Training the Next Generation of Infectious Disease Researchers: A Biorisk Management Module Embedded in a European Master Program.

Introduction: The current global situation with increasing zoonotic transmissions of pathogens, rapidly changing ecosystems due to the climate change and with it the distribution of potential vectors, demands new ways of teaching and educating students in the field of infectious disease research. Methods: The international master program "Infectious Diseases and One Health-IDOH" started its second application period in 2019. Biosafety is an integral part of IDOH, exemplified by a biosafety level 3 hands-on training at the Animal Health Research Center IRTA-Centre de Recerca en Sanitat Animal (CReSA), Barcelona. At Hanover Medical School, biosafety is expanded to a comprehensive biorisk management approach with focus on risk assessment, bioethics, and training in a mobile containment laboratory. This article illustrates in depth the intention and the individual teaching units of the biorisk management module within the third semester of the IDOH master. Risk assessment is taught based on the new WHO Laboratory Biosafety Manual 4th edition, which represents a paradigm shift toward a risk-based approach instead of a prescriptive definition of biosafety levels. This methodology will enable the international IDOH students to cope with different national legislations and to provide guidance on biosafety in their home countries. In the final unit, the students operate a mobile containment laboratory and simulated a viral hemorrhagic fever outbreak in Africa. Conclusion: In sum, this module combines theoretical risk assessment and its practical implementation in the mobile laboratory as a future direction for training infectiologists. In addition, our report may serve as a blue print for others to amend their education with the herewith mentioned pillars of biosafety teaching.

Susceptibility Evaluation to Fire Blight and Genome-Wide Associations within a Collection of Asturian Apple Accessions.

Fire blight, caused by Erwinia amylovora, is one of the most devastating apple diseases. The selection of cultivars of low susceptibility and the study of the genetic mechanisms of the disease play important roles in fire blight management. The susceptibility level to fire blight was evaluated in 102 accessions originating from Asturias, a cider-producing region located in the north of Spain with a wide apple germplasm. Evaluations took place under quarantine conditions using artificial inoculations of grafted plants. The results revealed wide variation in susceptibility responses and low-susceptible cultivars were identified. In addition, 91 cultivars were genotyped using the Affymetrix Axiom® Apple 480 K SNP array to conduct genome-wide association studies (GWAS). A statistically significant signal was detected on chromosome 10 using the multi-locus mixed model (MLMM). Two genes were identified as major putative candidate genes: a TIR-NBS-LRR class disease protein and a protein containing a development and cell death (DCD) domain. The outcomes of this study provide a promising source of information, particularly in the context of cider apples, and set a starting point for future genetic and breeding approaches.

Removal of the Erns RNase Activity and of the 3' Untranslated Region Polyuridine Insertion in a Low-Virulence Classical Swine Fever Virus Triggers a Cytokine Storm and Lethal Disease.

In this study, we assessed the potential synergistic effect of the Erns RNase activity and the poly-U insertion in the 3' untranslated region (UTR) of the low-virulence classical swine fever virus (CSFV) isolate Pinar de Rio (PdR) in innate and adaptive immunity regulation and its relationship with classical swine fever (CSF) pathogenesis in pigs. We knocked out the Erns RNase activity of PdR and replaced the long polyuridine sequence of the 3' UTR with 5 uridines found typically at this position, resulting in a double mutant, vPdR-H30K-5U. This mutant induced severe CSF in 5-day-old piglets and 3-week-old pigs, with higher lethality in the newborn (89.5%) than in the older (33.3%) pigs. However, the viremia and viral excretion were surprisingly low, while the virus load was high in the tonsils. Only alpha interferon (IFN-α) and interleukin 12 (IL-12) were highly and consistently elevated in the two groups. Additionally, high IL-8 levels were found in the newborn but not in the older pigs. This points toward a role of these cytokines in the CSF outcome, with age-related differences. The disproportional activation of innate immunity might limit systemic viral spread from the tonsils and increase virus clearance, inducing strong cytokine-mediated symptoms. Infection with vPdR-H30K-5U resulted in poor neutralizing antibody responses compared with results obtained previously with the parent and RNase knockout PdR. This study shows for the first time the synergistic effect of the 3' UTR and the Erns RNase function in regulating innate immunity against CSFV, favoring virus replication in target tissue and thus contributing to disease severity. IMPORTANCE CSF is one of the most relevant viral epizootic diseases of swine, with high economic and sanitary impact. Systematic stamping out of infected herds with and without vaccination has permitted regional virus eradication. However, the causative agent, CSFV, persists in certain areas of the world, leading to disease reemergence. Nowadays, low- and moderate-virulence strains that could induce unapparent CSF forms are prevalent, posing a challenge for disease eradication. Here, we show for the first time the synergistic role of lacking the Erns RNase activity and the 3' UTR polyuridine insertion from a low-virulence CSFV isolate in innate immunity disproportional activation. This might limit systemic viral spread to the tonsils and increase virus clearance, inducing strong cytokine-mediated symptoms, thus contributing to disease severity. These results highlight the role played by the Erns RNase activity and the 3' UTR in CSFV pathogenesis, providing new perspectives for novel diagnostic tools and vaccine strategies.

The new emerging ovine pestivirus can infect pigs and confers strong protection against classical swine fever virus.

Several emerging pestiviruses have been reported lately, some of which have proved to cause disease. Recently, a new ovine pestivirus (OVPV), isolated from aborted lambs, with high genetic identity to classical swine fever virus (CSFV), has proved to induce reproductive disorders in pregnant ewes. OVPV also generated strong serological and molecular cross-reaction with CSFV. To assess the capacity of OVPV to infect swine, twelve piglets were infected either by intranasal or intramuscular route. Daily clinical evaluation and weekly samplings were performed to determine pathogenicity, viral replication and excretion and induction of immune response. Five weeks later, two pigs from each group were euthanized and tissue samples were collected to study viral replication and distribution. OVPV generated only mild clinical signs in the piglets, including wasting and polyarthritis. The virus was able to replicate, as shown by the RNA levels found in sera and swabs and persisted in tonsil for at least 5 weeks. Viral replication activated the innate and adaptive immunity, evidenced by the induction of interferon-alpha levels early after infection and cross-neutralizing antibodies against CSFV, including humoural response against CSFV E2 and Erns glycoproteins. Close antigenic relation between OVPV and CSFV genotype 2.3 was detected. To determine the OVPV protection against CSFV, the OVPV-infected pigs were challenged with a highly virulent strain. Strong clinical, virological and immunological protection was generated in the OVPV-infected pigs, in direct contrast with the infection control group. Our findings show, for the first time, the OVPV capacity to infect swine, activate immunity, and the robust protection conferred against CSFV. In addition, their genetic and antigenic similarities, the close relationship between both viruses, suggest their possible coevolution as two branches stemming from a shared origin at the same time in two different hosts.

Rapid SARS-CoV-2 Inactivation in a Simulated Hospital Room Using a Mobile and Autonomous Robot Emitting Ultraviolet-C Light.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since 2019 has made mask-wearing, physical distancing, hygiene, and disinfection complementary measures to control virus transmission. Especially for health facilities, we evaluated the efficacy of an UV-C autonomous robot to inactivate SARS-CoV-2 desiccated on potentially contaminated surfaces. ASSUM (autonomous sanitary sterilization ultraviolet machine) robot was used in an experimental box simulating a hospital intensive care unit room. Desiccated SARS-CoV-2 samples were exposed to UV-C in 2 independent runs of 5, 12, and 20 minutes. Residual virus was eluted from surfaces and viral titration was carried out in Vero E6 cells. ASSUM inactivated SARS-CoV-2 by ≥ 99.91% to ≥ 99.99% titer reduction with 12 minutes or longer of UV-C exposure and onwards and a minimum distance of 100cm between the device and the SARS-CoV-2 desiccated samples. This study demonstrates that ASSUM UV-C device is able to inactivate SARS-CoV-2 within a few minutes.

The impact of CSFV on the immune response to control infection.

The severity of the acute form of CSF is responsible for the high mortality rate and has been the subject of many studies. Nevertheless, some animals are likely to develop a mild, chronic, or unapparent form of the disease. Paradoxically, this clinical form of the disease has not been well studied, especially regarding its pathogenesis. In this study, we investigated the infection in domestic pigs that is caused by the CSFV Cat01 strain, which is responsible for the 2001-2002 CSFV outbreak in Catalonia, Spain, and which caused mild and nonspecific clinical signs compared to the infection that is caused by another CSFV strain that is responsible for inducing severe clinical symptoms of disease. We assessed the impact of the CSFV infection in the immune system of domestic pigs, mainly on the kinetics of different cytokines, such as IFN-α (innate immunity) and IFN-γ (adaptive immune response), during the first weeks after infection. In addition, we evaluated the impact on the induction of the humoral response and its relation to the course of infection and the RNA CSFV viral load. The IFN-α levels in the serum samples from the pigs that developed a milder form of the CSF disease (infected with Cat01 strain) were lower than those that were detected in the pig with severe clinical CSF signs (Margarita strain). After infection with Cat01 strain, the IFN-γ levels in response to CSFV were detected in addition to the humoral response. Interestingly, in the serum samples of these animals, we detected the lowest load of CSFV RNA. Similarly, the lowest viral load levels were detected in the tonsils of these pigs. Both the T cells and the humoral response that were generated in most of the pigs that were infected with strain Cat01 may be related to the protection in the symptom progression of CSF against this viral strain. These results explain the antiviral role of IFN-γ in the absence of an antibody response. Likewise, these results corroborate the relevance and relationship that exists between the intensity of the T cell response and the protection against CSFV replication. Additionally, these results also explain how the failure to induce optimal levels of humoral and cellular responses after CSFV infection promotes the spread and persistence of the virus.

Éxito del tratamiento de la litotricia extracorpórea por ondas de choque en la práctica habitual: estudio observacional con litotriptores electromagnético y electrohidráulico / Treatment success of extracorporeal shock-wave lithotripsy in clinical practice: observational study with electrohydraulic and electromagnetic lithotripters

La litotricia extracorpórea por ondas de choque (LEOC) es un tratamiento útil para la litiasis urinaria. El tipo de generador y las características del cálculo pueden condicionar los resultados del tratamiento. Sujetos y Métodos Se estudiaron pacientes ambulatorios, mayores de edad y con diagnóstico de litiasis urinaria alta tratada con LEOC en los hospitales Eugenio Espejo y Metropolitano de la ciudad de Quito. Dos cohortes de estudio se diferenciaron según el tipo de generador utilizado: LTEM (litotriptor electromagnético) y LTEH (litotriptor electrohidráulico). Las características del cálculo consideradas ideales para fragmentación (densidad ≤1000 UH, tamaño ≤20 mm y distancia piel-cálculo ≤10 cm) se determinaron por tomografía simple. El resultado del tratamiento se califcó como éxito temprano (desaparición del cálculo luego de la LEOC, valorada con fluoroscopía) y tardío (fragmentos <4 mm en la radiografía de control a los cinco días y cuatro semanas del tratamiento). Resultados Entre las cohortes LTEM (n=40; edad 46.2 ± 11.5 años; 42.5% hombres) y LTEH (n=40; edad 46.0 ± 13.8 años; 65.0% hombres) no hubo diferencias de los litos en su densidad (1027.7 ± 371.4 vs. 999.1 ± 269.4 UH; p=ns) y distancia piel-cálculo (9.0 ± 1.7 vs. 9.7 ± 2.0 cm; p=ns), pero tuvieron mayor tamaño en el grupo LTEM (16.1 ± 5.7 vs. 12.6 ± 4.4 mm; p<0.01). El éxito de fragmentaciones (72.5%; IC95: 61.8% - 81.1%) fue exactamente igual con ambos litotriptores. Los éxitos tempranos (57.5% vs. 47.5%; p=ns) y tardíos (15.0% vs. 25.0%; p=ns) no fueron estadísticamente diferentes. La frecuencia de litos con características ideales en su densidad (55.0% vs. 50.0%; p=ns), distancia a la piel (70.0% vs. 65.0%; p=ns) y tamaño (82.5% vs. 97.5%; p=0.05) fue similar entre los grupos. Tres, dos, una o ninguna de estas condiciones ideales se encontraron en el 37.5%, 40.0%, 17.5% y 5.0% del total de pacientes investigados. Conclusión La tasa de éxito de la LEOC durante la práctica habitual es similar con litotriptores electromagnético y electrohidráulico, sobre todo cuando las características ideales de los cálculos se presentan de forma similar en los pacientes.

Extracorporeal shock wave lithotripsy (ESWL) is a useful treatment for urolithiasis. However, the generator used and some characteristics of the stones may affect the treatment outcome. Subjects and methods We studied adult outpatients with urolithiasis who underwent ESWL at the Eugenio Espejo and Metropolitano hospitals from Quito-Ecuador were studied. Two study cohorts were defned according to the type of generator: EMLT (electromagnetic lithotripter) and EHLT (electrohydraulic lithotripter). The calculi characteristics considered ideal for fragmentation (density ≤1000 HU, size ≤20 mm, and distance skin-to-stone ≤10 cm) were determined by non-contrast computed tomography. Treatment outcome was defined as early success (disappearance of stone after ESWL, assessed by fluoroscopy) and delayed success (fragments <4 mm in the X-ray control fve days and four weeks after treatment). Results There was no difference between the groups EMLT (n=40; age 46.2 ± 11.5 years; 42.5% males) and EHLT (n = 40; age 46.0 ± 13.8 years; 65.0% male) respect to the stones density (1027.7 ± 371.4 vs. 999.1 ± 269.4 HU; p=ns) and skin-to-stone distance (9.0 ± 1.7 vs. 9.7 ± 2.0 cm; p=ns), but stones had a larger size in the EMLT group (16.1 ± 5.7 vs. 12.6 ± 4.4 mm; p<0.01). The success rate of stone fragmentation (72.5%; 95%CI: 61.8% - 81.1%) was exactly the same with both lithotripters. Early success (57.5% vs. 47.5%; p=ns) and delayed success (15.0% vs. 25.0%; p=ns) were not statistically different. Stones with ideal characteristics in density (55.0% vs. 50.0%; p=ns), skin-to-stone distance (70.0% vs. 65.0%; p=ns) and size (82.5% vs. 97.5%; p=0.05) showed a similar frequency between groups. Three, two, one or none of these ideal conditions were found in 37.5%, 40.0%, 17.5% and 5.0% of patients investigated. Conclusion The success rate of ESWL in clinical practice is similar with electromagnetic and electrohydraulic lithotripters, especially when stones with ideal characteristics are present similarly in patients.

Microsomía Cráneofacial: A qué edad se debe intervenir?

La microsomía craneofacial es un complejo malformativo con alteración en estructuras derivadas de los arcos branquiales primero y segundo. Aunque existe gran variabilidad en la presentación clínica del complejo, las tres áreas de mayor alteración constituyen la auricular, maxilar y mandibular. De todas maneras la afección más conspicua (la alteración del cóndilo mandibular), es el signo patognomónico de la microsomía cráneofacial. El concepto de "matriz funcional" sustentado por Moss en 1968 y el conocimiento del crecimiento facial han constituido los cimientos de los cambios progresivos que se han dado en el tratamiento de la microsomía craneofacial. No obstante los grandes avances en las técnicas quirúrgicas craneofaciales, aún existe considerable controversia en cuanto a la edad que debe tener el paciente para realizar los diversos procedimientos operatorios. Mientras existen autores que aún defienden las osteotomías tempranas, otros prefieren esperar hasta que el paciente cumpla por lo menos 9 años de edad y finalmente algunos autores consideran que las osteotomías maxilomandibulares se realizarían después de los 12 años de edad. El objetivo de este trabajo es racionalizar un cronograma de tratamiento, abarcando todos los aspectos del complejo malformativo, en base de la experiencia de los programas de tratamiento que más se han difundido en el mundo.