Algunos aspectos clínicos y epidemiológicos relacionados con mordeduras de perro en niños / Some clinical and epidemiological aspects related to dog bites in children

Introducción: Las mordeduras de perro afectan fundamentalmente a pacientes en la edad pediátrica, estas engendran peligros como secuelas estéticas y la posibilidad implícita de transmisibilidad del virus de la rabia a seres humanos. Objetivo: caracterizar el comportamiento clínico-epidemiológico de las mordeduras de perro en niños. Métodos: Se realizó un estudio observacional, longitudinal y prospectivo en pacientes con diagnóstico de mordedura de perro con necesidad de inmunización pasiva y activa, ingresados en el Servicio de Miscelánea del Hospital Infantil Sur Antonio María Béguez César de Santiago de Cuba, del 1ro de enero de 2017 al 31 de diciembre de 2018. Resultados: En los pacientes incluidos en el estudio predominaron los del sexo masculino (57,7 por ciento), los grupos de edades de 1 a 5 años 11 meses y 29 días, y de 6 a 10 años 11 meses y 29 días fueron los de mayor incidencia. Aunque no existió asociación estadística entre la localización anatómica de las lesiones y la edad, fueron las de miembros inferiores las más frecuentes en todas las edades con 63 pacientes (40,4 por ciento); destacándose como complicaciones la celulitis, las lesiones óseas y las secuelas estéticas; ningún niño presentó infección rábica. En el 50 por ciento de los casos la mordedura ocurrió en el horario nocturno y los perros de otro domicilio causaron la mayor parte de las agresiones. Conclusiones: El diagnóstico de mordeduras de perro se realizó con mayor frecuencia en niños del sexo masculino de uno y más años, en el horario nocturno y la principal complicación fue la celulitis(AU)

Introduction: Dog bites mainly affect patients in pediatric ages. They pose dangers such as esthetic sequels and the implicit possibility of transmission of the rabies virus to human beings. Objective: Characterize the clinical-epidemiological behavior of dog bites in children. Methods: An observational longitudinal prospective study was conducted of patients diagnosed with dog bite requiring passive and active immunization admitted to the Miscellany Service of Antonio María Béguez César South Children's Hospital in Santiago de Cuba from 1 January 2017 to 31 December 2018. Results: Among the patients included in the study there was a predominance of the male sex (57.7 percent). The age groups 1 to 5 years 11 months and 29 days and 6 to 10 years 11 months and 29 days showed the greatest incidence. Though no statistical association was found between anatomical location of the injuries and age, bites on the lower limbs were the most common at all ages with 63 patients (40.4 percent). Outstanding complications were cellulitis, bone lesions and esthetic sequels. No child had rabies infection. In 50 percent of the cases the bite occurred in the night hours. Most of the attacks were performed by dogs from other households. Conclusions: Dog bite diagnosis was more common among male children aged one year and over. Most bites occurred in the night hours. The main complication was cellulitis(AU)

Evidencia neuroanatómica del transporte del virus de la rabia por la vía propioespinal de la médula espinal de ratones / Neuroanatomical evidence of the transport of the rabies virus through the propriospinal tract in the spinal cord of mice

Resumen Introducción. Es escasa la información sobre los detalles neuroanatómicos del transporte del virus de la rabia en su ascenso por la médula espinal. Objetivos. Identificar la ruta neuroanatómica de diseminación del virus de la rabia en cada uno de los niveles de la médula espinal de ratón, después de ser inoculado por vía intramuscular. Materiales y métodos. Se inocularon ratones en los músculos isquiotibiales, con virus de la rabia. A partir de las 24 horas después de la inoculación, cada ocho horas se sacrificaron cinco animales por perfusión con paraformaldehído, se les extrajo la médula espinal y se hicieron cortes transversales en los niveles lumbosacro, torácico y cervical. Estos se procesaron mediante inmunohistoquímica para detectar antígenos virales. Resultados. Los primeros antígenos de la rabia se observaron como partículas agregadas, en la médula espinal lumbar, a las 24 horas después de la inoculación, dentro del asta ventral ipsilateral a la extremidad inoculada. A las 32 horas después de la inoculación, se hicieron visibles las primeras motoneuronas inmunorreactivas al virus. A las 40 horas después de la inoculación, se revelaron las primeras neuronas inmunorreactivas en la médula torácica, localizadas en la lámina 8 y, a las 48 horas después de la inoculación en la médula cervical, también en la lámina 8. A las 56 horas después de la inoculación, el virus se había diseminado por toda la médula espinal pero los animales aún no revelaban signos de la enfermedad. Conclusión. En el modelo de ratón aquí utilizado, el virus de la rabia ingresó a la médula espinal por las motoneuronas y, probablemente, utilizó la vía propioespinal descendente para su transporte axonal retrógrado hasta el encéfalo.

Abstract Introduction: Information about the neuroanatomical details of the ascendant transport of the rabies virus through the spinal cord is scarce. Objective: To identify the neuroanatomical route of dissemination of the rabies virus at each of the levels of the spinal cord of mice after being inoculated intramuscularly. Materials and methods: Mice were inoculated with the rabies virus in the hamstrings. After 24 hours post-inoculation, every eight hours, five animals were sacrificed by perfusion with paraformaldehyde. Then, the spinal cord was removed, and transverse cuts were made at the lumbosacral, thoracic, and cervical levels. These were processed by immunohistochemistry for the detection of viral antigens. Results: The first antigens of rabies were observed as aggregated particles in the lumbar spinal cord at 24 hours post-inoculation, within the ventral horn in the same side of the inoculated limb. At 32 hours post inoculation the first motoneurons immunoreactive to the virus became visible. At 40 hours post-inoculation the first immunoreactive neurons were revealed in the thoracic level, located on lamina 8 and at 48 hours post-inoculation in the cervical cord, also on lamina 8. At 56 hours post-inoculation the virus had spread throughout the spinal cord, but the animals still did not show signs of the disease. Conclusion: In the mouse model we used, the rabies virus entered the spinal cord through the motoneurons and probably used the descending propriospinal pathway for its retrograde axonal transport to the encephalus.

Short interfering RNAs targeting a vampire-bat related rabies virus phosphoprotein mRNA

Abstract The aim of this study was to assess the in vitro and in vivo effects of short-interfering RNAs (siRNAs) against rabies virus phosphoprotein (P) mRNA in a post-infection treatment for rabies as an extension of a previous report (Braz J Microbiol. 2013 Nov 15;44(3):879-82). To this end, rabies virus strain RABV-4005 (related to the Desmodus rotundus vampire bat) were used to inoculate BHK-21 cells and mice, and the transfection with each of the siRNAs was made with Lipofectamine-2000™. In vitro results showed that siRNA 360 was able to inhibit the replication of strain RABV-4005 with a 1 log decrease in virus titter and 5.16-fold reduction in P mRNA, 24 h post-inoculation when compared to non-treated cells. In vivo, siRNA 360 was able to induce partial protection, but with no significant difference when compared to non-treated mice. These results indicate that, despite the need for improvement for in vivo applications, P mRNA might be a target for an RNAi-based treatment for rabies.

Fluorescent antibody test, quantitative polymerase chain reaction pattern and clinical aspects of rabies virus strains isolated from main reservoirs in Brazil

ABSTRACTRabies virus (RABV) isolated from different mammals seems to have unique characteristics that influence the outcome of infection. RABV circulates in nature and is maintained by reservoirs that are responsible for the persistence of the disease for almost 4000 years. Considering the different pattern of pathogenicity of RABV strains in naturally and experimentally infected animals, the aim of this study was to analyze the characteristics of RABV variants isolated from the main Brazilian reservoirs, being related to a dog (variant 2),Desmodus rotundus (variant 3), crab eating fox, marmoset, and Myotis spp. Viral replication in brain tissue of experimentally infected mouse was evaluated by two laboratory techniques and the results were compared to clinical evolution from five RABV variants. The presence of the RABV was investigated in brain samples by fluorescent antibody test (FAT) and real time polymerase chain reaction (qRT-PCR) for quantification of rabies virus nucleoprotein gene (N gene). Virus replication is not correlated with clinical signs and evolution. The pattern of FAT is associated with RABV replication levels. Virus isolates from crab eating fox and marmoset had a longer evolution period and higher survival rate suggesting that the evolution period may contribute to the outcome. RABV virus variants had independent characteristics that determine the clinical evolution and survival of the infected mice.

Medium-term cryopreservation of rabies virus samples

Introduction The cryopreservation of rabies virus has been described in detail in the literature. To date, little information is available on the use of cryoprotective agents for cold preservation of this virus, and the available data focus only on short-term virus preservation. In this study, we investigated the medium-term cryopreservation of samples of rabies virus using different cryopreservation protocols. Methods The cryopreservation protocols for the rabies virus samples were performed at -20°C and were divided according to the variables of time and cryoprotectant type used. The laboratory tests (intracerebral inoculation of mice, viral titration and direct immunofluorescence) were performed at regular intervals (360 and 720 days) to assess the viability of the viral samples according to the different preservation techniques used. Results After 1 year of cryopreservation, the fluorescence intensity of intracellular corpuscles of the rabies virus and the median survival time of the mice differed between the positive controls and the treatments with the cryoprotectants. After 2 years, most of the samples subjected to the cryopreservation protocols (including the controls) did not produce fluorescence. However, the virus samples exposed to the cryoprotectant sucrose (68% solution) responded positively in the direct immunofluorescence assay and in the intracerebral inoculation of the mice. Conclusions Medium-term cryopreservation of the rabies virus inactivates the viral sample. However, the cryoprotectant agent sucrose (68%) produces a preservative effect in cryopreserved rabies virus samples. .

In vitro and in vivo inhibition of rabies virus replication by RNA interference

Rabies is a zoonotic disease that affects all mammals and leads to more than 55,000 human deaths every year, caused by rabies virus (RABV) (Mononegavirales: Rhabdoviridae: Lyssavirus). Currently, human rabies treatment is based on the Milwaukee Protocol which consists on the induction of coma and massive antiviral therapy. The aim of this study was to assess the decrease in the titer of rabies virus both in vitro and in vivo using short-interfering RNAs. To this end, three siRNAs were used with antisense strands complementary to rabies virus nucleoprotein (N) mRNA. BHK-21 cells monolayers were infected with 1000 to 0.1 TCID50 of PV and after 2 hours the cells were transfected with each of tree RNAs in separate using Lipofectamine-2000. All three siRNAs reduced the titer of PV strain in a least 0.72 logTCID50/mL and no cytotoxic effect was observed in the monolayers treated with Lipofectamine-2000. Swiss albino mice infected with 10.000 to 1 LD of PV strain by the intracerebral route were also transfected after two hours of infection with a pool 3 siRNAs with Lipofectamine-2000 by the intracerebral route, resulting in a survival rate of 30% in mice inoculated with 100 LD50, while the same dose led to 100% mortality in untreated animals. Lipofectamine-2000 showed no toxic effect in control mice. These results suggest that intracerebral administration of siRNAs might be an effective antiviral strategy for rabies.

Current status of human rabies transmitted by dogs in Latin America

Latin American countries made the political decision to eliminate human rabies transmitted by dogs by the year 2005. The purpose of the current study is to evaluate to what extent this goal has been reached. The epidemiological situation and control measures were analyzed and broken down within the countries by georeferencing. The 27 human cases reported in 2003 occurred in some 0.2 percent of the second-level geopolitical units (municipalities or counties) in the region, suggesting that the disease is a local problem. Several areas within the countries reported no more transmission of rabies in dogs. Nearly 1 million people potentially exposed to rabies received treatment. On average, 34,383 inhabitants per health post receive anti-rabies treatment (range: 4,300-148,043). Nearly 42 million dogs are vaccinated annually. Surveillance is considered fair according to the epidemiological criteria adopted by the study. Samples sent for rabies testing represent 0.05 percent of the estimated canine population (range: 0.001 to 0.2 percent). The countries are quite close to achieving the goal.

Os países da América Latina tomaram a decisão política de eliminar a raiva humana transmitida por cão até 2005, e o objetivo deste estudo é analisar o cumprimento desta meta. A situação epidemiológica e as ações de controle foram analisadas de forma desagregada dentro dos países, utilizando-se georreferenciamento da informação. Os 27 casos humanos relatados em 2003 ocorreram em cerca de 0,2 por cento das unidades de segundo nível geopolítico (municípios) da região. Esse dado sugere que a doença atualmente é muito localizada. Vários países não reportam mais transmissão de raiva em cães. Cerca de 1 milhão de pessoas são potencialmente expostas ao risco da raiva e recebem atendimento médico. Existem em média 34.383 (classe: 4.300-148.043) habitantes por posto de saúde com tratamento anti-rábico. São vacinados cerca de 42 milhões de cães anualmente, 70 por cento deles no Brasil e México. A vigilância epidemiológica para a raiva foi considerada média pelos critérios estabelecidos no estudo, sendo enviada 0,05 por cento da população canina estimada de amostras para diagnostico de raiva. Foi considerado que os países estão muito próximos de alcançar a meta.

Ultrastructural description of rabies virus infection in cultured sensory neurons

Primary cultures were made from adult mouse spinal ganglia for depicting an ultrastructural description of rabies virus (RABV) infection in adult mouse sensory neuron cultures; they were infected with rabies virus for 24, 36, and 48 h. The monolayers were processed for transmission electron microscopy and immunochemistry studies at the end of each period. As previously reported, sensory neurons showed great susceptibility to infection by RABV; however, in none of the periods evaluated were assembled virions observed in the cytoplasm or seen to be associated with the cytoplasmic membrane. Instead, fibril matrices of aggregated ribonucleoprotein were detected in the cytoplasm. When infected culture lysate were inoculated into normal animals via intra-cerebral route it was observed that these animals developed clinical symptoms characteristic of infection and transmission electron microscopy revealed assembled virions in the cerebral cortex and other areas of the brain. Sensory neurons infected in vitro by RABV produced a large amount of unassembled viral ribonucleoprotein. However, this intracellular material was able to produce infection and virions on being intra-cerebrally inoculated. It can thus be suggested that the lack of intracellular assembly in sensory neurons forms part of an efficient dissemination strategy.

Lack of correlation between rabies virus replication in the brain and antibody isotype profile in genetically modified mice

The relationship among the phenotypes resistance to infection, virus replication in the brain and isotype production was investigated in genetically modified High (H) or Low (L) antibody responder mouse lines. Although they express the same innate susceptibility to rabies infection, these lines differ as to different viral replication rates in the central nervous system and L mice showed a higher permissible state. After intramuscular infection with the Pasteur rabies strain (PV), the H-L interline differences on the earlier stage of virus replication were 1000 and 80 folds on days 5 and 6, respectively. The isotype profile in sera of the experimentally infected mice reflected an interline difference of 25 folds for IgG2a throughout the infection period, and for the IgE production the H-L difference was highly significant only at the beginning of the process. These results confirm the multi-specific effect of antibody immune responsiveness and the general isotype distribution of antibodies in these genetically selected mice. Contrary to the clear correlation between antibody responsiveness and the acquired resistance to rabies infection, the present study demonstrates that the constitutive genetic character of High and Low responder individuals does not intervene in the degree of resistance following infection. Altogether, this study contributes to the knowledge of the protective role of the general innate responsiveness on the pathological pattern to rabies virus infection

Determinaçäo do perfil antigênico de 3 cepas de virus rabico, isoladas no Brasil, através da técnica dos anticorpos monoclonais antinucleocapside / Determination of nucleocapside antigenic characteristics of 3 rabies virus strains isolated in Brazil by anti-nucleocapside, monoclonal antibodies technique

Determinou-se, através da técnica dos anticorpos monoclonais, o perfil antigênico antinucleocapside de 3 cepas de virus rábico, isoladas no Brasil, com o auxilio de técnica de imunofluorescência indireta. Duas das cepas eram de origem de cäo, uma delas procedente da cidade de Jales, SP, e a outra oriunda da Nigéria, Africa. A terceira cepa era de origem de morcego, identificada com DR 19 e considerada como cepa intermediária entre as fixas e as naturais. Os resultados obtidos evidenciaram diferenças pronunciadas entre as 3 cepas, caracterizando-as como distintas antigenicamente, mas com perfil antigênico caracteristico das cepas rábicas. Os resultados confirmaram, também, a procedência da cepa Nigéria, uma vez que seu perfil antigênico coincidiu com o daquelas isoladas em seu pais de origem. As cepas Jales e Nigéria, embora originárias de uma mesma espécie animal, apresentaram os perfis antigênicos do nucleocapside distintos