Active shooter in emergency departments in Guanajuato State, Mexico.

Mass violence events, especially in healthcare settings, have devastating consequences and long-lasting effects on the victims and the community. The rate of violent events in Mexico, especially in hospital settings, has increased since 2006, but has become more evident in 2018. Guanajuato State, located in central Mexico, is among the states most affected by the wave of violence, especially active shooter events. The year 2019 had the highest number of incidents. Therefore, the Silver Code and the components of Safe Hospitals, in accordance with the Hartford consensus and PAHO guidelines, were implemented in the hospitals of the Institute of Public Health of the State of Guanajuato, with a focus on the actions of healthcare personnel to prevent collateral damage. Although subsequently there were still fatalities and injuries in the events involving active shooters in the hospitals, there were no casualties among healthcare personnel, according to data from the Institute of Public Health, Guanajuato State. This paper presents information from the data from General Directorate of Epidemiology to describe the hospital mass violence situation in the State of Guanajuato, Mexico and recounts the step taken to effectively manage and prevent these situations moving forward. Specific recommendations based on international consensus and our experience provided include increasing the level of security checks for people entering the hospital premises, training healthcare personnel on violence-related preparedness and improving management of active shooter events consistent with published evidence, to reduce the possibility of casualties.

Renal transplantation recipient patients survival.

INTRODUCTION: The Bajío High Specialty Regional Hospital started operating in 2007 to tackle the health demands of 5.8 million inhabitants. It has 184 beds and a transplant unit with 26 beds. In 2008, the renal transplant program launched activities. OBJECTIVE: To describe the survival of kidney transplant receptor patients and of the grafted kidney at the Bajío High Specialty Regional Hospital. METHODS: Retrospective cohort study, where consecutive transplants carried out between 2008 and 2016 were included. Statistical analysis was performed using the Kaplan-Meier method. RESULTS: A total of 837 transplants were analyzed. Graft survival censored for death, with a functional graft at 1 and 5 years, was 94.6% and 78.9%. Patient survival at 1 and 5 years was 95.4% and 88.1%. CONCLUSIONS: The renal transplant program is one of the the best programs established in Mexico, both for the number of deceased-donor kidney transplants performed and for the patient and graft survival achieved. These data indicate that the renal transplant program has had a sustained development.

INTRODUCCIÓN: El Hospital Regional de Alta Especialidad del Bajío inició sus funciones en 2007 para atender la demanda de salud de 5.8 millones de habitantes, cuenta con 184 camas y una unidad de trasplantes con 26 camas. En 2008 inició actividades el programa de trasplante renal. OBJETIVO: Presentar la supervivencia de los pacientes receptores de trasplante renal y del riñón injertado en el Hospital Regional de Alta Especialidad del Bajío, Guanajuato, México. MÉTODO: Estudio de cohorte retrospectivo en el que se incluyeron los trasplantes consecutivos realizados entre 2008 y 2016. El análisis estadístico se efectuó con el método de Kaplan-Meier. RESULTADOS: Se analizaron 837 trasplantes. La supervivencia del injerto censurada para muerte con injerto funcional a uno y cinco años fue de 94.6 y 78.9 %. La supervivencia del paciente a uno y cinco años fue de 95.4 y 88.1 %. CONCLUSIONES: El programa de trasplante renal constituye uno de los mejor establecidos en México, tanto por el número de trasplantes renales de donante fallecido realizados como por la supervivencia obtenida de paciente e injerto. Los datos indican que el programa de trasplante renal ha tenido un desarrollo sostenido.

Melittin and its potential in the destruction and inhibition of the biofilm formation by Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa isolated from bovine milk.

Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa stand out in veterinary and human medicine for their role in opportunistic infections and their pathogenic mechanisms, including the biofilms formation. It was investigated the antibacterial activity of melittin and antibiofilm of such bacteria. Twelve strains of these microorganisms isolated from bovine milk were used, as well as the strains S. aureus ATCC 12600, E. coli ATCC 8739 and Pseudomonas aeruginosa ATCC 15442. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) were determined by broth microdilution technique. The biofilms were formed in 96-well plates and melittin on these colonies was added at different concentrations and times. Bacteria previously exposed to melittin were evaluated for inhibition of biofilm production. The MIC and MBC were respectively in µg/mL: S. aureus (6-7 and 32-64), E. coli (40-42.5 and 64-128) and P. aeruginosa (65-70 and 64-128). S. aureus biofilms were more sensitive to the action of melittin, since upon exposure to a concentration 10 times lower than the MIC for 4 h, was completely destroyed. In Gram negative bacteria, the pre-formed biofilm was destroyed only when exposed for 4 h under the MIC. With respect to inhibition of biofilm production, S. aureus was the most sensitive again because produced only 37.2% of the biofilm formed by the control (without previous exposure to melittin), when exposed to the MIC, and at a concentration hundred times smaller than MIC, this microorganism produced 75.2% of the biofilm. E. coli was the most resistant bacteria and produced 56.3% of the biofilm, even if previously exposed to melittin MIC. Melittin presents desirable effects in combating microorganisms studied both at your disposal, biofilm destruction and inhibition of the formation, and maybe used in future studies of new strategies to combat infections caused by these pathogens.

Review and analysis of the results of kidney transplantation programs in Mexico.

OBJECTIVE: To know, analyze and compare kidney transplant programs; considering the survival of recipients at 1 and 5 years, from hospitals in Mexico. METHOD: A systematic review was carried out whose search focused on the survival of kidney transplant recipients. All publications found in PubMed and Google from 1963 to 2021 were included. The expectation-maximization algorithm was applied, proposing a mixture of normals, and hierarchical grouping to establish if there is any type of pattern and determine if there is a difference between the percentages. of survival at 1 and 5 years between the groups formed. RESULTS: Eight hospitals that published the survival of kidney transplant recipients were found. Survival rates ranged, at 1 year, from 94.7% to 100%, and at 5 years, from 85% to 96.2%. The methods used for their comparison indicated that there is a difference between survival at 1 and 5 years. CONCLUSIONS: In Mexico there is little information on the results of kidney transplant programs, and the information found shows great heterogeneity in said programs. Some strategies and actions are proposed to improve survival underreporting.

OBJETIVO: Conocer, analizar y comparar los programas de trasplante renal, considerando la supervivencia de los receptores a 1 y 5 años, en los hospitales en México. MÉTODO: Se realizó una revisión sistemática cuya búsqueda se centró en la supervivencia de los receptores de trasplante renal. Se incluyeron todas las publicaciones encontradas en PubMed y Google de 1963 a 2021. Se aplicó el algoritmo de expectation-maximization, proponiendo una mezcla de normales, y agrupamiento jerárquico para establecer si hay algún tipo de patrón y determinar si hay diferencia entre los porcentajes de supervivencia a 1 y 5 años entre los grupos formados. RESULTADOS: Se encontraron ocho hospitales que publicaron la supervivencia de los receptores de trasplante renal. Los rangos de las tasas de supervivencia fueron, a 1 año, del 94.7% al 100%, y a los 5 años, del 85% al 96.2%. Los métodos empleados para su comparación indican que hay diferencia entre la supervivencia a 1 y 5 años. CONCLUSIONES: En México se tiene poca información sobre los resultados de los programas de trasplante renal, y la información encontrada muestra gran heterogeneidad en dichos programas. Se proponen algunas estrategias y acciones para mejorar el subregistro de supervivencia.

Survival in a pediatric patient with cerebral aspergillosis: A case report.

Aspergillosis is an infrequent infection in the Central Nervous System with a mortality rate higher than 95 %. Early diagnosis is challenging and crucial. In this report, we present the case of a six-year-old female with an intense headache accompanied by left hemiparesis, gaze deviation, horizontal nystagmus, and vomiting of mucous content on five occasions. After several approaches, a cerebrospinal fluid PCR resulted positive for Aspergillus spp., and then management started with amphotericin B at 2.6 mg/kg/day and was managed to have voriconazole. She survived, and two years after her first hospital admission, she suffered from cerebral aspergillosis sequelae. An area of improvement is the coordination between the request and delivery of studies outside the institution. In this case, the patient´s mother did not report the analysis results on time, delaying the diagnosis.

Peroxidase-linked assay for detection of antibodies against bovine leukosis virus.

A peroxidase linked assay (PLA) was designed to screen bovine sera for the presence of specific antibodies against bovine leukosis virus (BLV). Out of 201 samples of bovine sera analyzed, 52.2% were considered positive by PLA, 26.4% by AGID, and 38.9% by ELISA. Western blotting analyses excluded 27 samples found to be positive by PLA. PLA showed 100% of sensitivity when compared with AGID and ELISA. Specificity was 64.8% and 78%, respectively (kappa coefficients were 0.70 and 0.83). These findings indicate that PLA can be used as an alternative method for the diagnosis of BLV infection in cattle.

Paediatric fibrosarcoma treatment.

Introduction: Soft tissue sarcomas make up 7%-15% of childhood solid tumours. The aetiology of this disease is unknown. It is a fast-growing, painless tumour; histologically similar to adult fibrosarcoma, but having a lesser risk of metastasis and a better prognosis. The treatment is aimed towards localised intervention; complete surgical resection is the appropriate treatment as long as it can be performed. Case report: An 11 years old female was referred for resection of a soft tissue tumour on the right elbow with significant peripheral vascularisation. Tumour resection was scheduled, with the placement of a partial thickness skin graft, and a piece was sent to pathology; a histological type consistent with paediatric fibrosarcoma was obtained with margins less than 1 mm from the lesion. Therefore, the patient was referred to the paediatric oncology unit. Further studies with positron emission tomography were requested, in which no evidence of macroscopic anatomy-metabolic tumour activity was found. Subsequently, treatment was started by paediatric oncology with 2 sessions of chemotherapy and 20 sessions of radiotherapy with sufficient progress; finally, assessment by plastic and reconstructive surgery was performed and an adequate quality of graft was observed, without the need for any other intervention by their service. Conclusion: The involvement of the vascular surgeon in performing the tumour resection permitted the preservation of the best circulation to the extremity, thereby, avoiding amputation. The difficult decision made by the reconstructive surgeon to place a partial thickness graft over the surgical site, and to start radiotherapy/chemotherapy by paediatric oncology, were key to the success in achieving the patient's satisfactory progress.

Multiresistant bacteria isolated from domestic and wild animals with skin lesions were susceptible to native plants from Southern Brazil.

We evaluated the chemical composition, toxicity, and antibacterial activity of Schinus terebinthifolia (SCH), Eugenia uniflora (EUG), Persicaria hydropiperoides (PER), Equisetum hyemale (EQU), Solidago chilensis (SOL), and Baccharis trimera (BRA). These plants were tested (7.5-0.01 mg/mL) against Gram-positive (G+; n = 32) and Gram-negative (G-; n = 26) isolates from animals (M07-A9, CLSI). Antibiogram (disk diffusion), chromatographic analysis (UPLC), and toxicity assay (HET-CAM) were also performed. A high incidence of resistance was noted, in which 18.4% (07/38) of G+ (Staphylococcus intermedius/Enterococcus faecium) and 17.7% (06/34) of G- (Pseudomonas aeruginosa/Escherichia coli/Proteus mirabilis) were multidrug-resistant. All bacteria were sensitive (MIC50) to SCH (both 3.75 mg/mL), EUG (3.75 mg/mL and 7.5 mg/mL, respectively) and PER (both 7.5 mg/mL). SCH/EUG/PER highlighted as antibacterial, probably due to the major compounds (ethyl gallate, quinic acid, quercetin). These extracts showed normal embryonic development (SCH/EUG: 7.5-0.94 mg/mL). These findings highlighted the promising use of native plants for therapeutic purposes.

COVID-19-Associated Case Fatality Rate in Subjects Under 18 Years Old in Mexico, up to December 31, 2020.

Background: The emergence of the SARS-CoV-2 and the COVID-19 have become a global health crisis. The infection has been present in all the social sectors. Subjects under 18 years are one of them. The objective was to analyze the case fatality ratio of COVID-19 cases in the Mexican population under 18 years of age registered in the National Epidemiological Surveillance System from March 2020 to December 31, 2020. Material and Methods: The design is cross-sectional, quantitative, and analytical. All the suspected cases of respiratory viral disease, with a real-time polymerase chain reaction (RT-PCR) test result, aged from 0 to 17 years, were included. Descriptive statistics are presented for all the variables. Epidemiological curves were designed. The chi-squared test and its P-values were obtained to show the relationship between comorbidities and death. The case fatality ratio was computed for each comorbidity, sex, and age group. Multivariable logistic regression models were fitted to study the effect between comorbidities with the fatality of cases, adjusting for sex and age group as potential confounders. The alpha value was fixed to 0.05 to assess significance. Results: The number of records for this study was 167,856. Among them, 48,505 were from SARS-CoV-2-positive patients (28.90%), and 119,351 (71.10%) were negative. Of those who died, males (55.29%) (P < 0.05) and those under 2 years of age (50.35%) (P < 0.05) predominated. Unlike in older populations, from the comorbidities considered risk factors for death by COVID-19, only immunosuppression showed a statistically significant effect on the fatality of cases after adjustment by the other related variables. Sex and age group were not confounders for the models in those under 18 years old. Pneumonia, being younger than 5 years, and immunosuppression are related to death. Conclusion: The case fatality ratio in those under 18 years old is low. Special attention must be paid to those children under 5 years. The development of pneumonia is a warning indicator while treating them. On the other hand, having an open database of cases allows the researchers to analyze the impact of COVID-19 in different population sectors, which has clear benefits for public health.

Green propolis phenolic compounds act as vaccine adjuvants, improving humoral and cellular responses in mice inoculated with inactivated vaccines.

Adjuvants play an important role in vaccine formulations by increasing their immunogenicity. In this study, the phenolic compound-rich J fraction (JFR) of a Brazilian green propolis methanolic extract stimulated cellular and humoral immune responses when co-administered with an inactivated vaccine against swine herpesvirus type 1 (SuHV-1). When compared to control vaccines that used aluminium hydroxide as an adjuvant, the use of 10 mg/dose of JFR significantly increased (p < 0.05) neutralizing antibody titres against SuHV-1, as well as the percentage of protected animals following SuHV-1 challenge (p < 0.01). Furthermore, addition of phenolic compounds potentiated the performance of the control vaccine, leading to increased cellular and humoral immune responses and enhanced protection of animals after SuHV-1 challenge (p < 0.05). Prenylated compounds such as Artepillin C that are found in large quantities in JFR are likely to be the substances that are responsible for the adjuvant activity.

Identification of Clade E Avipoxvirus in Brazil.

Avian poxvirus (APV) is an enveloped double-stranded DNA virus that affects many domestic and wild birds worldwide. APVs are classified into three clades (A to C), represented by fowlpox (FP) virus (clade A), canarypox virus (clade B), and psittacinepox virus (clade C), although two additional clades (D and E) have been proposed. In this study, a tumorlike skin lesion found in a domestic fowl was submitted for molecular diagnosis of Avipoxvirus by PCR and sequencing. The phylogenetic analysis revealed that the amplified segment of the corelike 4b protein and polymerase genes clustered in clade E. The APVs in clade E were previously reported from outbreaks in Hungary (flock of turkeys) and in Mozambique (layer chickens), associated with a possible vaccine failure to protect against clade E viruses. To our knowledge, this report is the first identification of clade E in this country, providing new information about host range and genetic diversity of APVs in Brazil, and may represent a potential risk of FP disease outbreaks in commercial poultry.

Reporte de caso- Identificación del Avipoxvirus clado E en Brasil. El poxvirus aviar (APV) es un virus de ADN bicatenario envuelto que afecta a muchas aves domésticas y silvestres en todo el mundo. Los poxvirus aviares se clasifican en tres clados (A, B y C), representados por el virus de la viruela aviar (FP) (clado A), el virus de la viruela del canario (clado B) y el virus de la viruela de los psitácidos (clado C), aunque dos clados adicionales (D y E) han sido propuestos. En este estudio, una lesión cutánea similar a un tumor encontrada en una gallina doméstica fue sometida a diagnóstico molecular de Avipoxvirus por PCR y secuenciación. El análisis filogenético reveló que el segmento amplificado de los genes de la proteína del centro 4b y de la polimerasa se agruparon en el clado E. Los poxvirus aviares en el clado E se reportaron previamente de brotes en Hungría (parvada de pavos) y en Mozambique (gallinas de postura), asociados con una posible falla de la vacuna para proteger contra los virus del clado E. De acuerdo con el conocimiento de los autores, este informe es la primera identificación del clado E en este país, brindando nueva información sobre el rango de hospedadores y la diversidad genética de poxvirus aviares en Brasil, y puede representar un riesgo potencial de brotes de viruela aviar en aves comerciales.

Multicentric Study to Evaluate Mexican Medical Students' Knowledge of Transplants and Organ Donation.

INTRODUCTION: Transplants and organ donation are greatly aided by future medical professionals having adequate knowledge of this topic. This study aimed to elucidate the level of Mexican medical students' knowledge in the field of transplants and organ donation. MATERIALS AND METHODS: The evaluation instrument was designed and validated. The design used simple sampling with replacement, selecting a random sample of 5 universities from among the institutional members of the Mexican Association of Departments and Schools of Medicine (Asociación Mexicana de Facultades y Escuelas de Medicina [AMFEM]). The sample was composed of 3214 medical students. Measures of central tendency were determined, and the mean scores obtained across the different universities were compared using a Kruskal-Wallis test. The odds ratio was calculated for the students whose school or department included instruction on transplants and donation within their curriculum. Kendall correlation was used for the students' academic grade level and score. All analyses considered a threshold of P < .05. RESULTS: A questionnaire was administered to a sample of 2563 students to evaluate their knowledge of transplants and organ donation. The average score was 4.02 on a scale of 0 to 10 (standard deviation 0.03), with a 95% confidence interval (3.96-4.08). Students whose school or department taught the subject of transplants and donations within their curriculum obtained an odds ratio of 1.44 (P = .0000822). CONCLUSIONS: The findings of this study suggest that medical students in Mexico do not have sufficient knowledge of transplants and organ donation.

Adjuvant effect of green propolis on humoral immune response of bovines immunized with bovine herpesvirus type 5.

Despite recent technological advances in vaccine production, most vaccines depend on the association with adjuvant substances. In this study, propolis, which has been attracting the attention of researchers due to its bioactive properties, was evaluated as an immunological adjuvant. The association of 40mg/dose of an ethanolic extract of green propolis with an inactivated oil vaccine against bovine herpesvirus type 5 (BoHV-5), resulted in a significant increase (P<0.01) in the neutralizing antibody levels, comparing to the bovines that received the same vaccine without propolis. Besides, propolis increased the percentage of animals with high antibody titers (above 32). Phenolic compounds such as artepillin C (3,5-diprenyl-4-hydroxycinnamic acid) and the derivatives of cinnamic acid besides other flavonoid substances were abundant in the propolis extract used, and they could be the main substances with adjuvant action. The effect of the green propolis extract on the humoral immune response can be exploited in the development of new vaccines.

Alternatives for obtaining a continuous cell line from Apis mellifera / Alternativas para a obtenção de uma linhagem celular contínua de abelhas Apis mellifera

ABSTRACT: In worldwide there are reports of a significant decrease in colonies of the species Apis mellifera, caused by several factors, including viral infections. In order to study and diagnose illnesses caused by viruses, in vitro cell culture is used as a valuable tool. Yet, there are still no immortalized cell lines of honey bee Apis mellifera. Primary cell cultures are promising for this purpose and can supply the lack of continuous strains, but their establishment is difficult and laborious, which often makes them unfeasible for many research centers. Through the use of cell immortalization techniques, it is possible to develop continuous cell lines and thus benefit, in different ways, research related to different species of bees. The choice of technique is challenging, since in addition to the ability to remain viable for countless passages, cells must keep the genotype and phenotype similar or identical to the original tissue. This review intends to present methodologies that can be used to immortalize Apis mellifera cells, aiming to establish a cell line. The genotypic and phenotypic implications of each technique are evaluated, and the purpose of the cell line to be developed.

RESUMO: Ao redor do mundo há relatos da diminuição significativa de colônias da espécie Apis mellifera, causada por diversos fatores, incluindo infecções virais. Para estudo e diagnóstico de enfermidades causadas por vírus utiliza-se, como uma ferramenta valiosa, o cultivo celular in vitro. Contudo, ainda não existem linhagens celulares imortalizadas de abelhas Apis mellifera. Os cultivos celulares primários são promissores para este fim e podem suprir a falta de linhagens contínuas, porém seu estabelecimento é difícil e laborioso o que, muitas vezes, os torna inviáveis para muitos centros de pesquisa. Através do uso de técnicas de imortalização celular é possível desenvolver linhagens contínuas de células e assim beneficiar, de diversas formas, as pesquisas relacionadas às diferentes espécies de abelhas. A escolha da técnica é desafiadora, visto que, além da capacidade de permanecer viável por inúmeras passagens, as células devem manter o genótipo e fenótipo semelhante ou idêntico ao tecido original. O objetivo deste trabalho é apresentar metodologias que podem ser utilizadas para imortalização de células de Apis mellifera, visando o estabelecimento de uma linhagem celular. São avaliadas as implicações genotípicas e fenotípicas de cada técnica, e a finalidade da linhagem celular a ser desenvolvida.

Supervivencia de los pacientes receptores de trasplante renal / Renal transplantation recipient patients survival

Resumen Introducción: El Hospital Regional de Alta Especialidad del Bajío inició sus funciones en 2007 para atender la demanda de salud de 5.8 millones de habitantes, cuenta con 184 camas y una unidad de trasplantes con 26 camas. En 2008 inició actividades el programa de trasplante renal. Objetivo: Presentar la supervivencia de los pacientes receptores de trasplante renal y del riñón injertado en el Hospital Regional de Alta Especialidad del Bajío, Guanajuato, México. Método: Estudio de cohorte retrospectivo en el que se incluyeron los trasplantes consecutivos realizados entre 2008 y 2016. El análisis estadístico se efectuó con el método de Kaplan-Meier. Resultados: Se analizaron 837 trasplantes. La supervivencia del injerto censurada para muerte con injerto funcional a uno y cinco años fue de 94.6 y 78.9 %. La supervivencia del paciente a uno y cinco años fue de 95.4 y 88.1 %. Conclusiones: El programa de trasplante renal constituye uno de los mejor establecidos en México, tanto por el número de trasplantes renales de donante fallecido realizados como por la supervivencia obtenida de paciente e injerto. Los datos indican que el programa de trasplante renal ha tenido un desarrollo sostenido.

Abstract Introduction: The Bajío High Specialty Regional Hospital started operating in 2007 to tackle the health demands of 5.8 million inhabitants. It has 184 beds and a transplant unit with 26 beds. In 2008, the renal transplant program launched activities. Objective: To describe the survival of kidney transplant receptor patients and of the grafted kidney at the Bajío High Specialty Regional Hospital. Methods: Retrospective cohort study, where consecutive transplants carried out between 2008 and 2016 were included. Statistical analysis was performed using the Kaplan-Meier method. Results: A total of 837 transplants were analyzed. Graft survival censored for death, with a functional graft at 1 and 5 years, was 94.6% and 78.9%. Patient survival at 1 and 5 years was 95.4% and 88.1%. Conclusions: The renal transplant program is one of the the best programs established in Mexico, both for the number of deceased-donor kidney transplants performed and for the patient and graft survival achieved. These data indicate that the renal transplant program has had a sustained development.

Antiviral activity of a Bacillus sp. P34 peptide against pathogenic viruses of domestic animals.

P34 is an antimicrobial peptide produced by a Bacillus sp. strain isolated from the intestinal contents of a fish in the Brazilian Amazon basin with reported antibacterial activity. The aim of this work was to evaluate the peptide P34 for its in vitro antiviral properties against canine adenovirus type 2 (CAV-2), canine coronavirus (CCoV), canine distemper virus (CDV), canine parvovirus type 2 (CPV-2), equine arteritis virus (EAV), equine influenza virus (EIV), feline calicivirus (FCV) and feline herpesvirus type 1 (FHV-1). The results showed that the peptide P34 exhibited antiviral activity against EAV and FHV-1. The peptide P34 inhibited the replication of EAV by 99.9% and FHV-1 by 94.4%. Virucidal activity was detected only against EAV. When P34 and EAV were incubated for 6 h at 37 °C the viral titer reduced from 10(4.5) TCID50 to 10(2.75) TCID50, showing a percent of inhibition of 98.6%. In conclusion, our results demonstrated that P34 inhibited EAV and FHV-1 replication in infected cell cultures and it showed virucidal activity against EAV. Since there is documented resistance to the current drugs used against herpesviruses and there is no treatment for equine viral arteritis, it is advisable to search for new antiviral compounds to overcome these infections.

Potencial antiviral e virucida da melitina e apamina contra herpesvírus bovino tipo 1 e vírus da diarreia viral bovina / Antiviral and virucidal potential of melittin and apamin against bovine herpesvirus type 1 and bovine viral diarrhea virus

A busca por alternativa aos fármacos sintéticos têm revelado descobertas no campo da farmacologia e, nesse sentido, melitina e apamina, dois constituintes do veneno de abelhas, foram descritas com várias ações farmacológicas. Este estudo objetivou avaliar in vitro as capacidades antiviral e virucida destes componentes. Para tanto, células MDBK (Madin Darby Bovine Kidney), após verificação das respectivas doses tóxicas por ensaio MTT ((3-(4,5 dimetiltiazol-2yl)-2-5-difenil-2H tetrazolato de bromo), foram cultivadas em microplacas e tratadas com diferentes concentrações de apamina, melitina e sua associação. Esse tratamento ocorreu antes e após a infecção com 0,1 MOI (multiplicidade de infecção) de cepas citopatogênicas de herpesvírus bovino tipo 1 (BoHV-1) cepa Los Angeles e vírus da diarreia viral bovina (BVDV) cepa NADL. Após incubação por 72 horas, 37oC, as células foram submetidas ao ensaio MTT para estimativa da viabilidade celular. Em experimento paralelo, placas que foram submetidas ao mesmo procedimento sofreram ciclo de congelamento e descongelamento das células, para rompimento das mesmas e mensuração dos títulos virais. O ensaio virucida foi realizado incubando-se suspensões de BoHV-1 e BVDV com as soluções de apamina, melitina e associação por 24 horas a 37oC e 22oC. O título viral foi avaliado às 0 horas, 1, 2, 4, 8 e 24 horas de incubação. A concentração citotóxica para 50% das células (CC50) de melitina foi 2,32 μg/ml e apamina não demonstrou toxicidade à maior concentração testada (100μg/ml). Houve efeito antiviral da melitina sobre BoHV-1, especialmente na concentração de 2μg/ml, onde observou-se 85,96% de viabilidade celular quando o tratamento foi realizado antes da infecção e 86,78% de viabilidade quando o tratamento foi realizado após a infecção. Houve ainda redução de 90% das partículas virais de BoHV-1. Em menores concentrações (1 e 1,5μg/ml) de melitina não houve atividade antiviral, pois a viabilidade celular foi baixa, demonstrando efeito citopático do vírus. Na associação das duas substâncias houve queda no título de BVDV e observou-se maior viabilidade celular quando comparados à ação isolada dos composto sobre este vírus. Isso se confirma na atividade virucida, uma vez que houve decréscimo de 90% das partículas virais de BVDV com a associação dos dois compostos do veneno de abelhas. Atuando individualmente, melitina apresentou efeito antiviral e virucida frente ao BoHV-1, zerando seu título em apenas 2 horas a 37oC. Conclui-se que melitina tem ação antiviral e virucida frente ao BoHV-1 e sua associação com apamina potencializou seus efeitos frente ao BVDV.(AU)

The search for an alternative to synthetic drugs have revealed discoveries in the field of pharmacology and, according to melittin and apamin, two components of bee venom which have been described were with various pharmacological actions.This study aimed to evaluate the in vitro antiviral and virucidal capabilities of these components. Therefore, after verification of their toxic doses by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, MDBK cells (Madin Darby Bovine Kidney) have been cultivated in microplates and treated with different concentrations of apamin, melittin and its association. This treatment occurred before and after infection with MOI (multiplicity of infection) 0.1 of cytopathogenic strains of bovine herpesvirus type 1 (BoHV-1) strain Los Angeles and bovine viral diarrhea virus (BVDV) strain NADL. After incubation for 72 hours, 37°C, the cells were submitted to MTT assay to estimate cell viability. In parallel experiments, plates were subjected to the same procedure suffered freezing and thawing cycle the cells to rupture the same and measurement of viral titers. The virucidal assay was performed by incubating suspension of bovine herpesvirus type-1 and BVDV with apamin solutions, melittin and association for 24 hours at 37°C and 22°C. The viral titer was evaluated at 0 hours, 1, 2, 4, 8 and 24 hours of incubation. The cytotoxic concentration to 50% of the cells (CC50) of melittin was 2.32g/mL and apamin did not show toxicity at the greater concentration tested (100μg/mL). There was antiviral effect of melittin on bovine herpesvirus type-1, especially at a concentration of 2μg/mL, where was observed 85.96% cell viability when treatment was performed before the infection and 86.78% viability when the treatment was carried out after infection. There was also a 90% reduction of viral particles of bovine herpesvirus type-1. In lower concentrations (1 and 1.5μg/mL) melittin no antiviral activity because cell viability was low, showing cytopathic effect of the virus. At the association two substances there were a decrease in the title of BVDV and there was higher cell viability when compared to the isolated action of the compounds of this virus. This is confirmed in the virucidal activity, since there was a decrease of 90% of the viral particles of BVDV with the combination of the two compounds of bee venom. Acting individually, melittin showed antiviral effect and virucidal against for BoHV-1, zeroing its title in just 2 hours at 37°C. It is concluded that melittin has antiviral and virucidal action against the BoHV-1 and its association with apamin potentiate its effects against BVDV.(AU)

Antigenic and immunogenic properties of the canine distemper virus nucleocapsid protein expressed in Escherichia coli employing codon optimized synthetic gene / Propriedades antigênicas e imunogênicas da proteína do nucleocapsídeo do virus da cinomose canina expressa em Escherichia coli empregando gene sintético com codons otimizados

Despite common occurrence and importance of canine distemper disease the majority of tests currently available for diagnosis are hampered by either low sensitivity or specificity. In this study it was evaluated antigenic and immunogenic characteristics of a conserved region of nucleocapsid protein of canine distemper virus (rCDV NP) expressed in Escherichia coli employing a codon optimized synthetic gene. The expression of rCDVNP in Star strain (mean 300μg/mL, purified) was confirmed by SDS-PAGE and Western blot analysis by using His-Tag monoclonal antibodies. Western blot and ELISA, employing positive and negative control dog sera, demonstrated the rCDVNP antigenicity. The rCDVNP was inoculated in hens and immunoglobulin Y (IgY) was purified from the egg yolk. The mean yield of IgY was 28.55mg/mL. IgY reacted with the recombinant protein as demonstrated by Western blot and ELISA assays. In summary, our findings demonstrated that rCDVNP is antigenic since CDV positive dog sera recognized the protein in vitro. Additionally, the rCDVNP proved to be immunogenic in hens being possible to isolate a high concentration of specific IgY antibodies from the egg yolk. Taken together, these results indicate that the rCDVNP along with the specific IgY could be useful tools for development of the canine distemper immunodiagnostic assays.(AU)

Apesar da ocorrência comum e importância da cinomose canina, a maioria dos testes atualmente disponíveis para diagnóstico são prejudicados pela baixa sensibilidade ou especificidade. Neste estudo foram avaliadas características antigênicas e imunogênicas de uma região conservada da proteína do nucleocapsídeo do virus da cinomose canina (rCDV NP) expressa em Escherichia coli empregando um gene sintético e codons otimizados. A expressão na cepa Star (média de 300μg/mL, purificada) foi confirmada por SDS-PAGE e Western blot utilizando anticorpos monoclonais anti-His-Tag. A antigenicidade da rCDVNP foi demonstrada por western blot e ELISA empregando soros de cães positivos e negativos. A rCDVNP foi inoculada em galinhas e imunoglobulina Y (gY) foi obtida e purificada a partir da gema. A produção média de IgY foi 28.55mg/mL. Anticorpos IgY reagiram com a proteína recombinante, quando analisados por Western blot e ELISA. Em resumo, nossos achados demonstram que a rCDVNP produzida é antigênica, uma vez que os anticorpos de soro de cães positivos para CDV reconheceram a proteína in vitro. Além disso, a rCDVNP foi imunogênica em galinhas, sendo possível isolar anticorpos IgY específicos a partir da gema do ovo em altas concentrações. Tomados em conjunto, estes resultados indicam que a rCDVNP juntamente com a IgY específica podem ser ferramentas úteis para elaborar ensaios de imunodiagnóstico de cinomose canina.(AU)

Atividade antiviral e virucida de extratos hidroalcoólicos de própolis marrom, verde e de abelhas Jataí (Tetragonisca angustula) frente ao herpersvírus bovino tipo 1 (BoHV-1) e ao vírus da diarreia viral bovina (BVDV) / Antiviral and virucidal activity of hydroalcoholic extracts of propolis brown, green and jataí bees (Tetragonisca angustula) against Bovine Herpesvirus Type-1 (BoHV-1) and Bovine Viral Diarrhea Virus (BVDV)

Dentre as propriedades biológicas da própolis, a atividade antimicrobiana tem merecido destacada atenção. No presente trabalho, descreve-se a ação antiviral e virucida de três extratos hidroalcoólicos de própolis (marrom, verde e de abelhas jataí (Tetragonisca angustula), frente ao Herpesvírus Bovino tipo (BoHV-1) e ao Vírus da Diarreia Viral Bovina (BVDV). Os três extratos hidroalcoólicos foram obtidos de extração etanólica e são oriundos do sul do Brasil. A composição química dos extratos de própolis foi determinada pela cromatografia líquida de alta eficiência acoplada a espectrômetro de massas (UFLC-PDA-ESI-TOF/MS) que identificou e quantificou compostos como: ácido cafeico e ácido p-cumárico, ácido clorogênico, ácido ferúlico, além de flavonoides como a rutina. A toxicidade celular bem como a atividade antiviral dos extratos de própolis em monocamadas de células MDBK (Madin-Darby Bovine Kidney) foi avaliada através de observação microscópica e quantificada pelo teste de MTT (3-(4,5 dimetiltiazol-2yl)-2-5-difenil-2H tetrazolato de bromo). O extrato de própolis de abelhas jataí demonstrou ser menos citotóxico (1,57µg/mL), quando comparado aos extratos verde (0,78µg/mL) e marrom (0,39µg/mL). Quanto a atividade antiviral, a própolis verde demostrou maior eficácia em ambos os tratamentos celulares (pós e pré-exposição) frente ao BoHV-1 em relação aos outros extratos, ou seja, houve maior viabilidade celular quando comparada aos controles de células e vírus. Já a de jataí apresentou atividade frente aos dois vírus (BoHV-1 e BVDV) no método pré-infecção, enquanto a própolis marrom demonstrou ação apenas frente ao BoHV-1 também no método pré-infecção. Para determinação da atividade virucida foram utilizadas diferentes diluições dos vírus, bem como temperaturas e tempos distintos de incubação. A própolis verde a 37°C propiciou a maior redução no título viral (4,33log) em relação a marrom (log = 3,5log) e de jataí (log = 3,24log). No entanto, frente ao BVDV a própolis jataí apresentou os melhores resultados em ambas as temperaturas (22oC e 37oC). Portanto, os extratos avaliados apresentaram atividade antiviral e virucida frente ao BoHV-1 e BVDV, o que os torna alvo para o desenvolvimento de novos biofármacos como alternativa ao uso de antivirais comerciais em Medicina Veterinária.(AU)

Among the biological properties of propolis, the antimicrobial activity has received prominent attention. In this paper, we describe the antiviral and virucidal effect of three hydroalcoholic extracts of propolis (brown, green and jataí bees (Tetragonisca angustula), against bovine herpesvirus type-1 (BoHV-1) and bovine viral diarrhea Virus (BVDV). All hydroalcoholic extracts were obtained from ethanol extraction. The chemical composition of propolis extracts was determined by high-performance liquid chromatography coupled to mass spectrometer (UFLC-PDA-ESI-TOF/MS) to identify and quantify compounds such as caffeic acid and p-coumaric acid, chlorogenic acid, ferulic, and flavonoids such as rutin. Cell toxicity and antiviral activity of propolis extracts in monolayers of MDBK cells (Madin-Darby Bovine Kidney) were assessed by microscopic observation and quantified by the MTT assay (3- (4.5 dimethylthiazol-2yl) -2- 5-diphenyl-2H-tetrazolato bromine). Propolis extract from Jataí bees proved to be less cytotoxic (1.57mg / ml) when compared to green extracts (0.78mg / ml) and brown (0.39mg/mL). Regarding antiviral activity, propolis has shown greater efficacy in both cellular treatments (post and pre-exposure) against BoHV-1 when compared to other extracts, ie, there was increased cell viability compared to cell and virus controls. Extracts from Jataí showed activity against both viruses (BoHV-1 and BVDV) infection in the pre-test, whereas brown propolis demonstrated action only against the BoHV-1 in the pre-infection method. To determine the virucidal activity, it were used different dilutions of virus, as well as different temperatures and incubation times. The green propolis at 37°C led to a greater reduction in viral titer (4.33log) compared to brown (3.5log) and jataí (3.24log). Jataí propolis showed the best results in both temperatures (22oC and 37oC) when tested against BVDV. In summary, the evaluated extracts showed antiviral and virucidal activity against BoHV-1 and BVDV, and may be important targets for the development of new compounds as an alternative to commercial antivirals.(AU)

Activity of the antimicrobial peptide P34 against bovine alphaherpesvirus type 1 / Atividade do peptídeo antimicrobiano P34 contra o alfaherpesvírus bovino tipo 1

ABSTRACT: Previous studies have demonstrated the antimicrobial activity of the peptide P34. In this study, the antiviral potential of P34 and the in vitro mechanism of action were investigated against bovine alphaherpesvirus type 1 (BoHV1). P34 exhibited low toxicity, a high selectivity index (22.9) and a percentage of inhibition of up to 100% in MDBK cells. Results from antiviral assays indicated that P34 did not interact with cell receptors, but it was able to inhibit the viral penetration immediately after pre-adsorption. In addition, BoHV1 growth curve in MDBK cells in the presence of P34 revealed a significant reduction in virus titer only 8h post-infection, also suggesting an important role at late stages of the replicative cycle. Virucidal effect was observed only in cytotoxic concentrations of the peptide. These findings showed that the antimicrobial peptide P34 may be considered as a potential novel inhibitor of in vitro herpesviruses and must encourage further investigation of its antiherpetic activity in animal models as well as against a wide spectrum of viruses.

RESUMO: A atividade antimicrobiana do peptídeo P34 já foi previamente demonstrada. Neste estudo, o potencial antiviral do P34 e o mecanismo de ação in vitro contra o alfaherpesvírus bovino tipo 1 (BoHV1) foram investigados. O P34 exibiu baixa toxicidade, alto índice de seletividade (22.9) e percentagem de inibição viral de até 100% em células MDBK. Os resultados dos ensaios antivirais indicaram que não interage com receptores celulares, mas é capaz de inibir a penetração viral, imediatamente após a pré-adsorção. Além disso, a curva de crescimento do BoHV1 em células MDBK na presença do P34 revelou uma significativa redução no título somente após 8h de infecção, sugerindo também uma importante atividade do peptídeo nas fases finais do ciclo replicativo. Efeito virucida frente / BoHV1 foi observado apenas em concentrações citotóxicas do peptídeo. Os dados obtidos indicam que o peptídeo antimicrobiano P34 pode ser considerado um potencial composto inibidor de herpesvírus, in vitro, e estimulam posteriores investigações sobre sua atividade anti-herpética em modelos animais, bem como contra outros vírus.