Development and Evaluation of a One-Step Multiplex Real-Time TaqMan\u3csup\u3e®\u3c/sup\u3e RT-qPCR Assay for the Detection and Genotyping of Equine G3 and G14 Rotaviruses in Fecal Samples

Background: Equine rotavirus A (ERVA) is the leading cause of diarrhea in neonatal foals and has a negative impact on equine breeding enterprises worldwide. Among ERVA strains infecting foals, the genotypes G3P[12] and G14P[12] are the most prevalent, while infections by strains with other genomic arrangements are infrequent. The identification of circulating strains of ERVA is critical for diagnostic and surveillance purposes, as well as to understand their molecular epidemiology. Current genotyping methods available for ERVA and rotaviruses affecting other animal species rely on Sanger sequencing and are significantly time-consuming, costly and labor intensive. Here, we developed the first one-step multiplex TaqMan® real-time reverse transcription polymerase chain reaction (RT-qPCR) assay targeting the NSP3 and VP7 genes of ERVA G3 and G14 genotypes for the rapid detection and G-typing directly from fecal specimens. Methods: A one-step multiplex TaqMan® RT-qPCR assay targeting the NSP3 and VP7 genes of ERVA G3 and G14 genotypes was designed. The analytical sensitivity was assessed using serial dilutions of in vitro transcribed RNA containing the target sequences while the analytical specificity was determined using RNA and DNA derived from a panel of group A rotaviruses along with other equine viruses and bacteria. The clinical performance of this multiplex assay was evaluated using a panel of 177 fecal samples and compared to a VP7-specific standard RT-PCR assay and Sanger sequencing. Limits of detection (LOD), sensitivity, specificity, and agreement were determined. Results: The multiplex G3 and G14 VP7 assays demonstrated high specificity and efficiency, with perfect linearity. A 100-fold difference in their analytical sensitivity was observed when compared to the singleplex assays; however, this difference did not have an impact on the clinical performance. Clinical performance of the multiplex RT-qPCR assay demonstrated that this assay had a high sensitivity/specificity for every target (100% for NSP3, \u3e 90% for G3 VP7 and \u3e 99% for G14 VP7, respectively) and high overall agreement (\u3e 98%) compared to conventional RT-PCR and sequencing. Conclusions: This new multiplex RT-qPCR assay constitutes a useful, very reliable tool that could significantly aid in the rapid detection and G-typing of ERVA strains circulating in the field

Development and evaluation of a one-step multiplex real-time TaqMan® RT-qPCR assay for the detection and genotyping of equine G3 and G14 rotaviruses in fecal samples

Abstract Background Equine rotavirus A (ERVA) is the leading cause of diarrhea in neonatal foals and has a negative impact on equine breeding enterprises worldwide. Among ERVA strains infecting foals, the genotypes G3P[12] and G14P[12] are the most prevalent, while infections by strains with other genomic arrangements are infrequent. The identification of circulating strains of ERVA is critical for diagnostic and surveillance purposes, as well as to understand their molecular epidemiology. Current genotyping methods available for ERVA and rotaviruses affecting other animal species rely on Sanger sequencing and are significantly time-consuming, costly and labor intensive. Here, we developed the first one-step multiplex TaqMan® real-time reverse transcription polymerase chain reaction (RT-qPCR) assay targeting the NSP3 and VP7 genes of ERVA G3 and G14 genotypes for the rapid detection and G-typing directly from fecal specimens. Methods A one-step multiplex TaqMan® RT-qPCR assay targeting the NSP3 and VP7 genes of ERVA G3 and G14 genotypes was designed. The analytical sensitivity was assessed using serial dilutions of in vitro transcribed RNA containing the target sequences while the analytical specificity was determined using RNA and DNA derived from a panel of group A rotaviruses along with other equine viruses and bacteria. The clinical performance of this multiplex assay was evaluated using a panel of 177 fecal samples and compared to a VP7-specific standard RT-PCR assay and Sanger sequencing. Limits of detection (LOD), sensitivity, specificity, and agreement were determined. Results The multiplex G3 and G14 VP7 assays demonstrated high specificity and efficiency, with perfect linearity. A 100-fold difference in their analytical sensitivity was observed when compared to the singleplex assays; however, this difference did not have an impact on the clinical performance. Clinical performance of the multiplex RT-qPCR assay demonstrated that this assay had a high sensitivity/specificity for every target (100% for NSP3, > 90% for G3 VP7 and > 99% for G14 VP7, respectively) and high overall agreement (> 98%) compared to conventional RT-PCR and sequencing. Conclusions This new multiplex RT-qPCR assay constitutes a useful, very reliable tool that could significantly aid in the rapid detection and G-typing of ERVA strains circulating in the field

Quadruplex Real-Time TaqMan^® RT-qPCR Assay for Differentiation of Equine Group A and B Rotaviruses and Identification of Group A G3 and G14 Genotypes

Equine rotavirus A (ERVA) is the leading cause of diarrhea in foals, with G3P[12] and G14P[12] genotypes being the most prevalent. Recently, equine G3-like RVA was recognized as an emerging infection in children, and a group B equine rotavirus (ERVB) was identified as an emergent cause of foal diarrhea in the US. Thus, there is a need to adapt molecular diagnostic tools for improved detection and surveillance to identify emerging strains, understand their molecular epidemiology, and inform future vaccine development. We developed a quadruplex TaqMan® RT-qPCR assay for differentiation of ERVA and ERVB and simultaneous G-typing of ERVA strains, evaluated its analytical and clinical performance, and compared it to (1) a previously established ERVA triplex RT-qPCR assay and (2) standard RT-PCR assay and Sanger sequencing of PCR products. This quadruplex RT-qPCR assay demonstrated high sensitivity (>90%)/specificity (100%) for every target and high overall agreement (>96%). Comparison between the triplex and quadruplex assays revealed only a slightly higher sensitivity for the ERVA NSP3 target using the triplex format (p-value 0.008) while no significant differences were detected for other targets. This quadruplex RT-qPCR assay will significantly enhance rapid surveillance of both ERVA and ERVB circulating and emerging strains with potential for interspecies transmission

Relevamiento serológico de anticuerpos contra enfermedades virales de interés sanitario en llamas (Lama glama) de la provincia de Jujuy, Argentina = Serological survey of antibodies against viral diseases of public health interest in llamas (Lama glama) from Jujuy province, Argentina

Las poblaciones de llamas de Argentina se concentran principalmente en la provincia de Jujuy; su explotación representa un importante recurso económico de las comunidades altoandinas. El objetivo de este trabajo fue evaluar la seroprevalencia de anticuerpos contra algunos agentes virales asociados a enfermedades de impacto productivo en rodeos de llamas de Jujuy. Se analizaron 349 sueros de llamas adultas de 6 departamentos de la puna jujeña ubicados por encima de los 3300 msnm. Se obtuvo una prevalencia del 100 % para rotavirus grupo A y del 70 % para el virus parainfluenza-3 bovino, mientras que no se detectaron reactores para herpesvirus bovino 1, virus de la diarrea viral bovina, influenza A humana (H1N1) e influenza equina (H3N8). Los resultados obtenidos confirman la amplia distribución de rotavirus y virus parainfluenza y la baja susceptibilidad a herpesvirus y pestivirus en las tropas de llamas de la puna jujeña.Llama population from Argentina is mainly concentrated in the Andean Puna, Jujuy. Llamas represent an important economic resource for the Andean communities. The aim of this study was to investigate the prevalence of antibodies against viral antigens associated to viral diseases of economic impact (neonatal diarrhea, reproductive and respiratory syndromes). A total of 349 serum samples from adult llamas were analyzed. The obtained antibody prevalence was 100 % for Rotavirus A and 70 % for Bovine parainfluenza virus 3. In contrast, no reactors were detected to Bovine herpesvirus 1, Bovine viral diarrhea virus 1, Human influenza A virus (H1N1) and Equine influenza virus (H3N8). These results confirm the wide circulation of rotavirus and parainfluenza virus in Argentinean llamas and suggest that susceptibility to infection with bovine herpesvirus, pestivirus and influenza A viruses is low. This serologic survey provides novel information regarding the epidemiology of viral diseases affecting llamas from the Argentinean Andean Puna.Instituto de VirologíaFil: Barbieri, Elena Susana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rodriguez, Daniela Vanesa. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; ArgentinaFil: Marín, Raúl E. Jujuy. Ministerio de Producción; ArgentinaFil: Setti, Walter Eduardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Abra Pampa; ArgentinaFil: Romero, Sandra Raquel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Abra Pampa; ArgentinaFil: Barrandeguy, Maria Edith. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina. Universidad del Salvador. Escuela de Veterinaria; ArgentinaFil: Parreño, Gladys Viviana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Novel vaccination approaches against equine alphavirus encephalitides

The current production of inactivated vaccines for the prevention of equine alphavirus encephalitides caused by Eastern, Western and Venezuelan Equine Encephalitis viruses (EEEV, WEEV, VEEV) involves the manipulation of large quantities of infectious viral particles under biosafety level 3 containment laboratories with the potential risk of transmission to the operators. Moreover, these vaccines are not capable of inducing a long-lasting immunity. Modified live vaccines, which were also attempted, maintain residual virulence and neurotropism, causing disease in both horses and humans. Therefore, the production of an efficacious second generation vaccine which could be used in the prevention of alphavirus infection without the need to manipulate infectious viral particles under high biocontainment conditions could be of great benefit for the worldwide horse industry. Furthermore, equine alphaviruses are considered as biological threat agents. Subunit, chimeric, gene-deleted live mutants, DNA and adenovirus-vectored alphavirus vaccines have been evaluated; such approaches are reviewed in this work. Climate changes, together with modifications in bird and vector ecology, are leading to the arise of emerging pathogens in new geographical locations, and these zoonotic New World arboviruses are gaining concern. Novel vaccine development does show a promising future for prevention of these infections in both horses and humans.Instituto de VirologíaFil: Carossino, Mariano. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virologia; Argentina. Universidad del Salvador. Escuela de Veterinaria; ArgentinaFil: Thiry, Etienne. University of Liege. Faculty of Veterinary Medicine. Veterinary Virology and Animal Viral Diseases and UREAR. Department of Infectious and Parasitic Diseases; BélgicaFil: Grandière, Ana de la. University of Liege. Faculty of Veterinary Medicine. Veterinary Virology and Animal Viral Diseases and UREAR. Department of Infectious and Parasitic Diseases; BélgicaFil: Barrandeguy, Maria Edith. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina. Universidad del Salvador. Escuela de Veterinaria; Argentin

The seed endosphere of Anadenanthera colubrina is inhabited by a complex microbiota, including Methylobacteriumspp. and Staphylococcus spp. with potential plant-growth promoting activities

Background and aims Plant seeds are emerging micro–habitats, whose importance as reservoir and vector of beneficial microbes just begins to be recognized. Here we aimed to characterize the bacterial microbiota of the Anadenanthera colubrina seed endosphere, with special focus to beneficial traits and to the colonization pattern. Methods Cultivation–dependent (isolation from surface–sterilized seeds) and cultivation–independent (pyrosequencing of 16S rRNA gene from metagenomic seed DNA) analyses, functional tests and microscopical investigations (fluorescence in situ hybridization coupled with confocal laser scanning microscopy (FISH-CLSM) were performed. Results We isolated several Methylobacterium and Staphylococcus spp., exhibiting both plant growth promotion and antimicrobial activities. The two taxonomic groups showed complementary traits, which supports a functional selection. Both genera were detected also by pyrosequencing, together with further taxa. The genera Friedmaniella, Bifidobacterium, Delftia, Anaerococcus and Actinomyces appeared here for the first time as seed endophytes. We detected bacterial cells and micro–colonies in seed cryosections by FISHCLSM. Alphaproteobacteria, Firmicutes and other bacteria colonized intercellular spaces of the parenchyma and associated to transport vessels. Conclusions This work sheds light onto the diversity, functions and colonization pattern of the Anadenanthera colubrina seed endophytes, and strongly suggest a role as beneficial partners for seed-associated microbiot