Hacia el control de la diarrea por rotavirus A: ¿podrían los nanoanticuerpos VHH marcar la diferencia? / Controlling rotavirus-associated diarrhea: could single-domain antibody fragments make the difference?

Los rotavirus del grupo A (RVA) constituyen la principal causa de diarrea grave y mortalidad infantil. La porción variable de los anticuerpos de cadena pesada derivados de camélidos presentan una amplia capacidad de unión antigénica (reconocen sitios antigénicos no accesibles a los anticuerpos tradicionales, con elevada afinidad) tienen bajos costos de producción y resultan ideales para las terapias orales. A la fecha, se desarrollaron 2 pares de nanoanticuerpos VHH contra RVA: ARP1-ARP3 y 2KD1-3B2. En este trabajo, exploramos el potencial de ambos grupos de nanoanticuerpos como estrategias de prevención complementarias a la vacunación y como una opción de tratamiento frente a la diarrea asociada a RVA en poblaciones de riesgo. Ambos pares de nanoanticuerpos fueron expresados en diferentes sistemas de producción y mostraron amplia capacidad neutralizante contra diversas cepas de RVA in vitro. También fueron usados en el modelo de ratón lactante, en el que evidenciaron distintos grados de éxito en la prevención o el tratamiento de la diarrea inducida por RVA. Es interesante destacar que la mitigación de los síntomas también se logró con ARP1 liofilizado y conservado, por lo que podría ser utilizado en áreas donde es difícil mantener la cadena de frío. Asimismo, 3B2 fue testeado en una prueba preclínica utilizando como modelo al cerdo gnotobiótico, al cual confirió completa protección contra la diarrea inducida por RVA. ARP1 fue usado en la primera prueba clínica de nanoanticuerpos VHH contra RVA, donde redujo significativamente las deposiciones en pacientes pediátricos con diarrea positivos para RVA, sin evidenciar ninguna reacción adversa

Group A Rotavirus (RVA) remains a leading cause of severe diarrhea and child mortality. The variable domain of camelid heavy chain antibodies (VHH) display potent antigen-binding capacity, have low production costs and are suitable for oral therapies. Two sets of anti-RVA VHHs have been developed: ARP1-ARP3; 2KD1-3B2. Here, we explore the potential of both sets as a prevention strategy complementary to vaccination and a treatment option against RVA-associated diarrhea in endangered populations. Both sets have been expressed in multiple production systems, showing extensive neutralizing capacity against strains of RVA in vitro. They were also tested in the neonatal mouse model with various degrees of success in preventing or treating RVA-induced diarrhea. Interestingly, mitigation of the symptoms was also achieved with freeze-dried ARP1, so that it could be applied in areas where cold chains are difficult to maintain. 3B2 was tested in a pre-clinical trial involving gnotobiotic piglets where it conferred complete protection against RVA-induced diarrhea. ARP1 was used in the first clinical trial for anti-RVA VHHs, successfully reducing stool output in infants with RVA diarrhea, with no detected side effects

Economic impact of a rotavirus vaccine in Brazil.

The study was done to evaluate the cost-effectiveness of a national rotavirus vaccination programme in Brazilian children from the healthcare system perspective. A hypothetical annual birth-cohort was followed for a five-year period. Published and national administrative data were incorporated into a model to quantify the consequences of vaccination versus no vaccination. Main outcome measures included the reduction in disease burden, lives saved, and disability-adjusted life-years (DALYs) averted. A rotavirus vaccination programme in Brazil would prevent an estimated 1,804 deaths associated with gastroenteritis due to rotavirus, 91,127 hospitalizations, and 550,198 outpatient visits. Vaccination is likely to reduce 76% of the overall healthcare burden of rotavirus-associated gastroenteritis in Brazil. At a vaccine price of US$ 7-8 per dose, the cost-effectiveness ratio would be US$ 643 per DALY averted. Rotavirus vaccination can reduce the burden of gastroenteritis due to rotavirus at a reasonable cost-effectiveness ratio.

In vitro antiviral activity of antimicrobial peptides against herpes simplex virus 1, adenovirus, and rotavirus

Peptides with broad-spectrum antimicrobial activity, known as antimicrobial peptides, have been isolated from distinct organisms. This paper describes the in vitro evaluation of the cytotoxicity and antiviral activity of nine peptides with different structures and origins against herpes simplex virus type 1, human adenovirus respiratory strain, and rotavirus SA11. Most of the evaluated peptides presented antiviral activity but they were only active near cytotoxic concentrations. Nevertheless, these results seem promising, and further modifications on the peptide's structures may improve their selectivity and reduce their cytotoxicity.

Association of rotavirus viroplasms with microtubules through NSP2 and NSP5

Rotavirus replication and virus assembly take place in electrodense spherical structures known as viroplasms whose main components are the viral proteins NSP2 and NSP5. The viroplasms are produced since early times after infection and seem to grow by stepwise addition of viral proteins and by fusion, however, the mechanism of viropIasms formation is unknown. In this study we found that the viroplasms surface colocalized with microtubules, and seem to be caged by a microtubule network. Moreover inhibition of microtubule assembly with nocodazole interfered with viroplasms growth in rotavirus infected cells. We searched for a physical link between viroplasms and microtubules by co-immunoprecipitation assays, and we found that the proteins NSP2 and NSP5 were co-immunoprecipitated with anti-tubulin in rotavirus infected cells and also when they were transiently co-expressed or individually expressed. These results indicate that a functional microtubule network is needed for viroplasm growth presumably due to the association of viroplasms with microtubules via NSP2 and NSP5.

Effect of isoprinosine on rotavirus replication in vitro

Isoprinosine (IPS) is a synthetic drug whose antiviral effect on rotavirus replication in vitro has been characterized in terms of the decrease in metachromasia after acridine orange staining. The present study describes the effect of IPS on the synthesis of viral RNA in vitro. MA-104 cell cultures infected with simian rotavirus strain SA-11 were incubated with zero, 250, 500 and 1,000 microg/ml IPS and 22, 24, 48, 52, 72 and 76 h after infection the cultures were submitted to a 1-h starvation period, followed by a 2-h pulse with 10 microCi/ml of [3H]-uridine. The homogenates of virus-infected cultures treated or not with IPS were submitted to phenol/chloroform extraction followed by polyacrylamide gel electrophoresis. The amount of radioactivity in viral RNA eluted from the gel strips was determined. Inhibition of viral RNA synthesis was highest at the IPS concentration of 1,000 microg/ml at 72 h after infection, corresponding to 78 percent inhibition. Although the results obtained in vitro suggest that IPS may be useful for the treatment of rotavirus infection, an in vivo demonstration of its efficacy is needed.

Teste de coaglutinaçäo para detecçäo de rotavirus / Coagglutination assay for detection of rotaviruses

Resumo: O teste de coaglutinaçäo foi utilizado para a detecçäo de rotavírus em fezes de origem humana e de suínos. Suspensäo de Staphylococcus aureos, produtor de proteina A, foi sensibilizada com uma diluiçäo seriada de antissoro anti-rotavirus do grupo A mostrando que quando foi utilizada a diluiçäo a 1:20, o teste foi capaz de detectar tanto antígeno SA11 como também o extrato fecal, ambos diluídos. Um total de 89 amostras de fezes absorvidas com S. aureus foram testadas por coaglutinaçäo e por um ensaio imunoenzimático. A análise estatística dos resultados obtidos mostrou uma concordância de 0,91 entre os dois testes o que levou-nos a conluir que a coaglutinaçäo é um método simples, rápido, sensível e pouco dispendioso para a detecçäo de rotavírus diretamente do material fecal. Além da utilizaçäo do soro diluído para a sensibilizaçäo de s> aureus ficou demonstrado também que, esta mistura, quando estocada a 4 graus C pode ser utilizada por até 6 meses após o seu preparo, sem implicar em resultados falso-positivos (au)

Rotavirus and bacterial enteropathogens causing acute diarrhea.

The etiology of rotavirus in acute diarrhoeal illness in children 0-5 years of age, admitted to the Pediatric wards of Kasturba Medical College Hospital, Manipal was studied, over a period of one year. Rotavirus in the faecal samples detected by the slide latex agglutination test accounted for 14.9% of the diarrheas with maximum incidence in the 7-12 months of age group (57.5%). Bacterial enteropathogens continued to play a significant role in diarrheal diseases. Salmonella enteritis was found more in the age group 0-6 months and shigellosis in 37-60 months. In a control study of 100 children who had no diarrhea, 2 were found positive for rotaviruses.

A reaçäo de imunofluorescência indireta no diagnóstico por rotavírus / The indirect immunofluorescence assay in the diagnosis of human rotavirus gastroenteritis

Utilizando a cepa de rotavírus SA-11, cultivada em células MA-104 e purificada em gradiente de CsCl, prepararam-se soros antirotavírus em cobaias para uso no diagnóstico de gastroenterites por rotavírus, através da reaçäo de imunofluorescência indireta. Num total de 268 casos, obteve-se uma porcentagem de positividade de 14.93 por cento (40 amostras). A comparaçäo desta prova com o ensaio imunoenzimático (EIERA) e a eletroforese em gel de poliacrilamida(EGPA), revelou boa concordância entre os três ensaios, com valores de kappa entre 0.72 e 0.76.

Inmunogenicidad e inocuidad de una vacuna contra rotavirus / Immnunogenicity and innocuid by rotavirus vaccine

Las enfermedades diarreicas están difundidas y causan todos los años la muerte de más de 4,5 millones de menores de 5 años, la importancia que presentan los rotavirus como agentes virales ha originado que se desarrollen estrategias inmunoprofilácticas, siendo de ellas la utilizadas en el desarrollo de una vacuna anti-rotavirus que consisten en el uso de una cepa animal relacionada antigenicamente y que sea capaz de crecer de manera eficiente en cultivos celulares. En este estudio fueron seleccionados 54 niños con edades entre 4 y 10 meses y recibieron 2 dosis de vacunas y el placebo. Fueron tomadas muestras de heces diariamente durante el período de observación. Los resultados preliminares de este estudio demostraron que la vacuna no produce reacciones secundarias en los diferentes grupos etarios estudiados, en especial en el grupo menor de 4 meses, en el grupo de niños de 2 a 3 meses se determinó la excreción viral en un 75