Lyssavirus M protein degrades neuronal microtubules by reprogramming mitochondrial metabolism.

Infection with neurotropic viruses may result in changes in host behavior, which are closely associated with degenerative changes in neurons. The lyssavirus genus comprises highly neurotropic viruses, including the rabies virus (RABV), which has been shown to induce degenerative changes in neurons, marked by the self-destruction of axons. The underlying mechanism by which the RABV degrades neuronal cytoskeletal proteins remains incomplete. In this study, we show that infection with RABV or overexpression of its M protein can disrupt mitochondrial metabolism by binding to Slc25a4. This leads to a reduction in NAD+ production and a subsequent influx of Ca2+ from the endoplasmic reticulum and mitochondria into the cytoplasm of neuronal cell lines, activating Ca2+-dependent proteinase calpains that degrade α-tubulin. We further screened the M proteins of different lyssaviruses and discovered that the M protein of the dog-derived RABV strain (DRV) does not degrade α-tubulin. Sequence analysis of the DRV M protein and that of the lab-attenuated RABV strain CVS revealed that the 57th amino acid is vital for M-induced microtubule degradation. We generated a recombinant RABV with a mutation at the 57th amino acid position in its M protein and showed that this mutation reduces α-tubulin degradation in vitro and axonal degeneration in vivo. This study elucidates the mechanism by which lyssavirus induces neuron degeneration.IMPORTANCEPrevious studies have suggested that RABV (rabies virus, the representative of lyssavirus) infection induces structural abnormalities in neurons. But there are few articles on the mechanism of lyssavirus' effect on neurons, and the mechanism of how RABV infection induces neurological dysfunction remains incomplete. The M protein of lyssavirus can downregulate cellular ATP levels by interacting with Slc25a4, and this decrease in ATP leads to a decrease in the level of NAD+ in the cytosol, which results in the release of Ca2+ from the intracellular calcium pool, the endoplasmic reticulum, and mitochondria. The presence of large amounts of Ca2+ in the cytoplasm activates Ca2+-dependent proteases and degrades microtubule proteins. The amino acid 57 of M protein is the key site determining its disruption of mitochondrial metabolism and subsequent neuron degeneration.

[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.

Longitudinal Tracing of Lyssavirus Infection in Mice via In Vivo Bioluminescence Imaging.

Bioluminescence imaging (BLI) is a technique that can be employed to quantify biological processes in living cells. When used in small animal models such as mice, BLI can provide both longitudinal and positional information regarding the biological process under investigation. Although perhaps best known for its utility in non-invasively quantifying tumor burden over time in experimental animals, BLI has also been applied in many pathogenesis models to track pathogen burden and responses to therapeutic interventions. In this chapter, we present a BLI-based method for tracing anatomical progression of lyssavirus infection in a mouse model. We also include validation methods to ensure that semiquantitative BLI data correlate well with viral load. Due to the longitudinal nature of this approach, lyssavirus pathogenesis and therapeutic intervention studies can be performed with far fewer animals than more traditional approaches, which typically require euthanasia of large animal groups at every data collection time point.

[Anti-rabies vaccines applied in the Russian Federation and perspectives for their improvement].

Rabies is almost ubiquitous (except in certain areas) and poses a significant danger to both animals and humans. Every year around 55,000 people die from this disease worldwide. In the Russian Federation alone 400,000- 450,000 patients annually apply for anti-rabies treatment. In the absolute majority of cases human infection is caused by contact with infected animals. In RF, a number of cultured inactivated anti-rabies vaccines for medical and veterinary purposes have been developed, registered and used for specific prevention of rabies. These vaccine preparations have shown high effectiveness in preventing infection in domestic and farm animals. At the same time, the main reservoir of the rabies virus (Mononegavirales: Rhabdoviridae: Lyssavirus) (RV) are wild carnivores (Mammalia: Carnivora). For the purpose of their oral immunization, live virus vaccines from attenuated (fixed) strains of RV that are little resistant in the external environment are used. In Western Europe and North America there is successful experience with recombinant anti-rabies vaccine preparations containing a viral glycoprotein gene (G-protein). Such vaccines are safe for humans and animals. In Russia also had been developed a vector anti-rabies vaccine based on adenovirus (Adenoviridae), which can be used to combat this infection. Currently, in addition to classical rabies, diseases caused by new, previously unknown lyssaviruses (Lyssavirus) are becoming increasingly important. Bats (Mammalia: Microchiroptera) are their vectors. Cases of illness and death after contact with these animals have been described. In the near future, we should expect the development of new vaccines that will provide protection not only against RV, but also against other lyssaviruses.

Educação em saúde para prevenção da raiva humana / Health education for prevention of human rabies / Educación en salud para prevención de la rabia humana

A raiva é uma antropozoonose viral que se desenvolve de forma progressiva e aguda podendo apresentar até 100% de letalidade. O seu agente etiológico é o vírus rábico do gênero Lyssavirus pertencente à família Rhabdoviridae. O presente trabalho teve como objetivo divulgar informações acerca da ocorrência da raiva em humanos em virtude da sua expressiva importância para saúde pública e analisar a percepção da população sobre a raiva humana, de forma a obter dados relacionados a conhecimentos básicos sobre a doença. Para isso, foi criada uma página informativa denominada "@contraraiva_" na rede social Instagram para a realização de postagens interativas sobre o tema abordado, e uso de um questionário criado a partir da plataforma Google Forms em diferentes mídias sociais para a obtenção e coleta de dados. Foram obtidas 1.075 respostas, provenientes de diferentes localidades. O questionário alcançou todas as regiões brasileiras, todos 26 estados e o Distrito Federal. As informações publicadas pela página criada ajudaram a sanar dúvidas relacionadas aos principais aspectos da doença. Os dados obtidos a partir do questionário contribuem para o planejamento de ações voltadas para a educação em saúde de forma mais estratégica, visando contribuir para os pontos em que a população tem menos conhecimentos.(AU)

Rabies is a viral anthropozoonosis that is developed in a progressive and acute way and can present up to 100% lethality. Its etiologic agent is the rabies virus of the Lyssavirus gene belonging to the Rhabdoviridae family. This study aimed at disseminating information about the occurrence of rabies in humans due to its expressive importance for public health, and at analyzing the population perception on human rabies in order to obtain data related to basic knowledge about the disease. For that purpose, an information page called "@ contraraiva _" was created on the social network Instagram for providing interactive posts on the topic, and a questionnaire was created from the Google Forms platform on different social media to obtain and collect data. A total of 1,075 responses were obtained from different locations. The questionnaire included all Brazilian regions, all 26 states and the Federal District. The information published on the created page helped to clarify doubts related to the main aspects of the disease. The data obtained from the questionnaire contribute towards the planning of actions aimed at health education in a more strategic way, aiming at contributing to the points where the population is less knowledgeable.(AU)

La rabia es una antropozoonosis viral que se desarrolla de forma progresiva y aguda y puede presentar hasta un 100% de letalidad. Su agente etiológico es el virus de la rabia del género Lyssavirus perteneciente a la familia Rhabdoviridae. Este estudio tuvo como objetivo difundir informaciones sobre la ocurrencia de la rabia en humanos en virtud de su importancia expresiva para la salud pública, y analizar la percepción de la población sobre la rabia humana, con el fin de obtener datos relacionados a los conocimientos básicos sobre la enfermedad. Para ello, se creó una página de información denominada "@ contraraiva _" en la red social Instagram para realizar publicaciones interactivas sobre el tema abordado, y utilizar un cuestionario creado a partir de la plataforma Google Forms en diferentes redes sociales para la obtención y recolección de datos. Se obtuvieron 1.075 respuestas de diferentes lugares. El cuestionario llegó a todas las regiones brasileñas, a los 26 estados y al Distrito Federal. Las informaciones publicadas en el sitio web creado, ayudaron a sanar dudas relacionadas a los principales aspectos de la enfermedad. Los datos obtenidos del cuestionario contribuyen a la planificación de acciones orientadas a la educación para la salud de forma más estratégica, con el objetivo de contribuir a los puntos donde la población tiene menos conocimientos.(AU)

Lyssavirus P-protein selectively targets STAT3-STAT1 heterodimers to modulate cytokine signalling.

Many viruses target signal transducer and activator of transcription (STAT) 1 to antagonise antiviral interferon signalling, but targeting of STAT3, a pleiotropic molecule that mediates signalling by diverse cytokines, is poorly understood. Here, using lyssavirus infection, quantitative live cell imaging, innate immune signalling and protein interaction assays, and complementation/depletion of STAT expression, we show that STAT3 antagonism is conserved among P-proteins of diverse pathogenic lyssaviruses and correlates with pathogenesis. Importantly, P-protein targeting of STAT3 involves a highly selective mechanism whereby P-protein antagonises cytokine-activated STAT3-STAT1 heterodimers, but not STAT3 homodimers. RT-qPCR and reporter gene assays indicate that this results in specific modulation of interleukin-6-dependent pathways, effecting differential antagonism of target genes. These data provide novel insights into mechanisms by which viruses can modulate cellular function to support infection through discriminatory targeting of immune signalling complexes. The findings also highlight the potential application of selective interferon-antagonists as tools to delineate signalling by particular STAT complexes, significant not only to pathogen-host interactions but also cell physiology, development and cancer.

Crystal structure of Mokola virus glycoprotein in its post-fusion conformation.

Mokola virus (MOKV) belongs to the lyssavirus genus. As other genus members-including rabies virus (RABV)-it causes deadly encephalitis in mammals. MOKV entry into host cells is mediated by its transmembrane glycoprotein G. First, G binds cellular receptors, triggering virion endocytosis. Then, in the acidic endosomal environment, G undergoes a conformational change from its pre- toward its post-fusion state that catalyzes the merger of the viral and endosomal membranes. Here, we have determined the crystal structure of a soluble MOKV G ectodomain in which the hydrophobic fusion loops have been replaced by more hydrophilic sequences. The crystal structure corresponds to a monomer that is similar to the protomer of the trimeric post-fusion state of vesicular stomatitis virus (VSV) G. However, by electron microscopy, we show that, at low pH, at the surface of pseudotyped VSV, MOKV spikes adopt the trimeric post-fusion conformation and have a tendency to reorganize into regular arrays. Sequence alignment between MOKV G and RABV G allows a precise location of RABV G antigenic sites. Repositioning MOKV G domains on VSV G pre-fusion structure reveals that antigenic sites are located in the most exposed part of the molecule in its pre-fusion conformation and are therefore very accessible to antibodies. Furthermore, the structure allows the identification of pH-sensitive molecular switches. Specifically, the long helix, which constitutes the core of the post-fusion trimer for class III fusion glycoproteins, contains many acidic residues located at the trimeric interface. Several of them, aligned along the helix, point toward the trimer axis. They have to be protonated for the post-fusion trimer to be stable. At high pH, when they are negatively charged, they destabilize the interface, which explains the conformational change reversibility. Finally, the present structure will be of great help to perform rational mutagenesis on lyssavirus glycoproteins.

The LC8-RavP ensemble Structure Evinces A Role for LC8 in Regulating Lyssavirus Polymerase Functionality.

The rabies and Ebola viruses recruit the highly conserved host protein LC8 for their own reproductive success. In vivo knockouts of the LC8 recognition motif within the rabies virus phosphoprotein (RavP) result in completely nonlethal viral infections. In this work, we examine the molecular role LC8 plays in viral lethality. We show that RavP and LC8 colocalize in rabies infected cells, and that LC8 interactions are essential for efficient viral polymerase functionality. NMR, SAXS, and molecular modeling demonstrate that LC8 binding to a disordered linker adjacent to an endogenous dimerization domain results in restrictions in RavP domain orientations. The resulting ensemble structure of RavP-LC8 tetrameric complex is similar to that of a related virus phosphoprotein that does not bind LC8, suggesting that with RavP, LC8 binding acts as a switch to induce a more active conformation. The high conservation of the LC8 motif in Lyssavirus phosphoproteins and its presence in other analogous proteins such as the Ebola virus VP35 evinces a broader purpose for LC8 in regulating downstream phosphoprotein functions vital for viral replication.

Molecular Function Analysis of Rabies Virus RNA Polymerase L Protein by Using an L Gene-Deficient Virus.

While the RNA-dependent RNA polymerase L protein of rabies virus (RABV), a member of the genus Lyssavirus of the family Rhabdoviridae, has potential to be a therapeutic target for rabies, the molecular functions of this protein have remained largely unknown. In this study, to obtain a novel experimental tool for molecular function analysis of the RABV L protein, we established by using a reverse genetics approach an L gene-deficient RABV (Nishi-ΔL/Nluc), which infects, propagates, and correspondingly produces NanoLuc luciferase in cultured neuroblastoma cells transfected to express the L protein. trans-Complementation with wild-type L protein, but not that with a functionally defective L protein mutant, efficiently supported luciferase production by Nishi-ΔL/Nluc, confirming its potential for function analysis of the L protein. Based on the findings obtained from comprehensive genetic analyses of L genes from various RABV and other lyssavirus species, we examined the functional importance of a highly conserved L protein region at positions 1914 to 1933 by a trans-complementation assay with Nishi-ΔL/Nluc and a series of L protein mutants. The results revealed that the amino acid sequence at positions 1929 to 1933 (NPYNE) is functionally important, and this was supported by other findings that this sequence is critical for binding of the L protein with its essential cofactor, P protein, and thus also for L protein's RNA polymerase activity. Our findings provide useful information for the development of an anti-RABV drug targeting the L-P protein interaction.IMPORTANCE To the best of our knowledge, this is the first report on the establishment of an L gene-deficient, reporter gene-expressing virus in all species of the order Mononegavirales, also highlighting its applicability to a trans-complementation assay, which is useful for molecular function analyses of their L proteins. Moreover, this study revealed for the first time that the NPYNE sequence at positions 1929 to 1933 in the RABV L protein is important for L protein's interaction with the P protein, consistent with and extending the results of a previous study showing that the P protein-binding domain in the L protein is located in its C-terminal region, at positions 1562 to 2127. This study indicates that the NPYNE sequence is a promising target for the development of an inhibitor of viral RNA synthesis, which has high potential as a therapeutic drug for rabies.

First detection of European bat lyssavirus type 2 (EBLV-2) in Norway.

BACKGROUND: In Europe, bat rabies is primarily attributed to European bat lyssavirus type 1 (EBLV-1) and European bat lyssavirus type 2 (EBLV-2) which are both strongly host-specific. Approximately thirty cases of infection with EBLV-2 in Daubenton's bats (Myotis daubentonii) and pond bats (M. dasycneme) have been reported. Two human cases of rabies caused by EBLV-2 have also been confirmed during the last thirty years, while natural spill-over to other non-flying mammals has never been reported. Rabies has never been diagnosed in mainland Norway previously. CASE PRESENTATION: In late September 2015, a subadult male Daubenton's bat was found in a poor condition 800 m above sea level in the southern part of Norway. The bat was brought to the national Bat Care Centre where it eventually displayed signs of neurological disease and died after two days. EBLV-2 was detected in brain tissues by polymerase chain reaction (PCR) followed by sequencing of a part of the nucleoprotein gene, and lyssavirus was isolated in neuroblastoma cells. CONCLUSIONS: The detection of EBLV-2 in a bat in Norway broadens the knowledge on the occurrence of this zoonotic agent. Since Norway is considered free of rabies, adequate information to the general public regarding the possibility of human cases of bat-associated rabies should be given. No extensive surveillance of lyssavirus infections in bats has been conducted in the country, and a passive surveillance network to assess rabies prevalence and bat epidemiology is highly desired.

Epidemiologia descritiva da raiva dos herbívoros notificados no estado do Paraná entre 1977 e 2012 / Epidemiology of rabies in large animals of Parana, Brazil, from 1977 to 2012

Realizou-se estudo epidemiológico descritivo da raiva dos herbívoros no estado do Paraná entre os anos de 1977 e 2012. Os casos confirmados de raiva e o total de amostras de encéfalo encaminhadas para o diagnóstico foram distribuídos por espécie, por ano, por meses, por mesorregião geográfica e por municípios, aplicando-se o teste de Qui-quadrado para verificar se havia associação com esses fatores. Modelo probabilístico foi ajustado à série histórica para verificação de padrões cíclico ou sazonal. Comprovou-se que a raiva é uma doença endêmica no PR, com ocorrência variável entre os anos, sem padrão sazonal e com ciclicidade aparente a cada 18 anos, acometendo, sobretudo, bovinos (86,9%) e equídeos (11,3%). Verificou-se grande difusão no estado (47,6% dos municípios), e a maior expansão geográfica aconteceu na última década. As áreas de ocorrência maior foram as mesorregiões Centro Oriental e de Curitiba, seguidas por Norte Pioneiro e Oeste. O número de casos de raiva por município se correlacionou, ainda que fracamente, com o número de abrigos de Desmodus rotundus (r=0,469; p<0,0001). Sugere-se que a imunização anual de bovinos e equídeos passe a ser adotada nas áreas de maior ocorrência (mesorregiões Centro Oriental e de Curitiba) e encorajada nas de ocorrência intermediária (mesorregiões Norte Pioneiro e Oeste).(AU)

A descriptive epidemiological survey of rabies in herbivorous reared in the state of Parana, Brazil, was carried out from 1977 to 2012. The positive cases and the total number of brain samples processed for diagnostic purposes were distributed according to species, year, month, geographical region and municipality. Chi-square test was used to verify if rabies was associated to these factors. Probabilistic model was applied to historical series in order to verify cyclic and seasonal patterns. In Parana, rabies is an endemic disease with variable yearly occurrence, without seasonal pattern and with a possible cyclic pattern every 18 years. Cattle (86.9%) and equides (11.3%) were mainly affected. Rabies was registered in 47.6% of all municipalities, indicating a great spread of this disease in Parana, mainly during the last decade. Middlewest and Curitiba regions, followed by Pioneer North and West regions, were the areas of most occurrence. The number of cases per municipality was weakly correlated with the number of shelters for Desmodus rotundus (r=0.469; p<0.0001). Therefore, we suggest that annual immunization of cattle and equides should be applied in the high occurrence areas (Middlewest and Curitiba regions) and encouraged in intermediate occurrence areas (Pioneer North and West regions).(AU)

Dual Combined Real-Time Reverse Transcription Polymerase Chain Reaction Assay for the Diagnosis of Lyssavirus Infection.

The definitive diagnosis of lyssavirus infection (including rabies) in animals and humans is based on laboratory confirmation. The reference techniques for post-mortem rabies diagnosis are still based on direct immunofluorescence and virus isolation, but molecular techniques, such as polymerase chain reaction (PCR) based methods, are increasingly being used and now constitute the principal tools for diagnosing rabies in humans and for epidemiological analyses. However, it remains a key challenge to obtain relevant specificity and sensitivity with these techniques while ensuring that the genetic diversity of lyssaviruses does not compromise detection. We developed a dual combined real-time reverse transcription polymerase chain reaction (combo RT-qPCR) method for pan-lyssavirus detection. This method is based on two complementary technologies: a probe-based (TaqMan) RT-qPCR for detecting the RABV species (pan-RABV RT-qPCR) and a second reaction using an intercalating dye (SYBR Green) to detect other lyssavirus species (pan-lyssa RT-qPCR). The performance parameters of this combined assay were evaluated with a large panel of primary animal samples covering almost all the genetic variability encountered at the viral species level, and they extended to almost all lyssavirus species characterized to date. This method was also evaluated for the diagnosis of human rabies on 211 biological samples (positive n = 76 and negative n = 135) including saliva, skin and brain biopsies. It detected all 41 human cases of rabies tested and confirmed the sensitivity and the interest of skin biopsy (91.5%) and saliva (54%) samples for intra-vitam diagnosis of human rabies. Finally, this method was successfully implemented in two rabies reference laboratories in enzootic countries (Cambodia and Morocco). This combined RT-qPCR method constitutes a relevant, useful, validated tool for the diagnosis of rabies in both humans and animals, and represents a promising tool for lyssavirus surveillance.

[Construction of a Recombinant Replication-defective Human Adenovirus Type 5 Expressing G Protein of Irkut Virus and the Immune Test in Mouse].

To develop a safe and effective new generation vaccine for IRKV-THChina12 prevention, we constructed a non-replicative recombinant human adenovirus carrying the IRKV-THChina12 G gene, named as rAd5-IRKV-G. The IRKV-THChina12 G protein expressed by the recombinant human adenovirus in 293AD cells was detected by western blot and indirect immunofluorescence test. To evaluate the immunogenicity of the recombinant, mice were immunized with rAd5-IRKV-G by intramuscular (i. m.) or intraperitoneal (i. p.) route and with non-exogenous gene expressing wild type adenovirus wt-rAd5 as a control. Results showed that the rAd5-IRKV-G could induce continuous and statistically significant (P ≤ 0.05) anti-IRKV neutralizing antibody (NA) production in immunized mice by i. m. or i. p. route. In particular, no significant difference (P > 0.05) of the NA titers between the two administration routes were observed, that provides an alternative choice for animal immunization method in the future application.

Australian bat lyssavirus infection in two horses.

In May 2013, the first cases of Australian bat lyssavirus infections in domestic animals were identified in Australia. Two horses (filly-H1 and gelding-H2) were infected with the Yellow-bellied sheathtail bat (YBST) variant of Australian bat lyssavirus (ABLV). The horses presented with neurological signs, pyrexia and progressing ataxia. Intra-cytoplasmic inclusion bodies (Negri bodies) were detected in some Purkinje neurons in haematoxylin and eosin (H&E) stained sections from the brain of one of the two infected horses (H2) by histological examination. A morphological diagnosis of sub-acute moderate non-suppurative, predominantly angiocentric, meningo-encephalomyelitis of viral aetiology was made. The presumptive diagnosis of ABLV infection was confirmed by the positive testing of the affected brain tissue from (H2) in a range of laboratory tests including fluorescent antibody test (FAT) and real-time PCR targeting the nucleocapsid (N) gene. Retrospective testing of the oral swab from (H1) in the real-time PCR also returned a positive result. The FAT and immunohistochemistry (IHC) revealed an abundance of ABLV antigen throughout the examined brain sections. ABLV was isolated from the brain (H2) and oral swab/saliva (H1) in the neuroblastoma cell line (MNA). Alignment of the genome sequence revealed a 97.7% identity with the YBST ABLV strain.

Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5.

BACKGROUND: Australian bat lyssavirus (ABLV), a rhabdovirus of the genus Lyssavirus which circulates in both pteropid fruit bats and insectivorous bats in mainland Australia, has caused three fatal human infections, the most recent in February 2013, manifested as acute neurological disease indistinguishable from clinical rabies. Rhabdoviruses infect host cells through receptor-mediated endocytosis and subsequent pH-dependent fusion mediated by their single envelope glycoprotein (G), but the specific host factors and pathways involved in ABLV entry have not been determined. METHODS: ABLV internalization into HEK293T cells was examined using maxGFP-encoding recombinant vesicular stomatitis viruses (rVSV) that express ABLV G glycoproteins. A combination of chemical and molecular approaches was used to investigate the contribution of different endocytic pathways to ABLV entry. Dominant negative Rab GTPases were used to identify the endosomal compartment utilized by ABLV to gain entry into the host cell cytosol. RESULTS: Here we show that ABLV G-mediated entry into HEK293T cells was significantly inhibited by the dynamin-specific inhibitor dynasore, chlorpromazine, a drug that blocks clathrin-mediated endocytosis, and the actin depolymerizing drug latrunculin B. Over expression of dominant negative mutants of Eps15 and Rab5 also significantly reduced ABLV G-mediated entry into HEK293T cells. Chemical inhibitors of caveolae-dependent endocytosis and macropinocytosis and dominant negative mutants of Rab7 and Rab11 had no effect on ABLV entry. CONCLUSIONS: The predominant pathway utilized by ABLV for internalization into HEK293T cells is clathrin-and actin-dependent. The requirement of Rab5 for productive infection indicates that ABLV G-mediated fusion occurs within the early endosome compartment.

Kinetics of rabies antibodies as a strategy for canine active immunization

Rabies, a zoonosis found throughout the globe, is caused by a virus of the Lyssavirus genus. The disease is transmitted to humans through the inoculation of the virus present in the saliva of infected mammals. Since its prognosis is usually fatal for humans, nationwide public campaigns to vaccinate dogs and cats against rabies aim to break the epidemiological link between the virus and its reservoirs in Brazil. During 12 months we evaluated the active immunity of dogs first vaccinated (booster shot at 30 days after first vaccination) against rabies using the Fuenzalida-Palácios modified vaccine in the urban area of Botucatu city, São Pauto state, Brazil. Of the analyzed dogs, 54.7% maintained protective titers (≥0.5 IU/mL) for 360 days after the first vaccination whereas 51.5% during all the study period. The present results suggest a new vaccination schedule for dogs that have never been vaccinated. In addition to the first dose of vaccine, two others are recommended: the second at 30 days after the first and the third dose at 180 days after the first for the maintenance of protective titers during 12 months.

Encefalitis rábica humana secundaria a mordedura por gato infectado por un virus rábico de origen silvestre / Encephalitis Due to Rabies Secondary to the Bite of a Cat Infected With a Rabies Virus of Silvester Origen

La rabia es una enfermedad viral zoonótica, producida por un virus del genero Lyssavirus de la Familia Rhabdoviridae, cuya principal fuente de transmisión es la mordedura de animales a humanos. Es una enfermedad fatal y se han descrito casos por ciclos urbanos y rurales. El caso que reportamos es el de una joven de 22 años, quien ingresa por un cuadro de dolor de características neuropáticas en el miembro superior derecho, con antecedente de mordedura por un gato de varios meses atrás, hospitalizada por el servicio de Neurología por sospecha de lesión de plejo braquial, con resonancia de columna cervical y líquido cefalorraquídeo (LCR) normales, quien posteriormente presenta deterioro clínico tórpido a un proceso encefalopático que en pocos días la llevó a la muerte. Se confirmó que la paciente presentó una encefalitis por un virus de rabia. Expondremos cómo fue el manejo de la paciente y todos los nexos epidemiológicos.

Rabies is a zoonotic viral disease, caused by a virus of the genus Lyssavirus of the Rhabdoviridae family. Its main source is transmission from animals to humans bite. The disease is fatal and has been reported to occur in rural and urban cycles. This reported case is a 22-year old, who was admitted with symptoms of neuropathic pain in the right arm, with a history of being bitten by a cat a few months earlier. The patient was hospitalized in the Neurology Department for suspected brachial plexopathy, and normal spinal MRI and cerebrospinal fluid (CSF) were found. The patient subsequently presented encephalopathic decline that resulted in death within a few days. It was confirmed that the patient had encephalitis due to the rabies virus. We present the management of the patient and all epidemiological links.

Host cell tropism mediated by Australian bat lyssavirus envelope glycoproteins.

Australian bat lyssavirus (ABLV) is a rhabdovirus of the lyssavirus genus capable of causing fatal rabies-like encephalitis in humans. There are two variants of ABLV, one circulating in pteropid fruit bats and another in insectivorous bats. Three fatal human cases of ABLV infection have been reported with the third case in 2013. Importantly, two equine cases also arose in 2013; the first occurrence of ABLV in a species other than bats or humans. We examined the host cell entry of ABLV, characterizing its tropism and exploring its cross-species transmission potential using maxGFP-encoding recombinant vesicular stomatitis viruses that express ABLV G glycoproteins. Results indicate that the ABLV receptor(s) is conserved but not ubiquitous among mammalian cell lines and that the two ABLV variants can utilize alternate receptors for entry. Proposed rabies virus receptors were not sufficient to permit ABLV entry into resistant cells, suggesting that ABLV utilizes an unknown alternative receptor(s).

RelAp43, a member of the NF-κB family involved in innate immune response against Lyssavirus infection.

NF-κB transcription factors are crucial for many cellular processes. NF-κB is activated by viral infections to induce expression of antiviral cytokines. Here, we identified a novel member of the human NF-κB family, denoted RelAp43, the nucleotide sequence of which contains several exons as well as an intron of the RelA gene. RelAp43 is expressed in all cell lines and tissues tested and exhibits all the properties of a NF-κB protein. Although its sequence does not include a transactivation domain, identifying it as a class I member of the NF-κB family, it is able to potentiate RelA-mediated transactivation and stabilize dimers comprising p50. Furthermore, RelAp43 stimulates the expression of HIAP1, IRF1, and IFN-ß - three genes involved in cell immunity against viral infection. It is also targeted by the matrix protein of lyssaviruses, the agents of rabies, resulting in an inhibition of the NF-κB pathway. Taken together, our data provide the description of a novel functional member of the NF-κB family, which plays a key role in the induction of anti-viral innate immune response.