Application prospects of the 2BS cell-adapted China fixed rabies virus vaccine strain 2aG4-B40.

BACKGROUND: Rabies is a fatal zoonotic disease whose pathogenesis has not been fully elucidated, and vaccination is the only effective method for protecting against rabies virus infection. Most inactivated vaccines are produced using Vero cells, which are African green monkey kidney cells, to achieve large-scale production. However, there is a potential carcinogenic risk due to nonhuman DNA contamination. Thus, replacing Vero cells with human diploid cells may be a safer strategy. In this study, we developed a novel 2BS cell-adapted rabies virus strain and analysed its sequence, virulence and immunogenicity to determine its application potential as a human diploid cell inactivated vaccine. METHODS AND RESULTS: The 2BS cell-adapted rabies virus strain 2aG4-B40 was established by passage for 40 generations and selection of plaques in 2BS cells. RNA sequence analysis revealed that mutations in 2BS cell-adapted strains were not located at key sites that regulate the production of neutralizing antibodies or virulence in the aG strain (GQ412744.1). The gradual increase in virulence (remaining above 7.0 logLD50/ml from the 40th to 55th generation) and antigen further indicated that these mutations may increase the affinity of the adapted strains for human diploid cells. Identification tests revealed that the 2BS cell-adapted virus strain was neutralized by anti-rabies serum, with a neutralization index of 19,952. PrEP and PEP vaccination and the NIH test further indicated that the vaccine prepared with the 2aG4-B40 strain had high neutralizing antibody levels (2.24 to 46.67 IU/ml), immunogenicity (protection index 270) and potency (average 11.6 IU/ml). CONCLUSIONS: In this study, a 2BS cell-adapted strain of the 2aG4 rabies virus was obtained by passage for 40 generations. The results of sequencing analysis and titre determination of the adapted strain showed that the mutations in the adaptive process are not located at key sequence regions of the virus, and these mutations may enhance the affinity of the adapted strain for human diploid cells. Moreover, vaccines made from the adapted strain 2aG4-B40 had high potency and immunogenicity and could be an ideal candidate rabies virus strain for inactivated vaccine preparation.

Rabies Virus Infection Causes Pyroptosis of Neuronal Cells.

Rabies virus (RABV) is a neurotropic virus that causes fatal neurological disease, raising serious public health issues and attracting extensive attention in society. To elucidate the molecular mechanism of RABV-induced neuronal damage, we used hematoxylin-eosin staining, transmission electron microscopy, transcriptomics analysis, and immune response factor testing to investigate RABV-infected neurons. We successfully isolated the neurons from murine brains. The specificity of the isolated neurons was identified by a monoclonal antibody, and the viability of the neurons was 83.53-95.0%. We confirmed that RABV infection induced serious damage to the neurons according to histochemistry and transmission electron microscope (TEM) scanning. In addition, the transcriptomics analysis suggested that multiple genes related to the pyroptosis pathway were significantly upregulated, including gasdermin D (Gsdmd), Nlrp3, caspase-1, and IL-1ß, as well as the chemokine genes Ccl2, Ccl3, Ccl4, Ccl5, Ccl7, Ccl12, and Cxcl10. We next verified this finding in the brains of mice infected with the rRC-HL, GX074, and challenge virus standard strain-24 (CVS-24) strains of RABV. Importantly, we found that the expression level of the Gsdmd protein was significantly upregulated in the neurons infected with different RABV strains and ranged from 691.1 to 5764.96 pg/mL, while the basal level of mock-infected neurons was less than 100 pg/mL. Taken together, our findings suggest that Gsdmd-induced pyroptosis is involved in the neuron damage caused by RABV infection.

RTP4 restricts lyssavirus rabies infection by binding to viral genomic RNA.

Rabies, caused by lyssavirus rabies (Rabies lyssavirus, RABV), is a fatal disease among humans and almost all warm-blooded animals. In this study, we found that RABV infection induces the up-regulation of receptor transporter protein 4 (RTP4) in mouse brains and different cells of nervous tissue. Over-expression of RTP4 reduces the viral titer of RABV in different neuronal cells. Furthermore, a recombinant RABV expressing RTP4, named rRABV-RTP4, was constructed and displayed a lower viral titer in different neuronal cells due to the expression of RTP4. Moreover, the survival rates of mice infected with rRABV-RTP4 were significantly higher than those of mice infected with parent virus rRABV or control virus rRABV-RTP4(-). In terms of mechanism, RTP4 could bind viral genomic RNA (vRNA) of RABV, and suppress the whole viral genome amplification. In addition, we found that the zinc finger domain (ZFD) of RTP4 exerts the antiviral function by truncation analysis, and an important amino acids site (C95) in the RTP4 3CxxC motif which is essential for its antiviral function was identified by mutation analysis. This study contributes to our understanding of how RTP4 or other RTP proteins play a role in defense against the invasion of RABV or other viruses.

Intracellular Pathogens: Infection, Immunity, and Intervention.

Intracellular pathogens comprise a diverse group of pathogens that all share a required location in a host cell to infect, survive, and replicate. Intracellular location allows pathogens to hide from host immune responses, avoid competition with other pathogens, mediate host cellular functions, replicate safely, and cause infection that is difficult to target with therapeutics. All intracellular pathogens have varying routes of infiltration into host cells and different host cell preferences. For example, bacteria Mycobacterium tuberculosis chooses to invade antigen-presenting cells, which allows them to moderate host antigen presentation to memory cells, whereas rabies virus prefers to invade neurons because they have pre-existing innate immunity protection systems. Regardless of the pathway that each intracellular pathogen follows, all share the capacity to cause disease if they succeed in entering host cells. Here, we give an overview of selected intracellular pathogens and infections they cause, immune responses they induce, and intervention strategies used to treat and control them.

Single Amino Acid Substitution in the Matrix Protein of Rabies Virus Is Associated with Neurovirulence in Mice.

Rabies is a fatal encephalitic infectious disease caused by the rabies virus (RABV). RABV is highly neurotropic and replicates in neuronal cell lines in vitro. The RABV fixed strain, HEP-Flury, was produced via passaging in primary chicken embryonic fibroblast cells. HEP-Flury showed rapid adaptation when propagated in mouse neuroblastoma (MNA) cells. In this study, we compared the growth of our previously constructed recombinant HEP (rHEP) strain-based on the sequence of the HEP (HEP-Flury) strain-with that of the original HEP strain. The original HEP strain exhibited higher titer than rHEP and a single substitution at position 80 in the matrix (M) protein M(D80N) after incubation in MNA cells, which was absent in rHEP. In vivo, intracerebral inoculation of the rHEP-M(D80N) strain with this substitution resulted in enhanced viral growth in the mouse brain and a significant loss of body weight in the adult mice. The number of viral antigen-positive cells in the brains of adult mice inoculated with the rHEP-M(D80N) strain was significantly higher than that with the rHEP strain at 5 days post-inoculation. Our findings demonstrate that a single amino acid substitution in the M protein M(D80N) is associated with neurovirulence in mice owing to adaptation to mouse neuronal cells.

Comparative analysis of rabies pathogenic and vaccine strains detection by RIG-I-like receptors.

Rabies virus (RABV) is a lethal neurotropic virus that causes 60,000 human deaths every year globally. RABV infection is characterized by the suppression of the interferon (IFN)-mediated antiviral response. However, molecular mechanisms leading to RABV sensing by RIG-I-like receptors (RLR) that initiates IFN signaling currently remain elusive. Here, we showed that RABV RNAs are primarily recognized by the RIG-I RLR, resulting in an IFN response in the infected cells, but this response varied according to the type of RABV used. Pathogenic RABV strain RNAs, Tha, were poorly detected in the cytosol by RIG-I and therefore caused a weak antiviral response. However, we revealed a strong IFN activity triggered by the attenuated RABV vaccine strain RNAs, SAD, mediated by RIG-I. We characterized two major 5' copy-back defective interfering (5'cb DI) genomes generated during SAD replication. Furthermore, we identified an interaction between 5'cb DI genomes, and RIG-I correlated with a high stimulation of the type I IFN signaling. This study indicates that wild-type RABV RNAs poorly activate the RIG-I pathway, while the presence of 5'cb DIs in the live-attenuated vaccine strain serves as an intrinsic adjuvant that strengthens its efficiency by enhancing RIG-I detection thus strongly stimulates the IFN response.

Historia de la rabia en Chile: tres tesis médicas / History of rabies in Chile: three medical theses

En la antigüedad ya se describía la rabia como una enfermedad zoonótica fatal cuyo pronóstico inexorable superaba todas las alter-nativas terapéuticas de los más célebres médicos. La realidad chilena sobre esta enfermedad a fines del siglo XIX fue descrita certeramente por el médico mártir Pedro Videla Órdenes en su tesis "La rabia" de 1879, destacando la descripción clínica de la rabia, su pronóstico fatal y la ausencia de tratamientos eficaces. Tan sólo seis años después, en 1885, el aclamado químico y microbiólogo Louis Pasteur desarrolló la vacuna antirrábica, logrando por primera vez en la historia de la humanidad prevenir esta terrible enfermedad. En Chile, se inició rápidamente la implementación de la vacuna Pasteur, vacunando al primer chileno el 7 de julio de 1896. Los doctores Milcíades Espinosa y Arturo Atria, en sus tesis "Generalidades sobre la rabia" (1898) y "Sobre la rabia y su profilaxia en Chile" (1905), respectivamente, abordaron esta primera etapa del desarrollo de la vacuna antirrábica en el país.

In antiquity, rabies was already described as a fatal zoonotic disease whose inexorable prognosis exceeded all the therapeutic alternatives of the most famous doctors. The Chilean reality about this disease at the end of the 19th century was accurately described by the martyred doctor Pedro Videla Ordenes in his thesis "La rabia" of 1879, highlighting in it his description about the unknown etiological agent, the fatal prognosis of the disease and the absence of effective treatments. Just six years later, in 1885, the acclaimed chemist and microbiologist Louis Pasteur developed the rabies vaccine, managing to prevent this terrible disease for the first time in human history. In Chile, the implementation of the Pasteur vaccine began rapidly, vaccinating the first Chilean on July 7, 1896. Doctors Milcíades Espinosa and Arturo Atria, in their theses "Generalidades sobre la rabia" (1898) and "Sobre la rabia y su profilaxia en Chile" (1905), respectively, addressed this first stage of the development of the rabies vaccine in the country.

The Amino Acid at Position 95 in the Matrix Protein of Rabies Virus Is Involved in Antiviral Stress Granule Formation in Infected Cells.

Stress granules (SGs) are dynamic structures that store cytosolic messenger ribonucleoproteins. SGs have recently been shown to serve as a platform for activating antiviral innate immunity; however, several pathogenic viruses suppress SG formation to evade innate immunity. In this study, we investigated the relationship between rabies virus (RABV) virulence and SG formation, using viral strains with different levels of virulence. We found that the virulent Nishigahara strain did not induce SG formation, but its avirulent offshoot, the Ni-CE strain, strongly induced SG formation. Furthermore, we demonstrated that the amino acid at position 95 in the RABV matrix protein (M95), a pathogenic determinant for the Nishigahara strain, plays a key role in inhibiting SG formation, followed by protein kinase R (PKR)-dependent phosphorylation of the α subunit of eukaryotic initiation factor 2α (eIF2α). M95 was also implicated in the accumulation of RIG-I, a viral RNA sensor protein, in SGs and in the subsequent acceleration of interferon induction. Taken together, our findings strongly suggest that M95-related inhibition of SG formation contributes to the pathogenesis of RABV by allowing the virus to evade the innate immune responses of the host. IMPORTANCE Rabies virus (RABV) is a neglected zoonotic pathogen that causes lethal infections in almost all mammalian hosts, including humans. Recently, RABV has been reported to induce intracellular formation of stress granules (SGs), also known as platforms that activate innate immune responses. However, the relationship between SG formation capacity and pathogenicity of RABV has remained unclear. In this study, by comparing two RABV strains with completely different levels of virulence, we found that the amino acid mutation from valine to alanine at position 95 of matrix protein (M95), which is known to be one of the amino acid mutations that determine the difference in virulence between the strains, plays a major role in SG formation. Importantly, M95 was involved in the accumulation of RIG-I in SGs and in promoting interferon induction. These findings are the first report of the effect of a single amino acid substitution associated with SGs on viral virulence.

A spatial and cellular distribution of rabies virus infection in the mouse brain revealed by fMOST and single-cell RNA sequencing.

BACKGROUND: Neurotropic virus infection can cause serious damage to the central nervous system (CNS) in both humans and animals. The complexity of the CNS poses unique challenges to investigate the infection of these viruses in the brain using traditional techniques. METHODS: In this study, we explore the use of fluorescence micro-optical sectioning tomography (fMOST) and single-cell RNA sequencing (scRNA-seq) to map the spatial and cellular distribution of a representative neurotropic virus, rabies virus (RABV), in the whole brain. Mice were inoculated with a lethal dose of a recombinant RABV encoding enhanced green fluorescent protein (EGFP) under different infection routes, and a three-dimensional (3D) view of RABV distribution in the whole mouse brain was obtained using fMOST. Meanwhile, we pinpointed the cellular distribution of RABV by utilizing scRNA-seq. RESULTS: Our fMOST data provided the 3D view of a neurotropic virus in the whole mouse brain, which indicated that the spatial distribution of RABV in the brain was influenced by the infection route. Interestingly, we provided evidence that RABV could infect multiple nuclei related to fear independent of different infection routes. More surprisingly, our scRNA-seq data revealed that besides neurons RABV could infect macrophages and the infiltrating macrophages played at least three different antiviral roles during RABV infection. CONCLUSION: This study draws a comprehensively spatial and cellular map of typical neurotropic virus infection in the mouse brain, providing a novel and insightful strategy to investigate the pathogenesis of RABV and other neurotropic viruses.

Comprehensive Analysis of Protein Acetylation and Glucose Metabolism in Mouse Brains Infected with Rabies Virus.

Rabies, caused by rabies virus (RABV), is a widespread zoonosis that is nearly 100% fatal. Alteration of the metabolic environment affects viral replication and the immune response during viral infection. In this study, glucose uptake was increased in mouse brains at the late stage of infection with different RABV strains (lab-attenuated CVS strain and wild-type DRV strain). To illustrate the mechanism underlying glucose metabolism alteration, comprehensive analysis of lysine acetylation and target analysis of energy metabolites in mouse brains infected with CVS and DRV strains were performed. A total of 156 acetylated sites and 115 acetylated proteins were identified as significantly different during RABV infection. Compared to CVS- and mock-infected mice, the lysine acetylation levels of glycolysis and tricarboxylic acid (TCA) cycle enzymes were decreased, and enzyme activity was upregulated in DRV-infected mouse brains. Metabolomic analysis revealed high levels of oxaloacetate (OAA) in RABV-infected mouse brains. Specifically, the OAA level in CVS-infected mouse brains was higher than that in DRV-infected mouse brains, which contributed to the enhancement of the metabolic rate at the substrate level. Finally, we confirmed that OAA could reduce excessive neuroinflammation in CVS-infected mouse brains by inhibiting JNK and P38 phosphorylation. Taken together, this study provides fresh insight into the different strategies the host adapts to regulate glucose metabolism for energy requirements after different RABV strain infections and suggests that OAA treatment is a strategy to prevent neural damage during RABV infection. IMPORTANCE Both viral replication and the host immune response are highly energy dependent. It is important to understand how the rabies virus affects energy metabolism in the brain. Glucose is the direct energy source for cell metabolism. Previous studies have revealed that there is some association between acetylation and metabolic processes. In this study, comprehensive protein acetylation and glucose metabolism analysis were conducted to compare glucose metabolism in mouse brains infected with different RABV strains. Our study demonstrates that the regulation of enzyme activity by acetylation and OAA accumulation at the substrate level are two strategies for the host to respond to energy requirements after RABV infection. Our study also indicates the role OAA could play in neuronal protection by suppressing excessive neuroinflammation.

Occurrence of mesocarnivores in montane sky islands: How spatial and temporal overlap informs rabies management in a regional hotspot.

Interspecific interactions among mesocarnivores can influence community dynamics and resource partitioning. Insights into these interactions can enhance understanding of local ecological processes that have impacts on pathogen transmission, such as the rabies lyssavirus. Host species ecology can provide an important baseline for disease management strategies especially in biologically diverse ecosystems and heterogeneous landscapes. We used a mesocarnivore guild native to the southwestern United States, a regional rabies hotspot, that are prone to rabies outbreaks as our study system. Gray foxes (Urocyon cinereoargenteus), striped skunks (Mephitis mephitis), bobcats (Lynx rufus), and coyotes (Canis latrans) share large portions of their geographic ranges and can compete for resources, occupy similar niches, and influence population dynamics of each other. We deployed 80 cameras across two mountain ranges in Arizona, stratified by vegetation type. We used two-stage modeling to gain insight into species occurrence and co-occurrence patterns. There was strong evidence for the effects of elevation, season, and temperature impacting detection probability of all four species, with understory height and canopy cover also influencing gray foxes and skunks. For all four mesocarnivores, a second stage multi-species co-occurrence model better explained patterns of detection than the single-species occurrence model. These four species are influencing the space use of each other and are likely competing for resources seasonally. We did not observe spatial partitioning between these competitors, likely due to an abundance of cover and food resources in the biologically diverse system we studied. From our results we can draw inferences on community dynamics to inform rabies management in a regional hotspot. Understanding environmental factors in disease hotspots can provide useful information to develop more reliable early-warning systems for viral outbreaks. We recommend that disease management focus on delivering oral vaccine baits onto the landscape when natural food resources are less abundant, specifically during the two drier seasons in Arizona (pre-monsoon spring and autumn) to maximize intake by all mesocarnivores.

Establishment of Human-Induced Pluripotent Stem Cell-Derived Neurons-A Promising In Vitro Model for a Molecular Study of Rabies Virus and Host Interaction.

Rabies is a deadly viral disease caused by the rabies virus (RABV), transmitted through a bite of an infected host, resulting in irreversible neurological symptoms and a 100% fatality rate in humans. Despite many aspects describing rabies neuropathogenesis, numerous hypotheses remain unanswered and concealed. Observations obtained from infected primary neurons or mouse brain samples are more relevant to human clinical rabies than permissive cell lines; however, limitations regarding the ethical issue and sample accessibility become a hurdle for discovering new insights into virus-host interplays. To better understand RABV pathogenesis in humans, we generated human-induced pluripotent stem cell (hiPSC)-derived neurons to offer the opportunity for an inimitable study of RABV infection at a molecular level in a pathologically relevant cell type. This study describes the characteristics and detailed proteomic changes of hiPSC-derived neurons in response to RABV infection using LC-MS/MS quantitative analysis. Gene ontology (GO) enrichment of differentially expressed proteins (DEPs) reveals temporal changes of proteins related to metabolic process, immune response, neurotransmitter transport/synaptic vesicle cycle, cytoskeleton organization, and cell stress response, demonstrating fundamental underlying mechanisms of neuropathogenesis in a time-course dependence. Lastly, we highlighted plausible functions of heat shock cognate protein 70 (HSC70 or HSPA8) that might play a pivotal role in regulating RABV replication and pathogenesis. Our findings acquired from this hiPSC-derived neuron platform help to define novel cellular mechanisms during RABV infection, which could be applicable to further studies to widen views of RABV-host interaction.

The role of interferon regulatory factor 7 in the pathogenicity and immunogenicity of rabies virus in a mouse model.

Rabies is a zoonotic disease caused by the rabies virus (RABV). RABV can lead to fatal encephalitis and is still a serious threat in most parts of the world. Interferon regulatory factor 7 (IRF7) is the main transcriptional regulator of type I IFN, and it is crucial for the induction of IFNα/ß and the type I IFN-dependent immune response. In this study, we focused on the role of IRF7 in the pathogenicity and immunogenicity of RABV using an IRF7-/- mouse model. The results showed that the absence of IRF7 made mice more susceptible to RABV, because IRF7 restricted the replication of RABV in the early stage of infection. IRF7 deficiency affected the recruitment of plasmacytoid dendritic cells to the draining lymph nodes (dLNs), reduced the production of type I IFN and expression of IFN-stimulated genes. Furthermore, we found that the ability to produce specific RABV-neutralizing antibody was impaired in IRF7-/- mice. Consistently, IRF7 deficiency affected the recruitment of germinal-centre B cells to dLNs, and the generation of plasma cells and RABV-specific antibody secreting cells. Moreover, the absence of IRF7 downregulated the induction of IFN-γ and reduced type 1 T helper cell (Th1)-dependent antibody production. Collectively, our findings demonstrate that IRF7 promotes humoral immune responses and compromises the pathogenicity of RABV in a mouse model.

Glycoproteins of Predicted Amphibian and Reptile Lyssaviruses Can Mediate Infection of Mammalian and Reptile Cells.

Lyssaviruses are neurotropic rhabdoviruses thought to be restricted to mammalian hosts, and to originate from bats. The identification of lyssavirus sequences from amphibians and reptiles by metatranscriptomics thus comes as a surprise and challenges the mammalian origin of lyssaviruses. The novel sequences of the proposed American tree frog lyssavirus (ATFLV) and anole lizard lyssavirus (ALLV) reveal substantial phylogenetic distances from each other and from bat lyssaviruses, with ATFLV being the most distant. As virus isolation has not been successful yet, we have here studied the functionality of the authentic ATFLV- and ALLV-encoded glycoproteins in the context of rabies virus pseudotype particles. Cryogenic electron microscopy uncovered the incorporation of the plasmid-encoded G proteins in viral envelopes. Infection experiments revealed the infectivity of ATFLV and ALLV G-coated RABV pp for a broad spectrum of cell lines from humans, bats, and reptiles, demonstrating membrane fusion activities. As presumed, ATFLV and ALLV G RABV pp escaped neutralization by human rabies immune sera. The present findings support the existence of contagious lyssaviruses in poikilothermic animals, and reveal a broad cell tropism in vitro, similar to that of the rabies virus.

Data-Driven Management-A Dynamic Occupancy Approach to Enhanced Rabies Surveillance Prioritization.

Rabies lyssavirus (RABV) is enzootic in raccoons across the eastern United States. Intensive management of RABV by oral rabies vaccination (ORV) has prevented its spread westward and shown evidence of local elimination in raccoon populations of the northeastern US. The USDA, Wildlife Services, National Rabies Management Program (NRMP) collaborates with other agencies to implement broad-scale ORV and conducts extensive monitoring to measure the effectiveness of the management. Enhanced Rabies Surveillance (ERS) was initiated during 2005 and updated in 2016 to direct surveillance efforts toward higher-value specimens by assigning points to different methods of encountering specimens for collection (strange-acting, roadkill, surveillance-trapped, etc.; specimen point values ranged from 1 to 15). We used the 2016-2019 data to re-evaluate the point values using a dynamic occupancy model. Additionally, we used ERS data from 2012-2015 and 2016-2019 to examine the impact that the point system had on surveillance data. Implementation of a point system increased positivity rates among specimens by 64%, indicating a substantial increase in the efficiency of the ERS to detect wildlife rabies. Our re-evaluation found that most points accurately reflect the value of the surveillance specimens. The notable exception was that samples from animals found dead were considerably more valuable for rabies detection than originally considered (original points = 5, new points = 20). This work demonstrates how specimen prioritization strategies can be used to refine and improve ERS in support of wildlife rabies management.

Murine Ifit3 restricts the replication of Rabies virus both in vitro and in vivo.

Rabies virus (RABV) infection can initiate the host immune defence response and induce an antiviral state characterized by the expression of interferon (IFN)-stimulated genes (ISGs), among which the family of genes of IFN-induced protein with tetratricopeptide repeats (Ifits) are prominent representatives. Herein, we demonstrated that the mRNA and protein levels of Ifit1, Ifit2 and Ifit3 were highly increased in cultured cells and mouse brains after RABV infection. Recombinant RABV expressing Ifit3, designated rRABV-Ifit3, displayed a lower pathogenicity than the parent RABV in C57BL/6 mice after intramuscular administration, and Ifit3-deficient mice exhibited higher susceptibility to RABV infection and higher mortality during RABV infection. Moreover, compared with their individual expressions, co-expression of Ifit2 and Ifit3 could more effectively inhibit RABV replication in vitro. These results indicate that murine Ifit3 plays an essential role in restricting the replication and reducing the pathogenicity of RABV. Ifit3 acts synergistically with Ifit2 to inhibit RABV replication, providing further insight into the function and complexity of the Ifit family.

Analyses of cell death mechanisms related to amino acid substitution at position 95 in the rabies virus matrix protein.

We previously reported that the avirulent fixed rabies virus strain Ni-CE induces a clear cytopathic effect in mouse neuroblastoma cells, whereas its virulent progenitor, the Nishigahara strain, does not. Infection with Nishigahara and Ni-CE mutants containing a single amino acid substitution in the matrix protein (M) demonstrated that the amino acid at position 95 of M (M95) is a cytopathic determinant. The characteristics of cell death induced by Ni-CE infection resemble those of apoptosis (rounded and shrunken cells, DNA fragmentation), but the intracellular signalling pathway for this process has not been fully investigated. In this study, we aimed to elucidate the mechanism by which M95 affects cell death induced by human neuroblastoma cell infection with the Nishigahara, Ni-CE and M95-mutated strains. We demonstrated that the Ni-CE strain induced DNA fragmentation, cell membrane disruption, exposure of phosphatidylserine (PS), activation of caspase-3/7 and anti-poly (ADP-ribose) polymerase 1 (PARP-1) cleavage, an early apoptosis indicator, whereas the Nishigahara strain did not induce DNA fragmentation, caspase-3/7 activation, cell membrane disruption, or PARP-1 cleavage, but did induce PS exposure. We also demonstrated that these characteristics were associated with M95 using M95-mutated strains. However, we found that Ni-CE induced cell death despite the presence of a caspase inhibitor, Z-VAD-FMK. In conclusion, our data suggest that M95 mutation-related cell death is caused by both the caspase-dependent and -independent pathways.

The role of a mechanistic host in maintaining arctic rabies variant distributions: Assessment of functional genetic diversity in Alaskan red fox (Vulpes vulpes).

Populations are exposed to different types and strains of pathogens across heterogeneous landscapes, where local interactions between host and pathogen may present reciprocal selective forces leading to correlated patterns of spatial genetic structure. Understanding these coevolutionary patterns provides insight into mechanisms of disease spread and maintenance. Arctic rabies (AR) is a lethal disease with viral variants that occupy distinct geographic distributions across North America and Europe. Red fox (Vulpes vulpes) are a highly susceptible AR host, whose range overlaps both geographically distinct AR strains and regions where AR is absent. It is unclear if genetic structure exists among red fox populations relative to the presence/absence of AR or the spatial distribution of AR variants. Acquiring these data may enhance our understanding of the role of red fox in AR maintenance/spread and inform disease control strategies. Using a genotyping-by-sequencing assay targeting 116 genomic regions of immunogenetic relevance, we screened for sequence variation among red fox populations from Alaska and an outgroup from Ontario, including areas with different AR variants, and regions where the disease was absent. Presumed neutral SNP data from the assay found negligible levels of neutral genetic structure among Alaskan populations. The immunogenetically-associated data identified 30 outlier SNPs supporting weak to moderate genetic structure between regions with and without AR in Alaska. The outliers included SNPs with the potential to cause missense mutations within several toll-like receptor genes that have been associated with AR outcome. In contrast, there was a lack of genetic structure between regions with different AR variants. Combined, we interpret these data to suggest red fox populations respond differently to the presence of AR, but not AR variants. This research increases our understanding of AR dynamics in the Arctic, where host/disease patterns are undergoing flux in a rapidly changing Arctic landscape, including the continued northward expansion of red fox into regions previously predominated by the arctic fox (Vulpes lagopus).

Relationships between fox populations and rabies virus spread in northern Canada.

Rabies spreads in both Arctic (Vulpes lagopus) and red foxes (Vulpes vulpes) throughout the Canadian Arctic but limited wildlife disease surveillance, due to the extensive landmass of the Canadian north and its small widely scattered human population, undermines our knowledge of disease transmission patterns. This study has explored genetic population structure in both the rabies virus and its fox hosts to better understand factors that impact rabies spread. Phylogenetic analysis of 278 samples of the Arctic lineage of rabies virus recovered over 40 years identified four sub-lineages, A1 to A4. The A1 lineage has been restricted to southern regions of the Canadian province of Ontario. The A2 lineage, which predominates in Siberia, has also spread to northern Alaska while the A4 lineage was recovered from southern Alaska only. The A3 sub-lineage, which was also found in northern Alaska, has been responsible for virtually all cases across northern Canada and Greenland, where it further differentiated into 18 groups which have systematically evolved from a common predecessor since 1975. In areas of Arctic and red fox sympatry, viral groups appear to circulate in both hosts, but both mitochondrial DNA control region sequences and 9-locus microsatellite genotypes revealed contrasting phylogeographic patterns for the two fox species. Among 157 Arctic foxes, 33 mitochondrial control region haplotypes were identified but little genetic structure differentiating localities was detected. Among 162 red foxes, 18 control region haplotypes delineated three groups which discriminated among the Churchill region of Manitoba, northern Quebec and Labrador populations, and the coastal Labrador locality of Cartwright. Microsatellite analyses demonstrated some genetic heterogeneity among sampling localities of Arctic foxes but no obvious pattern, while two or three clusters of red foxes suggested some admixture between the Churchill and Quebec-Labrador regions but uniqueness of the Cartwright group. The limited population structure of Arctic foxes is consistent with the rapid spread of rabies virus subtypes throughout the north, while red fox population substructure suggests that disease spread in this host moves most readily down certain independent corridors such as the northeastern coast of Canada and the central interior. Interestingly the evidence suggests that these red fox populations have limited capacity to maintain the virus over the long term, but they may contribute to viral persistence in areas of red and Arctic fox sympatry.

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.