Síndrome diarreico en potrillos, diagnóstico y caracterización de los agentes virales involucrados

Neonatal diarrhea is one of the most relevant and frequent syndromes in foals younger than six month of age. Group A Rotavirus (RVA) is the main viral agent associated to diarrhea in foals, worldwide. However, around 50% of the diarrhea episodes remain without an etiological diagnosis. The aims of the present work were, to go in detail into the molecular epidemiology, the evolutive and structural characteristics of equine RVA and to investigate the presence and role of other viral agents as potential causes of diarrhea in Argentinean foals. The study in equine RVA started with the evaluation of the sensitivity and specificity of tree diagnosis tests. Secondly, we studied the molecular epidemiology of the RVA circulating in Argentinean foals from 2009 to 2014. The G3P[12] and G14P[12] genotypes were detected with a cyclic prevalence among the years under study. Considering all the genome sequences of the equine RVA strains available an evolutive study allowed us to determine the monophyletic clustering of the equine RVA detected in Argentina. The E12 genotype -encoding the viral enterotoxin NSP4- was defined as a geographical marker which allows describing the evolution of the equine RVA present in South America since the first foundation of Buenos Aires in 1536. The obtained results led to the hypothesis that an inter-species transmission gave rise to new RVA genome constellations as a result of gene reassortment between the RVA circulating in American autochthonous species (Lama guanicoe) and the RVA of the introduced species (equine and bovine). The E12 gene from the autochthonous RVA to the equine and bovine RVA backbones were transferred and fixed. The study of equine RVA also included a structural analysis that showed the genetic linkage between the two genotypes of the VP7 and VP6 structural proteins. Finally, the virome of diarrheic samples from foals were studied by ?next generation sequencing? technology. In conclusion the present work contributed new information regarding equine RVA diagnosis and addressed aspects related to evolutive, phylodynamic and structural processes of RVA that might be extrapolated to other viruses carrying segmented genomes. In addition, the analysis of the virome from samples from diarrheic foals allowed us to detect other potential viral agents associated to diarrhea in equines. Our results remark the important of conducting the continuous surveillance of diarrhea in foals, the identification and characterization of the associated viral agents with the final goal of the rationale design of new control and preventive tools.Fil: Miño, Orlando Samuel. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Buenos Aires, ArgentinaLas diarreas neonatales representan uno de los síndromes clínicos más relevantes y frecuentes en potrillos menores de 6 meses de edad. Rotavirus grupo A (RVA) es el principal agente viral asociado a diarreas en potrillos, a nivel mundial. Sin embargo, en aproximadamente el 50% de los casos de diarrea no se puede establecer su etiología. Por estos motivos, los objetivos de este trabajo fueron, profundizar el conocimiento de la epidemiología molecular y, de las características evolutivas y estructurales de RVA equino, y estudiar la presencia de otros agentes virales como posibles causantes de diarreas en potrillos de nuestro país.\nEl estudio de RVA equino comenzó con la determinación de la sensibilidad y especificidad de tres metodos de diagnóstico. En segundo lugar, se estudió la epidemiología molecular de los RVA circulantes en equinos durante el período 2009 ? 2014. Las variantes G3P[12] y G14P[12] fueron las cepas detectadas con una prevalencia cíclica y alternada entre los años bajo estudio. Con todas las cepas disponibles se realizó un estudio evolutivo, determinándose la monofilia de los RVA equinos de Argentina. Se identificó el genotipo E12 -correspondiente al gen codificante de la enterotoxina viral NSP4- como un indicador geográfico que permitió trazar la evolución de los RVA equino de Sudamérica, desde la primera fundación de Buenos Aires en 1536. El resultado obtenido permite plantear la hipótesis de una infección inter especie que dio origen a una cepa emergente resultado de la reasociación de genes entre las cepas de RVA de animales autóctonos (guanacos) y las cepas de RVA de los animales introducidos (equinos y bovinos). Se detectó el fenómeno de transferencia y fijación del genotipo E12 autóctono en las cepas de RVA circulantes en equinos y bovinos de Sudamérica. El estudio de RVA equino incluyó también un análisis estructural que permitió explicar el ligamiento genético observado entre las variantes de las proteínas estructurales VP7 y VP6. Finalmente, mediante la tecnología de ?next generation sequencing?, se estudió el viroma de la materia fecal de potrillos con diarrea. Así se determinó la presencia de otros agentes virales, potenciales causantes de diarrea.En conclusión, este trabajo profundizó aspectos relacionados con el diagnóstico de RVA equino, aportando datos novedosos respecto a los procesos evolutivos, filodinámicos y estructurales que podrían extrapolarse a otros virus de genoma segmentado. Asimismo, se describió por primera vez el viroma de materia fecal de potrillos con diarrea, sugiriendo otros posibles agentes causales. Nuestros resultados enfatizan la importancia de realizar una vigilancia epidemiológica continua de las diarreas en potrillos, profundizar en la identificación y caracterización de los agentes virales asociados, con el fin último de realizar el diseño racional de nuevas herramientas de prevención y control

Zoonotic RVA: State of the Art and Distribution in the Animal World

Rotavirus species A (RVA) is a pathogen mainly affecting children under five years old and young animals. The infection produces acute diarrhea in its hosts and, in intensively reared livestock animals, can cause severe economic losses. In this study, we analyzed all RVA genomic constellations described in animal hosts. This review included animal RVA strains in humans. We compiled detection methods, hosts, genotypes and complete genomes. RVA was described in 86 animal species, with 52% (45/86) described by serology, microscopy or the hybridization method; however, strain sequences were not described. All of these reports were carried out between 1980 and 1990. In 48% (41/86) of them, 9251 strain sequences were reported, with 28% being porcine, 27% bovine, 12% equine and 33% from several other animal species. Genomic constellations were performed in 80% (32/40) of hosts. Typical constellation patterns were observed in groups such as birds, domestic animals and artiodactyls. The analysis of the constellations showed RVA’s capacity to infect a broad range of species, because there are RVA genotypes (even entire constellations) from animal species which were described in other studies. This suggests that this virus could generate highly virulent variants through gene reassortments and that these strains could be transmitted to humans as a zoonotic disease, making future surveillance necessary for the prevention of future outbreaks.Fil: Díaz Alarcón, Ricardo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Universidad Nacional de Misiones; ArgentinaFil: Liotta, Domingo Javier. Universidad Nacional de Misiones; Argentina. Dirección Nacional de Instituto de Investigación.Administración Nacional de Laboratorios e Institutos de Salud "Dr. Carlos G. Malbrán"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; ArgentinaFil: Miño, Orlando Samuel. Universidad Nacional de Misiones; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Misiones. Estación Experimental Agropecuaria Cerro Azul; Argentin

A new molecular method for the rapid subtyping of bovine herpesvirus 1 field isolates

Bovine herpesvirus 1 (BoHV-1) causes several clinical syndromes in cattle worldwide. There are 3 subtypes of BoHV-1: 1.1, 1.2a, and 1.2b. Several molecular methods are commonly used in the detection and characterization of BoHV-1. Among them, restriction endonuclease analysis (REA) and single-nucleotide polymorphism (SNP) analysis of the complete viral genome allow classification of BoHV-1 into different subtypes. However, developing countries need simpler and cheaper screening assays for routine testing. We designed a standard multiplex PCR followed by a REA assay allowing straightforward subclassification of all BoHV-1 isolates tested into 1.1, 1.2a, and 1.2b subtypes based on the analysis of fragment length polymorphism. Our standard multiplex PCR-REA was used to analyze 33 field strains of BoHV-1 isolated from various tissues. The assay confirmed the subtype identified previously by REA. In addition, non-polymorphic or undigested fragments were sequenced in order to confirm the mutation affecting the RE HindIII site. Our PCR-REA method is an affordable and rapid test that will subtype all BoHV-1 strains.Fil: Maidana, Silvina Soledad. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Virología E Innovaciones Tecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Virología E Innovaciones Tecnológicas; ArgentinaFil: Miño, Orlando Samuel. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Apóstolo, Romina María. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; ArgentinaFil: De Stefano, Gabriel A.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; ArgentinaFil: Romera, Sonia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Virología E Innovaciones Tecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Virología E Innovaciones Tecnológicas; Argentin

Complete molecular genome analyses of equine rotavirus a strains from different continents reveal several novel genotypes and a largely conserved genotype constellation

In this study, the complete genome sequences of seven equine group A rotavirus (RVA) strains (RVA/Horse-tc/GBR/L338/1991/G13P[18], RVA/Horse-wt/IRL/03V04954/2003/G3P[12] and RVA/Horse-wt/IRL/04V2024/2004/G14P[12] from Europe; RVA/Horse-wt/ARG/E30/1993/ G3P[12], RVA/Horse-wt/ARG/E403/2006/G14P[12] and RVA/Horse-wt/ARG/E4040/2008/ G14P[12] from Argentina; and RVA/Horse-wt/ZAF/EqRV-SA1/2006/G14P[12] from South Africa) were determined. Multiple novel genotypes were identified and genotype numbers were assigned by the Rotavirus Classification Working Group: R9 (VP1), C9 (VP2), N9 (NSP2), T12 (NSP3), E14 (NSP4), and H7 and H11 (NSP5). The genotype constellation of L338 was unique: G13-P[18]-I6- R9-C9-M6-A6-N9-T12-E14-H11. The six remaining equine RVA strains showed a largely conserved genotype constellation: G3/G14-P[12]-I2/I6-R2-C2-M3-A10-N2-T3-E2/E12-H7, which is highly divergent from other known non-equine RVA genotype constellations. Phylogenetic analyses revealed that the sequences of these equine RVA strains are related distantly to nonequine RVA strains, and that at least three lineages exist within equine RVA strains. A small number of reassortment events were observed. Interestingly, the three RVA strains from Argentina possessed the E12 genotype, whereas the three RVA strains from Ireland and South Africa possessed the E2 genotype. The unusual E12 genotype has until now only been described in Argentina among RVA strains collected from guanaco, cattle and horses, suggesting geographical isolation of this NSP4 genotype. This conserved genetic configuration of equine RVA strains could be useful for future vaccine development or improvement of currently used equine RVA vaccines.Fil: Matthijnssens, Jelle. Katholikie Universiteit Leuven; BélgicaFil: Miño, Orlando Samuel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Papp, Hajnalka. Hungarian Academy of Sciences; HungríaFil: Potgieter, Christiaan. Ondersterpoort Veterinary Institute; SudáfricaFil: Novo, Luis. Katholikie Universiteit Leuven; BélgicaFil: Heylen, Elisabeth. Katholikie Universiteit Leuven; BélgicaFil: Zeller, Mark. Katholikie Universiteit Leuven; BélgicaFil: Garaicoechea, Lorena Laura. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Badaracco, Alejandra. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lengyel, György. Dr György Radó Military Medical Centre; HungríaFil: Kisfali, Péter. University Of Pécs; HungríaFil: Cullinane, Ann. Irish Equine Centre; IrlandaFil: Collins, P. J.. Cork Ins Of Technology; IrlandaFil: Ciarlet, Max. Novartis Vaccines and Diagnostics; Estados UnidosFil: O'Shea, Helen. Cork Ins Of Technology; IrlandaFil: Parreño, Gladys Viviana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bányai, Krisztián. Hungarian Academy of Sciences; HungríaFil: Barrandeguy, María Edith. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Van Ranst, Marc. Katholikie Universiteit Leuven; Bélgic

Matriz de resultados del acoplamiento molecular en VP4 de RVA

Se presentan las energías de unión en kcal/mol obtenidas como resultado del acoplamiento molecular entre los compuestos del subconjunto de ZINC aprobado por FDA y los P-tipos P[4], P[6] y P[8] de RVA como receptores. El acoplamiento molecular se realizó en el programa AutoDock Vina en el marco de la búsqueda de candidatos a antivirales contra VP4 de RVA mediante cribado virtual

Genetic linkage of capsid protein-encoding RNA segments in group A equine rotaviruses

Rotavirus virions are formed by three concentric protein layers that enclose the 11 dsRNA genome segments and the viral proteins VP1 and VP3. Interactions amongst the capsid proteins (VP2, VP6, VP7 and VP4) have been described to play a major role in viral fitness, whilst restricting the reassortment of the genomic segments during co-infection with different rotavirus strains. In this work we describe and characterize the linkage between VP6 and VP7 proteins based on structural and genomic analyses of group A rotavirus strains circulating in Argentinean horses. Strains with the VP7 genotype G3 showed a strong association with the VP6 genotype I6, whilst strains with G14 were associated with the I2 genotype. Most of the differences on the VP6 and VP7 proteins were observed in interactive regions between the two proteins, suggesting that VP6: VP7 interactions may drive the co-evolution and co-segregation of their respective gene segments.Fil: Miño, Orlando Samuel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Barrandeguy, María. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Universidad del Salvador. Escuela de Veterinaria; ArgentinaFil: Parreño, Gladys Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Parra, Gabriel. Universidad Nacional de Asunción; Paraguay. National Institutes of Health; Estados Unido

Comparison of two commercial kits and an in-house ELISA for the detection of equine rotavirus in foal feces

Group A rotaviruses (RVA) are important infectious agents associated with diarrhea in the young of several animal species including foals. Currently, a variety of diagnosis methods are commercially available, like ELISA, latex agglutination and immunochromatographic assays. These commercial tests are mainly designed for the detection of human RVA; its applicability in veterinary diagnosis has been poorly studied. The aim of this study was to compare the sensitivity and specificity of two commercial diagnostic kits, Pathfinder™ Rotavirus and FASTest Rota® strip, with an in-house KERI ELISA, for the detection of equine RVA.A total of 172 stool samples from Thoroughbred foals with diarrhea were analyzed. The presence of equine RVA in samples in which only one of the three methods showed positive results was confirmed by RT-PCR. A sample was considered "true positive" when RVA was detected by at least two of the methods, and "true negative" when it tested negative by the three assays. Following these criteria, 50 samples were found positive and 122 were found negative, and were handled as reference population for the assay validation.Pathfinder™ Rotavirus assay showed 32% sensitivity and 97% specificity, FASTest Rota® strip, 92% sensitivity and 97% specificity, and KERI ELISA, 76% sensitivity and 93% specificity. Pathfinder™ Rotavirus showed 77%, FASTest Rota® strip 95%, and KERI ELISA 88% accuracy to correctly classify the samples as equine RVA positive or negative. Pathfinder failed specifically to detect equine RVA G3P12I6 genotype; such performance might be related to the specificity of the monoclonal antibody included in this kit. According to our results, differences among VP6 genotypes could influence the sensitivity to detect equine RVA in foal feces, and thus assay validation of diagnostic kits for each species is necessary. In conclusion, FASTest Rota® strip is more suitable than ELISA Pathfinder™ Rotavirus for the screening of rotavirus infection in foals. The KERI ELISA showed an acceptable performance, and could be considered a proper economic alternative for equine RVA diagnosis.Fil: Miño, Orlando Samuel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Kern, A.. Megacor Diagnostk; AustriaFil: Barrandeguy, M.. Universidad del Salvador. Escuela de Veterinaria; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Parreño, Gladys Viviana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

The first caprine rotavirus detected in Argentina displays genomic features resembling virus strains infecting members of the Bovidae and Camelidae

Rotavirus group A (RVA) is a major cause of diarrhea in humans and young animals including small ruminants. The purpose of this study was to identify RVA in dairy goat kids, and to characterize the complete genomic constellation and genetic relatedness with other RVA strains. Four out of twenty fecal samples from diarrheic and non-diarrheic goat kids were positive for RVA by ELISA. A representative sample was selected for further genome analyses. The RVA strain RVA/Goat-wt/ARG/0040/2011/G8P[1] displayed the following genomic constellation: G8-P[1]-I2-R5-C2-M2-A3-N2-T6-E12-H3, reminiscent to guanaco and other bovine-like RVA strains detected in Argentina. Phylogenetic analyses revealed that most of the genome segments had a rather close relatedness with RVA strains typically obtained from cattle, sheep, South American camelids and goats. Interestingly, strain 0040 possessed the R5 and E12 genotypes which have up to date only been found in different animal species from Argentina. Overall, these findings suggest that strain 0040 could represent a typical goat RVA genome constellation similar to those previously found in other animal species within the order Artiodactyla.EEA BalcarceFil: Louge Uriarte, Enrique Leopoldo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Area de Producción Animal; ArgentinaFil: Badaracco, Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; ArgentinaFil: Matthijnssens, Jelle. University of Leuven. Department of Microbiology and Immunology. Rega Institute for Medical Research. Laboratory of Viral Metagenomics; Bélgica.Fil: Zeller, Mark. University of Leuven. Department of Microbiology and Immunology. Rega Institute for Medical Research. Laboratory of Viral Metagenomics; BélgicaFil: Heylen, Elisabeth. University of Leuven. Department of Microbiology and Immunology. Rega Institute for Medical Research. Laboratory of Viral Metagenomics; BélgicaFil: Manazza, Jorge Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Miño, Samuel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; ArgentinaFil: Ranst, Marc Van. University of Leuven. Department of Microbiology and Immunology. Rega Institute for Medical Research. Laboratory of Viral Metagenomics; BélgicaFil: Odeon, Anselmo Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Parreño, Gladys Viviana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentin

Anti-VP6 VHH: An Experimental Treatment for Rotavirus A-Associated Disease

Species A Rotaviruses (RVA) remain a leading cause of mortality in children under 5 years of age. Current treatment options are limited. We assessed the efficacy of two VP6-specific llama-derived heavy chain antibody fragments (VHH) -2KD1 and 3B2- as an oral prophylactic and therapeutic treatment against RVA-induced diarrhea in a neonatal mouse model inoculated with virulent murine RVA (ECw, G16P[16]I7). Joint therapeutic administration of 2KD1+3B2 (200 μg/dose) successfully reduced diarrhea duration, RVA infection severity and virus shedding in feces. While the same dose of 2KD1 or 3B2 (200 μg) significantly reduced duration of RVA-induced diarrhea, 2KD1 was more effective in diminishing the severity of intestinal infection and RVA shedding in feces, perhaps because 2KD1 presented higher binding affinity for RVA particles than 3B2. Neither prophylactic nor therapeutic administration of the VHH interfered with the host’s humoral immune response against RVA. When 2KD1 (200 μg) was administered after diarrhea development, it also significantly reduced RVA intestinal infection and fecal shedding. Host antibody responses against the oral VHH treatment were not detected, nor did viral escape mutants. Our findings show that oral administration of anti-VP6 VHH constitute, not only an effective prophylactic treatment against RVA-associated diarrhea, but also a safe therapeutic tool against RVA infection, even once diarrhea is present. Anti-VP6 VHH could be used complementary to ongoing vaccination, especially in populations that have shown lower immunization efficacy. These VHH could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea.Fil: Maffey, Lucía. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vega, Celina Guadalupe. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Miño, Orlando Samuel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Garaicoechea, Lorena Laura. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Parreño, Gladys Viviana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin