shed viable virus in saliva, while lyssavirus RNA was detected by RT-PCR for five individuals. CONCLUSION: This study demonstrated that the lyssavirus infection in the serotine maternity colony was followed by a high rate of bat rabies immunity after circulation of the virus in the colony. The ratio of seropositive bats is probably indicative of an efficient virus transmission coupled to a rapid circulation of EBLV-1 in the colony.

Molecular Characterization of Canine Rabies Virus, Mali, 2006-2013.

We genetically characterized 32 canine rabies viruses isolated in Mali during 2006-2013 and identified 3 subgroups that belonged to the Africa 2 lineage. We also detected subgroup F rabies virus. This information should be useful for development of mass vaccination campaigns for dogs and eventual large-scale control programs in this country.

Isolation of Bokeloh bat lyssavirus in Myotis nattereri in France.

Bokeloh bat lyssavirus (BBLV) was found in Myotis nattereri for the first time in northeastern France in July 2012. The complete genome sequence of the virus from the infected Natterer's bat was determined by whole-genome sequencing and compared to that of the first BBLV strain isolated in 2010 in Germany and with those of all currently identified lyssaviruses. The French isolate [KC169985] showed 98.7 % nucleotide sequence identity to the German BBLV strain [JF311903]. Several organs of the infected French bat were examined by classical rabies diagnostic methods: fluorescent antibody test, cell culture inoculation test and RT-qPCR. Antigen, infectious virus and high viral RNA levels were found in both the brain and salivary glands. Traces of genomic RNA were detected in the bladder, kidney and lung tissue. The results of an investigation of the distribution of lyssaviruses with the detection of infectious virus in the salivary glands suggest a possible mode of transmission of the virus.

Molecular characterisation of rabies virus strains in the Republic of Macedonia.

Rabies, a worldwide zoonosis, remains a public-health concern despite oral wildlife vaccination in Europe. After a ten-year break, Macedonia reported eight rabies cases in 2011-2012. Two countries (Serbia and Bulgaria) bordering Macedonia are reporting cases in domestic and wild animals. This report describes the genetic characterisation of eight isolates from Macedonia compared with representative samples from neighbouring countries. All of the isolates tested belong to the Eastern European group, with a high degree of nucleotide sequence identity in the nucleoprotein gene. The close genetic relationship between isolates from the three bordering countries suggests that wildlife is responsible for rabies movements in the region.

Assessment of virus and Leptospira carriage in bats in France.

With over 1,400 species worldwide, bats represent the second largest order of mammals after rodents, and are known to host major zoonotic pathogens. Here, we estimate the presence of pathogens in autochthonous bat populations. First, we set out to check our samples for PCR amplification efficiency by assessing the occurrence of inhibited PCR reactions from different types of bat samples with amplifying the housekeeping gene ß-actin. Second, we investigated the presence of five targeted pathogens in a French bat population using PCR. We targeted viral RNA of Canine distemper virus, Alphacoronavirus, Lyssavirus, Rotavirus and bacterial Leptospira DNA. To do so, we screened for these viruses in bat faecal samples as well as in oropharyngeal swab samples. The presence of Leptospira was assessed in urine, kidney, lung and faecal samples. Results showed a frequency of inhibited reactions ranging from 5 to 60% of samples, varying according to the sample itself and also suspected to vary according to sampling method and the storage buffer solution used, demonstrating the importance of the sampling and storage on the probability of obtaining negative PCR results. For pathogen assessment, rotavirus and alphacoronavirus RNA were detected in Myotis myotis, Myotis daubentonii, Myotis emarginatus and Rhinolophus ferrumequinum bats. Rotaviruses were also detected in Barbastella barbastellus. The presence of alphacoronavirus also varied seasonally, with higher frequencies in late summer and October, suggesting that juveniles potentially play an important role in the dynamics of these viruses. Leptospira DNA was detected in M. myotis and M. daubentonii colonies. The 16S rRNA sequences obtained from Leptospira positive samples showed 100% genetic identity with L. borgpetersenii. Neither canine distemper virus nor lyssavirus RNA were detected in any of the tested samples. This study is the first to show the presence of Leptospira in autochthonous French bats in addition to coronavirus and rotavirus RNA previously reported in European autochthonous bats.

Investigations on SARS-CoV-2 and other coronaviruses in mink farms in France at the end of the first year of COVID-19 pandemic.

Soon after the beginning of the COVID-19 pandemic in early 2020, the Betacoronavirus SARS-CoV-2 infection of several mink farms breeding American minks ( Neovison vison ) for fur was detected in several countries of Europe. The risk of a new reservoir formation and of a reverse zoonosis from minks was then a major concern. The aim of this study was to investigate the four French mink farms for the circulation of SARS-CoV-2 at the end of 2020. The investigations took place during the slaughtering period thus facilitating different types of sampling (swabs and blood). In one of the four mink farms, 96.6% of serum samples were positive in SARS-CoV-2 ELISA coated with purified N protein recombinant antigen and 54 out of 162 (33%) pharyngo-tracheal swabs were positive by RT-qPCR. The genetic variability among 12 SARS-CoV-2 genomes sequenced in this farm indicated the co-circulation of several lineages at the time of sampling. All SARS-CoV-2 genomes detected were nested within the 20A clade (Nextclade), together with SARS-CoV-2 genomes from humans sampled at the same period. The percentage of SARS-CoV-2 seropositivity by ELISA varied between 0.5 and 1.2% in the three other farms. Interestingly, among these three farms, 11 pharyngo-tracheal swabs and 3 fecal pools from two farms were positive by end-point RT-PCR for an Alphacoronavirus highly similar to a mink coronavirus sequence observed in Danish farms in 2015. In addition, a mink Caliciviridae was identified in one of the two positive farms for Alphacoronavirus . The clinical impact of these unapparent viral infections is not known. The co-infection of SARS-CoV-2 with other viruses in mink farms could contribute to explain the diversity of clinical symptoms noted in different infected farms in Europe. In addition, the co-circulation of an Alphacoronavirus and SARS-CoV-2 within a mink farm would increase potentially the risk of viral recombination between alpha and betacoronaviruses already suggested in wild and domestic animals, as well as in humans. Author summary: France is not a country of major mink fur production. Following the SARS-CoV-2 contamination of mink farms in Denmark and the Netherlands, the question arose for the four French farms.The investigation conducted at the same time in the four farms revealed the contamination of one of them by a variant different from the one circulating at the same time in Denmark and the Netherlands mink farms. Investigation of three other farms free of SARS-CoV-2 contamination revealed the circulation of other viruses including a mink Alphacoronavirus and Caliciviridae , which could modify the symptomatology of SARS-CoV-2 infection in minks.

Investigations into SARS-CoV-2 and other coronaviruses on mink farms in France late in the first year of the COVID-19 pandemic.

Soon after the beginning of the COVID-19 pandemic in early 2020, the Betacoronavirus SARS-CoV-2 infection of several mink farms breeding American minks (Neovison vison) for fur was detected in various European countries. The risk of a new reservoir being formed and of a reverse zoonosis from minks quickly became a major concern. The aim of this study was to investigate the four French mink farms to see whether SARS-CoV-2 was circulating there in late 2020. The investigations took place during the slaughtering period, thus facilitating different types of sampling (swabs and blood). On one of the four mink farms, 96.6% of serum samples were positive when tested with a SARS-CoV-2 ELISA coated with purified N protein recombinant antigen, and 54 out of 162 (33%) pharyngo-tracheal swabs were positive by RT-qPCR. The genetic variability among 12 SARS-CoV-2 genomes sequenced from this farm indicated the co-circulation of several lineages at the time of sampling. All the SARS-CoV-2 genomes detected were nested within the 20A clade (Nextclade), together with SARS-CoV-2 genomes from humans sampled during the same period. The percentage of SARS-CoV-2 seropositivity by ELISA varied between 0.3 and 1.1% on the other three farms. Interestingly, among these three farms, 11 pharyngo-tracheal swabs and 3 fecal pools from two farms were positive by end-point RT-PCR for an Alphacoronavirus very similar to a mink coronavirus sequence observed on Danish farms in 2015. In addition, a mink Caliciviridae was identified on one of the two farms positive for Alphacoronavirus. The clinical impact of these inapparent viral infections is not known. The co-infection of SARS-CoV-2 with other viruses on mink farms could help explain the diversity of clinical symptoms noted on different infected farms in Europe. In addition, the co-circulation of an Alphacoronavirus and SARS-CoV-2 on a mink farm would potentially increase the risk of viral recombination between alpha and betacoronaviruses as already suggested in wild and domestic animals, as well as in humans.

Whole genome sequencing and phylogenetic characterisation of rabies virus strains from Moldova and north-eastern Romania.

BACKGROUND: Rabies is the oldest fatal zoonotic disease recognised as a neglected tropical disease and is caused by an RNA virus belonging to the genus Lyssavirus, family Rhabdoviridae. METHODOLOGY/PRINCIPAL FINDINGS: A deep molecular analysis was conducted on full-length nucleoprotein (N) gene and whole genome sequences of rabies virus from 37 animal brain samples collected between 2012 and 2017 to study the circulation of rabies virus (RABV) variants. The overall aim was to better understand their distribution in Moldova and north-eastern Romania. Both Sanger and high throughput sequencing on Ion Torrent and Illumina platforms were performed. Phylogenetic analysis of the RABV sequences from both Moldova and Romania revealed that all the samples (irrespective of the year of isolation and the species) belonged to a single phylogenetic group: north-eastern Europe (NEE), clustering into three assigned lineages: RO#5, RO#6 and RO#7. CONCLUSIONS/SIGNIFICANCE: High throughput sequencing of RABV samples from domestic and wild animals was performed for the first time for both countries, providing new insights into virus evolution and epidemiology in this less studied region, expanding our understanding of the disease.

OVX033, a nucleocapsid-based vaccine candidate, provides broad-spectrum protection against SARS-CoV-2 variants in a hamster challenge model.

Spike-based COVID-19 vaccines induce potent neutralizing antibodies but their efficacy against SARS-CoV-2 variants decreases. OVX033 is a recombinant protein composed of the full-length nucleocapsid (N) protein of SARS-CoV-2 genetically fused to oligoDOM®, a self-assembling domain which improves antigen immunogenicity. OVX033 including N as an antigenic target is proposed as new vaccine candidate providing broad-spectrum protection against sarbecoviruses. OVX033 demonstrated its ability to trigger cross-reactive T cell responses and cross-protection against three variants of SARS-CoV-2 (B.1 Europe, Delta B.1.617.2, and Omicron B.1.1.529) in a hamster challenge model, as evidenced by lower weight loss, lower lung viral loads, and reduced lung histopathological lesions.

Evaluation of a Rapid Immunochromatographic Diagnostic Test for the detection of rabies from brain material of European mammals.

The surveillance of rabies relies on investigations conducted on dead suspected animals or animals showing clinical signs suggestive of rabies. An immunochromatographic method based on lateral flow principle has been evaluated against a collection of brain samples mainly of European mammals including bats. The performance of this new test has been compared to the conventional gold standard methods: the fluorescent Antibody Test (FAT) and the Rapid Tissue Culture Infection Test (RTCIT). This test enabled the detection of various rabies strains belonging to rabies species 1, 5, 6 and 7 and demonstrated an overall specificity of 100% and a sensitivity of more than 88% when compared to FAT and RTCIT. A total agreement between the Rapid Immunochromatographic Diagnostic Test and conventional technique results have been obtained for European bat samples.

Genetic identification of bat species for pathogen surveillance across France.

With more than 1400 chiropteran species identified to date, bats comprise one-fifth of all mammalian species worldwide. Many studies have associated viral zoonoses with 45 different species of bats in the EU, which cluster within 5 families of bats. For example, the Serotine bats are infected by European Bat 1 Lyssavirus throughout Europe while Myotis bats are shown infected by coronavirus, herpesvirus and paramyxovirus. Correct host species identification is important to increase our knowledge of the ecology and evolutionary pattern of bat viruses in the EU. Bat species identification is commonly determined using morphological keys. Morphological determination of bat species from bat carcasses can be limited in some cases, due to the state of decomposition or nearly indistinguishable morphological features in juvenile bats and can lead to misidentifications. The overall objective of our study was to identify insectivorous bat species using molecular biology tools with the amplification of the partial cytochrome b gene of mitochondrial DNA. Two types of samples were tested in this study, bat wing punches and bat faeces. A total of 163 bat wing punches representing 22 species, and 31 faecal pellets representing 7 species were included in the study. From the 163 bat wing punches tested, a total of 159 were genetically identified from amplification of the partial cyt b gene. All 31 faecal pellets were genetically identified based on the cyt b gene. A comparison between morphological and genetic determination showed 21 misidentifications from the 163 wing punches, representing ~12.5% of misidentifications of morphological determination compared with the genetic method, across 11 species. In addition, genetic determination allowed the identification of 24 out of 25 morphologically non-determined bat samples. Our findings demonstrate the importance of a genetic approach as an efficient and reliable method to identify bat species precisely.

Multi-annual performance evaluation of laboratories in post-mortem diagnosis of animal rabies: Which techniques lead to the most reliable results in practice?

Rabies diagnosis proficiency tests on animal specimens using four techniques (FAT, RTCIT, conventional RT-PCR and real-time RT-PCR) were organised over 10 years (2009-2019). Seventy-three laboratories, of which 59% were from Europe, took part. As the panels were prepared with experimentally-infected samples, the error rate of laboratories on positive and negative samples was accurately estimated. Based on fitted values produced by mixed modelling including the variable "laboratory" as a random variable to take into account the longitudinal design of our dataset, the technique that provided the most concordant results was conventional RT-PCR (99.3%; 95% CI 99.0-99.6), closely followed by FAT (99.1%; 95% CI 98.7-99.4), real-time RT-PCR (98.7%; 95% CI 98.1-99.3) and then RTCIT (96.8%; 95% CI 95.8-97.7). We also found that conventional RT-PCR provided a better diagnostic sensitivity level (99.3% ±4.4%) than FAT (98.7% ±1.6%), real-time RT-PCR (97.9% ±0.8%) and RTCIT (95.3% ±5.1%). Regarding diagnostic specificity, RTCIT was the most specific technique (96.4% ±3.9%) followed closely by FAT (95.6% ±3.8%), real-time RT-PCR (95.0% ±1.8%) and conventional RT-PCR (92.9% ±0.5%). Due to multiple testing of the samples with different techniques, the overall diagnostic conclusion was also evaluated, and found to reach an inter-laboratory concordance level of 99.3%. The concordance for diagnostic sensitivity was 99.6% ±2.0% and for diagnostic specificity, 98.0% ±8.5%. Molecular biology techniques were, however, found to be less specific than expected. The potential reasons for such findings are discussed herein. The regular organisation of performance tests has contributed to an increase in the performance of participating laboratories over time, demonstrating the benefits of such testing. Maintaining a high-quality rabies diagnosis capability on a global scale is key to achieving the goal of eliminating dog-mediated human rabies deaths. The regular organisation of exercises on each continent using selected local strains to be tested according to the local epidemiological situation is one factor that could help increase reliable diagnosis worldwide. Rabies diagnosis capabilities could indeed be enhanced by providing adequate and sustainable proficiency testing on a large scale and in the long term.

Viral Metagenomic Profiling of Croatian Bat Population Reveals Sample and Habitat Dependent Diversity.

To date, the microbiome, as well as the virome of the Croatian populations of bats, was unknown. Here, we present the results of the first viral metagenomic analysis of guano, feces and saliva (oral swabs) of seven bat species (Myotis myotis, Miniopterus schreibersii, Rhinolophus ferrumequinum, Eptesicus serotinus, Myotis blythii, Myotis nattereri and Myotis emarginatus) conducted in Mediterranean and continental Croatia. Viral nucleic acids were extracted from sample pools, and analyzed using Illumina sequencing. The presence of 63 different viral families representing all seven Baltimore groups were confirmed, most commonly insect viruses likely reflecting the diet of insectivorous bats. Virome compositions of our samples were largely impacted by the sample type: invertebrate-infecting viruses were most frequently found in feces, bacterial viruses in guano, whereas vertebrate-infecting viruses were most common in swabs. Most vertebrate-infecting virus sequences were assigned to retroviruses, parvoviruses, iridoviruses, and poxviruses. We further report the complete genome sequence of a novel adeno-associated virus, densovirus and a near complete length genome sequence of a novel iflavirus. Additionally, one of the most interesting findings in this study was the difference in viromes between two contrasting habitats, the continental and Mediterranean Croatia.