[Competitive ELISA test system for the detection of antibodies to the rabies virus in animals (Rhabdoviridae: Lyssavirus)].

INTRODUCTION: The main approach to the rabies prevention is the vaccination of domestic and wild carnivores. For the routine evaluation the anti-rabies vaccination effectiveness, World Organization for Animal Health (OIE) recommends various enzyme-linked immunosorbent assays (ELISA).The aim of the study was to design and validate a competitive ELISA (cELISA) test system for the detection of antibodies to the rabies virus (RABV). MATERIALS AND METHODS: The development of the cELISA was carried out following the OIE recommendations. RESULTS: The repeatability of the cELISA results within one laboratory was satisfactory (coefficient of variation 7.95-13.61%). The coefficient of determination (CD) between the results of the virus neutralization reaction (FAVN) and cELISA was 0.988, p < 0.001. The lower threshold for antibody detection was less than 0.02 IU/ml. The cELISA did not demonstrate cross-reactivity against antibodies to canine distemper virus, parainfluenza virus, parvovirus, coronavirus, and canine adenovirus (types I and II). During the study of 137 dog blood sera, diagnostic specificity (DSp) and diagnostic sensitivity (DSe) for the cELISA were 83.1% and 94.9%, respectively, and CD between the cELISA and FAVN results was 0.968, p < 0.001. DISCUSSION: Indirect ELISA test systems for determining the level of antibodies to RABV are not sensitive enough compared to reference tests, unlike cELISA. The developed test system is not inferior for its DSp and DSe to the commercial cELISA BioPro ELISA Rabies Ab (DSp 66.7%, DSe 94.4%). CONCLUSION: The developed cELISA test system can be used to detect antibodies to RABV in the blood serum of dogs for evaluating the effectiveness of mass vaccination programs.

An inactivated recombinant rabies virus chimerically expressed RBD induces humoral and cellular immunity against SARS-CoV-2 and RABV.

Many studies suggest that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect various animals and transmit among animals, and even to humans, posing a threat to humans and animals. There is an urgent need to develop inexpensive and efficient animal vaccines to prevent and control coronavirus disease 2019 (COVID-19) in animals. Rabies virus (RABV) is another important zoonotic pathogen that infects almost all warm-blooded animals and poses a great public health threat. The present study constructed two recombinant chimeric viruses expressing the S1 and RBD proteins of the SARS-CoV-2 Wuhan01 strain based on a reverse genetic system of the RABV SRV9 strain and evaluated their immunogenicity in mice, cats and dogs. The results showed that both inactivated recombinant viruses induced durable neutralizing antibodies against SARS-CoV-2 and RABV and a strong cellular immune response in mice. Notably, inactivated SRV-nCoV-RBD induced earlier antibody production than SRV-nCoV-S1, which was maintained at high levels for longer periods. Inactivated SRV-nCoV-RBD induced neutralizing antibodies against both SARS-CoV-2 and RABV in cats and dogs, with a relatively broad-spectrum cross-neutralization capability against the SARS-CoV-2 pseudoviruses including Alpha, Beta, Gamma, Delta, and Omicron, showing potential to be used as a safe bivalent vaccine candidate against COVID-19 and rabies in animals.

RVG Peptide-Functionalized Favipiravir Nanoparticle Delivery System Facilitates Antiviral Therapy of Neurotropic Virus Infection in a Mouse Model.

Neurotropic viruses severely damage the central nervous system (CNS) and human health. Common neurotropic viruses include rabies virus (RABV), Zika virus, and poliovirus. When treating neurotropic virus infection, obstruction of the blood-brain barrier (BBB) reduces the efficiency of drug delivery to the CNS. An efficient intracerebral delivery system can significantly increase intracerebral delivery efficiency and facilitate antiviral therapy. In this study, a rabies virus glycopeptide (RVG) functionalized mesoporous silica nanoparticle (MSN) packaging favipiravir (T-705) was developed to generate T-705@MSN-RVG. It was further evaluated for drug delivery and antiviral treatment in a VSV-infected mouse model. The RVG, a polypeptide consisting of 29 amino acids, was conjugated on the nanoparticle to enhance CNS delivery. The T-705@MSN-RVG caused a significant decrease in virus titers and virus proliferation without inducing substantial cell damage in vitro. By releasing T-705, the nanoparticle promoted viral inhibition in the brain during infection. At 21 days post-infection (dpi), a significantly enhanced survival ratio (77%) was observed in the group inoculated with nanoparticle compared with the non-treated group (23%). The viral RNA levels were also decreased in the therapy group at 4 and 6 dpi compared with that of the control group. The T-705@MSN-RVG could be considered a promising system for CNS delivery for treating neurotropic virus infection.

Transferrin Receptor Protein 1 Cooperates with mGluR2 To Mediate the Internalization of Rabies Virus and SARS-CoV-2.

Identification of bona fide functional receptors and elucidation of the mechanism of receptor-mediated virus entry are important to reveal targets for developing therapeutics against rabies virus (RABV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our previous studies suggest that metabotropic glutamate receptor subtype 2 (mGluR2) functions as an entry receptor for RABV in vitro, and is an important internalization factor for SARS-CoV-2 in vitro and in vivo. Here, we demonstrate that mGluR2 facilitates RABV internalization in vitro and infection in vivo. We found that transferrin receptor 1 (TfR1) interacts with mGluR2 and internalizes with mGluR2 and RABV in the same clathrin-coated pit. Knockdown of TfR1 blocks agonist-triggered internalization of mGluR2. Importantly, TfR1 also interacts with the SARS-CoV-2 spike protein and is important for SARS-CoV-2 internalization. Our findings identify a novel axis (mGluR2-TfR1 axis) used by RABV and SARS-CoV-2 for entry, and reveal TfR1 as a potential target for therapeutics against RABV and SARS-CoV-2. IMPORTANCE We previously found that metabotropic glutamate receptor subtype 2 (mGluR2) is an entry receptor for RABV in vitro, and an important internalization factor for SARS-CoV-2 in vitro and in vivo. However, whether mGluR2 is required for RABV infection in vivo was unknown. In addition, how mGluR2 mediates the internalization of RABV and SARS-CoV-2 needed to be resolved. Here, we found that mGluR2 gene knockout mice survived a lethal challenge with RABV. To our knowledge, mGluR2 is the first host factor to be definitively shown to play an important role in RABV street virus infection in vivo. We further found that transferrin receptor protein 1 (TfR1) directly interacts and cooperates with mGluR2 to regulate the endocytosis of RABV and SARS-CoV-2. Our study identifies a novel axis (mGluR2-TfR1 axis) used by RABV and SARS-CoV-2 for entry and opens a new door for the development of therapeutics against RABV and SARS-CoV-2.

Drivers of Spatial Expansions of Vampire Bat Rabies in Colombia.

Spatial expansions of vampire bat-transmitted rabies (VBR) are increasing the risk of lethal infections in livestock and humans in Latin America. Identifying the drivers of these expansions could improve current approaches to surveillance and prevention. We aimed to identify if VBR spatial expansions are occurring in Colombia and test factors associated with these expansions. We analyzed 2336 VBR outbreaks in livestock reported to the National Animal Health Agency (Instituto Colombiano Agropecuario-ICA) affecting 297 municipalities from 2000-2019. The area affected by VBR changed through time and was correlated to the reported number of outbreaks each year. Consistent with spatial expansions, some municipalities reported VBR outbreaks for the first time each year and nearly half of the estimated infected area in 2010-2019 did not report outbreaks in the previous decade. However, the number of newly infected municipalities decreased between 2000-2019, suggesting decelerating spatial expansions. Municipalities infected later had lower cattle populations and were located further from the local reporting offices of the ICA. Reducing the VBR burden in Colombia requires improving vaccination coverage in both endemic and newly infected areas while improving surveillance capacity in increasingly remote areas with lower cattle populations where rabies is emerging.

Characterization of the Immune Response of MERS-CoV Vaccine Candidates Derived from Two Different Vectors in Mice.

Middle East respiratory syndrome (MERS) is an acute, high-mortality-rate, severe infectious disease caused by an emerging MERS coronavirus (MERS-CoV) that causes severe respiratory diseases. The continuous spread and great pandemic potential of MERS-CoV make it necessarily important to develop effective vaccines. We previously demonstrated that the application of Gram-positive enhancer matrix (GEM) particles as a bacterial vector displaying the MERS-CoV receptor-binding domain (RBD) is a very promising MERS vaccine candidate that is capable of producing potential neutralization antibodies. We have also used the rabies virus (RV) as a viral vector to design a recombinant vaccine by expressing the MERS-CoV S1 (spike) protein on the surface of the RV. In this study, we compared the immunological efficacy of the vaccine candidates in BALB/c mice in terms of the levels of humoral and cellular immune responses. The results show that the rabies virus vector-based vaccine can induce remarkably earlier antibody response and higher levels of cellular immunity than the GEM particles vector. However, the GEM particles vector-based vaccine candidate can induce remarkably higher antibody response, even at a very low dose of 1 µg. These results indicate that vaccines constructed using different vaccine vector platforms for the same pathogen have different rates and trends in humoral and cellular immune responses in the same animal model. This discovery not only provides more alternative vaccine development platforms for MERS-CoV vaccine development, but also provides a theoretical basis for our future selection of vaccine vector platforms for other specific pathogens.

A Single Dose of the Deactivated Rabies-Virus Vectored COVID-19 Vaccine, CORAVAX, Is Highly Efficacious and Alleviates Lung Inflammation in the Hamster Model.

Without sufficient herd immunity through either vaccination or natural infection, the coronavirus disease 2019 pandemic is unlikely to be controlled. Waning immunity with the currently approved vaccines suggests the need to evaluate vaccines causing the induction of long-term responses. Here, we report the immunogenicity and efficacy of our adjuvanted single-dose Rabies-vectored SARS-CoV-2 S1 vaccine, CORAVAX, in hamsters. CORAVAX induces high SARS-CoV-2 S1-specific and virus-neutralizing antibodies (VNAs) that prevent weight loss, viral loads, disease, lung inflammation, and the cytokine storm in hamsters. We also observed high Rabies VNA titers. In summary, CORAVAX is a promising dual-antigen vaccine candidate for clinical evaluation against SARS-CoV-2 and Rabies virus.

Making genomic surveillance deliver: A lineage classification and nomenclature system to inform rabies elimination.

The availability of pathogen sequence data and use of genomic surveillance is rapidly increasing. Genomic tools and classification systems need updating to reflect this. Here, rabies virus is used as an example to showcase the potential value of updated genomic tools to enhance surveillance to better understand epidemiological dynamics and improve disease control. Previous studies have described the evolutionary history of rabies virus, however the resulting taxonomy lacks the definition necessary to identify incursions, lineage turnover and transmission routes at high resolution. Here we propose a lineage classification system based on the dynamic nomenclature used for SARS-CoV-2, defining a lineage by phylogenetic methods for tracking virus spread and comparing sequences across geographic areas. We demonstrate this system through application to the globally distributed Cosmopolitan clade of rabies virus, defining 96 total lineages within the clade, beyond the 22 previously reported. We further show how integration of this tool with a new rabies virus sequence data resource (RABV-GLUE) enables rapid application, for example, highlighting lineage dynamics relevant to control and elimination programmes, such as identifying importations and their sources, as well as areas of persistence and routes of virus movement, including transboundary incursions. This system and the tools developed should be useful for coordinating and targeting control programmes and monitoring progress as countries work towards eliminating dog-mediated rabies, as well as having potential for broader application to the surveillance of other viruses.

The Importance of Accurate Host Species Identification in the Framework of Rabies Surveillance, Control and Elimination.

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.

Interferon Inhibition Enhances the Pilot-Scale Production of Rabies Virus in Human Diploid MRC-5 Cells.

Inactivated vaccines based on cell culture are very useful in the prevention and control of many diseases. The most popular strategy for the production of inactivated vaccines is based on monkey-derived Vero cells, which results in high productivity of the virus but has a certain carcinogenic risk due to non-human DNA contamination. Since human diploid cells, such as MRC-5 cells, can produce a safer vaccine, efforts to develop a strategy for inactivated vaccine production using these cells have been investigated using MRC-5 cells. However, most viruses do not replicate efficiently in MRC-5 cells. In this study, we found that rabies virus (RABV) infection activated a robust interferon (IFN)-ß response in MRC-5 cells but almost none in Vero cells, suggesting that the IFN response could be a key limiting factor for virus production. Treatment of the MRC-5 cells with IFN inhibitors increased RABV titers by 10-fold. Additionally, the RABV titer yield was improved five-fold when using IFN receptor 1 (IFNAR1) antibodies. As such, we established a stable IFNAR1-deficient MRC-5 cell line (MRC-5IFNAR1-), which increased RABV production by 6.5-fold compared to normal MRC-5 cells. Furthermore, in a pilot-scale production in 1500 square centimeter spinner flasks, utilization of the MRC-5IFNAR1- cell line or the addition of IFN inhibitors to MRC cells increased RABV production by 10-fold or four-fold, respectively. Thus, we successfully established a human diploid cell-based pilot scale virus production platform via inhibition of IFN response for rabies vaccines, which could also be used for other inactivated virus vaccine production.

The impact of the COVID-19 pandemic on rabies reemergence in Latin America: The case of Arequipa, Peru.

In Latin America, there has been tremendous progress towards eliminating canine rabies. Major components of rabies elimination programs leading to these successes have been constant and regular surveillance for rabid dogs and uninterrupted yearly mass dog vaccination campaigns. Unfortunately, vital measures to control COVID-19 have had the negative trade-off of jeopardizing these rabies elimination and prevention activities. We aimed to assess the effect of interrupting canine rabies surveillance and mass dog vaccination campaigns on rabies trends. We built a deterministic compartment model of dog rabies dynamics to create a conceptual framework for how different disruptions may affect rabies virus transmission. We parameterized the model for conditions found in Arequipa, Peru, a city with active rabies virus transmission. We examined our results over a range of plausible values for R0 (1.36-2.0). Also, we prospectively evaluated surveillance data during the pandemic to detect temporal changes. Our model suggests that a decrease in canine vaccination coverage as well as decreased surveillance could lead to a sharp rise in canine rabies within months. These results were consistent over all plausible values of R0. Surveillance data from late 2020 and early 2021 confirms that in Arequipa, Peru, rabies cases are on an increasing trajectory. The rising rabies trends in Arequipa, if indicative to the region as whole, suggest that the achievements made in Latin America towards the elimination of dog-mediated human rabies may be in jeopardy.

Innate Inhibiting Proteins Enhance Expression and Immunogenicity of Self-Amplifying RNA.

Self-amplifying RNA (saRNA) is a cutting-edge platform for both nucleic acid vaccines and therapeutics. saRNA is self-adjuvanting, as it activates types I and III interferon (IFN), which enhances the immunogenicity of RNA vaccines but can also lead to inhibition of translation. In this study, we screened a library of saRNA constructs with cis-encoded innate inhibiting proteins (IIPs) and determined the effect on protein expression and immunogenicity. We observed that the PIV-5 V and Middle East respiratory syndrome coronavirus (MERS-CoV) ORF4a proteins enhance protein expression 100- to 500-fold in vitro in IFN-competent HeLa and MRC5 cells. We found that the MERS-CoV ORF4a protein partially abates dose nonlinearity in vivo, and that ruxolitinib, a potent Janus kinase (JAK)/signal transducer and activator of transcription (STAT) inhibitor, but not the IIPs, enhances protein expression of saRNA in vivo. Both the PIV-5 V and MERS-CoV ORF4a proteins were found to enhance the percentage of resident cells in human skin explants expressing saRNA and completely rescued dose nonlinearity of saRNA. Finally, we observed that the MERS-CoV ORF4a increased the rabies virus (RABV)-specific immunoglobulin G (IgG) titer and neutralization half-maximal inhibitory concentration (IC50) by ∼10-fold in rabbits, but not in mice or rats. These experiments provide a proof of concept that IIPs can be directly encoded into saRNA vectors and effectively abate the nonlinear dose dependency and enhance immunogenicity.

Inactivated rabies virus vectored SARS-CoV-2 vaccine prevents disease in a Syrian hamster model.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent coronavirus that has caused a worldwide pandemic. Although human disease is often asymptomatic, some develop severe illnesses such as pneumonia, respiratory failure, and death. There is an urgent need for a vaccine to prevent its rapid spread as asymptomatic infections accounting for up to 40% of transmission events. Here we further evaluated an inactivated rabies vectored SARS-CoV-2 S1 vaccine CORAVAX in a Syrian hamster model. CORAVAX adjuvanted with MPLA-AddaVax, a TRL4 agonist, induced high levels of neutralizing antibodies and generated a strong Th1-biased immune response. Vaccinated hamsters were protected from weight loss and viral replication in the lungs and nasal turbinates three days after challenge with SARS-CoV-2. CORAVAX also prevented lung disease, as indicated by the significant reduction in lung pathology. This study highlights CORAVAX as a safe, immunogenic, and efficacious vaccine that warrants further assessment in human trials.

Novel and Diverse Non-Rabies Rhabdoviruses Identified in Bats with Human Exposure, South Dakota, USA.

Bats are a host and reservoir for a large number of viruses, many of which are zoonotic. In North America, the big brown bat (Eptesicus fuscus) is widely distributed and common. Big brown bats are a known reservoir for rabies virus, which, combined with their propensity to roost in human structures, necessitates testing for rabies virus following human exposure. The current pandemic caused by severe acute respiratory syndrome coronavirus 2, likely of bat origin, illustrates the need for continued surveillance of wildlife and bats for potentially emerging zoonotic viruses. Viral metagenomic sequencing was performed on 39 big brown bats and one hoary bat submitted for rabies testing due to human exposure in South Dakota. A new genotype of American bat vesiculovirus was identified in seven of 17 (41%) heart and lung homogenates at high levels in addition to two of 23 viscera pools. A second rhabdovirus, Sodak rhabdovirus 1 (SDRV1), was identified in four of 23 (17%) viscera pools. Phylogenetic analysis placed SDRV1 in the genus Alphanemrhavirus, which includes two recognized species that were identified in nematodes. Finally, a highly divergent rhabdovirus, Sodak rhabdovirus 2 (SDRV2), was identified in two of 23 (8.7%) big brown bats. Phylogenetic analysis placed SDRV2 as ancestral to the dimarhabdovirus supergroup and Lyssavirus. Intracranial inoculation of mouse pups with rhabdovirus-positive tissue homogenates failed to elicit clinical disease. Further research is needed to determine the zoonotic potential of these non-rabies rhabdoviruses.

A Novel Vaccine Employing Non-Replicating Rabies Virus Expressing Chimeric SARS-CoV-2 Spike Protein Domains: Functional Inhibition of Viral/Nicotinic Acetylcholine Receptor Complexes.

The emergence of the novel ß-coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in a global pandemic of coronavirus disease 2019 (COVID-19). Clinical studies have documented that potentially severe neurological symptoms are associated with SARS-CoV-2 infection, thereby suggesting direct CNS penetration by the virus. Prior studies have demonstrated that the destructive neurological effects of rabies virus (RABV) infections are mediated by CNS transport of the virus tightly bound to the nicotinic acetylcholine receptor (nAChR). By comparison, it has been hypothesized that a similar mechanism exists to explain the multiple neurological effects of SARS-CoV-2 via binding to peripheral nAChRs followed by orthograde or retrograde transport into the CNS. Genetic engineering of the RABV has been employed to generate novel vaccines consisting of non-replicating RABV particles expressing chimeric capsid proteins containing human immunodeficiency virus 1 (HIV-1), Middle East respiratory syndrome (MERS-CoV), Ebolavirus, and hepatitis C virus (HCV) sequences. Accordingly, we present a critical discussion that integrates lessons learned from prior RABV research and vaccine development into a working model of a SARS-CoV-2 vaccine that selectively targets and neutralizes CNS penetration of a tightly bound viral nAChR complex.