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.

Potency of Veterinary Rabies Vaccines Marketed in Sri Lanka.

Seven brands of veterinary rabies vaccines are commercially available in Sri Lanka, but there is no established procedure to test the potency of the vaccines at the local level, especially prior to their release. The aim of this study was to test the potency of these vaccines using a mouse challenge test in collaboration with the EU/WOAH/WHO Reference Laboratory for Rabies, ANSES-Nancy, France. Based on the European Pharmacopoeia, the inactivated rabies vaccines complied with the mouse potency test if the estimated potency is ≥1.0 IU in the smallest prescribed dose. Among the eight tested vaccines, four single-dose preparations (Rabisin™, Raksharab™, Nobivac™ RL, and Nobivac™ Rabies) were compliant, with potencies of 12 IU/dose, 7.2 IU/dose, 4.4 IU/dose, and 3.4 IU/dose, respectively. Three of the single-dose preparations (Canvac™ R, Defensor™ 3, and Rabies killed vaccine) were not compliant, with potency values <1.0 IU/dose. One multidose preparation (Raksharab™ multidose) had a potency of 1.3 IU/dose, even though the test was not validated. Based on these results, it appears that some rabies vaccine batches that are currently available in the local market do not comply with the mouse potency test. Testing the vaccines' potency before registration and release to the market appears to be an important step to allow good immunization to animals during pre-exposure vaccination programs.

EPAC1 Pharmacological Inhibition with AM-001 Prevents SARS-CoV-2 and Influenza A Virus Replication in Cells.

The exceptional impact of the COVID-19 pandemic has stimulated an intense search for antiviral molecules. Host-targeted antiviral molecules have the potential of presenting broad-spectrum antiviral activity and are also considered as less likely to select for resistant viruses. In this study, we investigated the antiviral activity exerted by AM-001, a specific pharmacological inhibitor of EPAC1, a host exchange protein directly activated by cyclic AMP (cAMP). The cAMP-sensitive protein, EPAC1 regulates various physiological and pathological processes but its role in SARS-CoV-2 and influenza A virus infection has not yet been studied. Here, we provide evidence that the EPAC1 specific inhibitor AM-001 exerts potent antiviral activity against SARS-CoV-2 in the human lung Calu-3 cell line and the African green monkey Vero cell line. We observed a concentration-dependent inhibition of SARS-CoV-2 infectious viral particles and viral RNA release in the supernatants of AM-001 treated cells that was not associated with a significant impact on cellular viability. Furthermore, we identified AM-001 as an inhibitor of influenza A virus in Calu-3 cells. Altogether these results identify EPAC1 inhibition as a promising therapeutic target against viral infections.

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.

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.

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.

Further Evidence of Inadequate Quality in Lateral Flow Devices Commercially Offered for the Diagnosis of Rabies.

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.

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.

Proficiency test for rabies serology: A design complying with international standards for a reliable assessment of participating laboratories.

BACKGROUND: Domestic carnivores can introduce rabies into disease-free countries or areas if they are incubating the disease and transported during the pre-symptomatic period. For pets moved into the European Union, the European Commission decided to establish a system of community approval of laboratories willing to carry out the rabies serological controls to guarantee an effective control system. As the specific institute to coordinate the approval of the laboratories, designated by the European Commission in 2000, our laboratory organizes annual proficiency tests (PT) for laboratories already agreed or willing to be agreed to perform rabies serological controls (by detecting rabies virus neutralizing antibodies only) in the frame of international trade. METHODOLOGY/PRINCIPAL FINDINGS: The assessment criteria of this PT rely on the analysis of the specificity and the intra-laboratory consistency. The approach used to evaluate the degree of laboratory consistency is based on the use of compiled data obtained from previous PT campaigns, and is measured by the quality of a regression model. By using historical data for calculating assigned values and associated standard deviations, instead of values obtained from only one campaign, they became robust without any additional statistical treatment. In the present paper, more than 800 historical values were compiled for each of the regression parameters. CONCLUSIONS/SIGNIFICANCE: Since the beginning of these PT schemes in 1999, the overall percentage of failing laboratories remained stable over the years (4.1%) while the number of participants increased to 79 in 2018. This highlighted the robustness and the consistency of the statistical analyses used to assess the laboratory's performance over the years. The improvements carried out and the consistency of our statistical analyses have resulted in the compliance of the rabies serology PT with the ISO/IEC 17043 and ISO 13528:2015 International Standards.

Cross-Protection of Inactivated Rabies Vaccines for Veterinary Use against Bat Lyssaviruses Occurring in Europe.

Human rabies vaccines have been shown to induce partial protection against members of phylogroup I bat lyssaviruses. Here, we investigated the capacity of a widely used rabies inactivated vaccine (Rabisin, Boehringer-Ingelheim) for veterinary use to cross-protect mice experimentally infected with European bat lyssavirus 1 (EBLV-1b), European bat lyssavirus 2 (EBLV-2), and Bokeloh bat lyssavirus (BBLV) occurring in Europe. For each lyssavirus, we investigated the efficacy of two different doses of vaccine against two viral doses administrated by either central or peripheral routes. In parallel, seroconversion following pre-exposure vaccination was investigated. In this study, we demonstrated that the three investigated bat isolates were pathogenic, even at low dose, when inoculated by the central route but were not/less pathogenic when administrated peripherally. The Rabisin vaccine was capable of significantly cross-protecting mice inoculated intramuscularly with EBLV-1b and EBLV-2 and intracerebrally with BBLV. The level of rabies neutralizing antibodies induced by the Rabisin was quite high against the bat lyssaviruses, but with no significant differences between immunization with 1 and 5 IU/dose. The study emphasizes that the quality of rabies-inactivated vaccines for veterinary use is of utmost importance to optimize the cross-protection of pets against phylogroup I bat lyssaviruses occurring in Europe.

An inter-laboratory comparison to evaluate the technical performance of rabies diagnosis lateral flow assays.

As in previous years, the European Union Reference Laboratory (EURL) for rabies organised in 2018 an Inter-laboratory trial (ILT) on rabies diagnosis. Contrarily to past years, the 2018 ILT did not aim to evaluate the performance of participating laboratories, but the technical performance of new rapid tests. Two lateral Flow Assays (LFA), namely the Anigen® and the CDIA™ Rabies Virus Antigen Rapid Test" (commercialized by Bionote and Creative Diagnostics Cie respectively), were evaluated together with the Fluorescent Antibody Test (FAT). One panel of virus samples (including RABV as well as EBLV1a, EBLV-1b, and EBLV2 strains) was sent to participating laboratories to compare results obtained with these different techniques. The study revealed that the FAT provided a good agreement toward expected results for both negative/positive samples (99.1%). The Anigen® test produced similar results to the FAT, with only one false negative result (0.5%) reported by all participants and a concordance of 100% for all but one sample demonstrating a good inter-laboratory reproducibility of the Anigen® batch. The CDIA™ test produced reproducible results for Rabies Virus (RABV) samples only. However, it hardly detected the Bokeloh Bat Lyssavirus (BBLV) and the European Bat Lyssaviruses types 1b and 2 (EBLV-1b and EBLV-2) in most laboratories resulting in a moderate inter-laboratory concordance (58.4%-82.7%) for these lyssaviruses. The two LFAs provided reliable and reproducible results on all RABV samples (100%) but lead to heterogeneous performances with other lyssaviruses leading to different levels of diagnostic/analytical sensitivity, specificity. The study confirmed that LFAs should be used with caution and that their validation are of upmost importance before any use in laboratories.

Lleida Bat Lyssavirus isolation in Miniopterus schreibersii in France.

Bat rabies cases are attributed in Europe to five different Lyssavirus species of 16 recognized Lyssavirus species causing rabies. One of the most genetically divergent Lyssavirus spp. has been detected in a dead Miniopterus schreibersii bat in France. Brain samples were found positive for the presence of antigen, infectious virus and viral RNA by classical virological methods and molecular methods respectively. The complete genome sequence was determined by next-generation sequencing. The analysis of the complete genome sequence confirmed the presence of Lleida bat lyssavirus (LLEBV) in bats in France with 99.7% of nucleotide identity with the Spanish LLEBV strain (KY006983).

Additional Progress in the Development and Application of a Direct, Rapid Immunohistochemical Test for Rabies Diagnosis.

Laboratory-based surveillance is fundamental to effective rabies prevention and control. The direct fluorescent antibody (AB) test (FAT) is the gold standard for rabies diagnosis. Recently, additional tests besides the FAT have been developed, such as the direct rapid immunohistochemical test (DRIT). In this study, our objective was to further refine technical aspects of the DRIT using a combination of two monoclonal ABs (MABs), 502 and 802, conduct additional testing among rabies reference laboratories using a diversity of animal species and rabies virus (RV) variants and compare the potential utility of the DRIT for end users via proficiency testing (PT) against the FAT. Considering the ideal molar ratios of biotin to AB in formulation of the DRIT conjugate, 3.9 was found to be superior to 7.4, for detection of RV antigens in the brain of a naturally infected raccoon. Optimization of the DRIT conjugate may also be dependent upon the apparent choice of specific viral antigens for testing, as a gray fox RV variant reacted less strongly than a raccoon RV variant in determining the working dilution of the MAB cocktail. Using the same MABs and protocol, the DRIT was compared to the FAT using more than 800 samples of mammalian brains, representative of more than 25 taxa, including in excess of 250 animal rabies cases from Europe and North America. Sensitivity was determined at 98% (96⁻100%, 95% CI) and specificity was calculated at 95% (92⁻96%, 95% CI). In a comparison among end users, PT of laboratory personnel resulted in values of 77⁻100% sensitivity and 86-100% specificity. Based upon these and previously reported results, the DRIT appears to be a suitable alternative to the FAT for use in lyssavirus diagnosis.

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.

Longitudinal survey of two serotine bat (Eptesicus serotinus) maternity colonies exposed to EBLV-1 (European Bat Lyssavirus type 1): Assessment of survival and serological status variations using capture-recapture models.

This study describes two longitudinal serological surveys of European Bat Lyssavirus type 1 (EBLV-1) antibodies in serotine bat (Eptesicus serotinus) maternity colonies located in the North-East of France. This species is currently considered as the main EBLV-1 reservoir. Multievent capture-recapture models were used to determine the factors influencing bat rabies transmission as this method accounts for imperfect detection and uncertainty in disease states. Considering the period of study, analyses revealed that survival and recapture probabilities were not affected by the serological status of individuals, confirming the capacity of bats to be exposed to lyssaviruses without dying. Five bats have been found with EBLV-1 RNA in the saliva at the start of the study, suggesting they were caught during virus excretion period. Among these bats, one was interestingly recaptured one year later and harbored a seropositive status. Along the survey, some others bats have been observed to both seroconvert (i.e. move from a negative to a positive serological status) and serorevert (i.e. move from a positive to a negative serological status). Peak of seroprevalence reached 34% and 70% in site A and B respectively. On one of the 2 sites, global decrease of seroprevalence was observed all along the study period nuanced by oscillation intervals of approximately 2-3 years supporting the oscillation infection dynamics hypothesized during a previous EBLV-1 study in a Myotis myotis colony. Seroprevalence were affected by significantly higher seroprevalence in summer than in spring. The maximum time observed between successive positive serological statuses of a bat demonstrated the potential persistence of neutralizing antibodies for at least 4 years. At last, EBLV-1 serological status transitions have been shown driven by age category with higher seroreversion frequencies in adults than in juvenile. Juveniles and female adults seemed indeed acting as distinct drivers of the rabies virus dynamics, hypothesis have been addressed but their exact role in the EBLV-1 transmission still need to be specified.

Comparison of G protein sequences of South African street rabies viruses showing distinct progression of the disease in a mouse model of experimental rabies.

Rabies is a fatal zoonotic disease and infections generally lead to a fatal encephalomyelitis in both humans and animals. In South Africa, domestic (dogs) and the wildlife (yellow mongoose) host species maintain the canid and mongoose rabies variants respectively. In this study, pathogenicity differences of South African canid and mongoose rabies viruses were investigated in a murine model, by assessing the progression of clinical signs and survivorship. Comparison of glycoprotein gene sequences revealed amino acid differences that may underpin the observed pathogenicity differences. Cumulatively, our results suggest that the canid rabies virus may be more neurovirulent in mice than the mongoose rabies variant.

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.