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The Reoviridae family represents the largest family of double-stranded RNA viruses, and members have been isolated from a wide range of mammals, birds, reptiles, fishes, insects, and plants. Orthoreoviruses, one of the 15 recognized genera in the Reoviridae family, can infect humans and nearly all mammals and birds. Genomic characterization of reoviruses has not been adopted on a large scale because of the complexity of obtaining sequences for all 10 segments. In this study, we develop a time-efficient and practical method to enrich reovirus sequencing reads from isolates that allows for full-genome recovery using a single-primer amplification method coupled with next-generation sequencing. We refer to this protocol as reovirus–single-primer amplification (R-SPA). Our results demonstrate that most of the genes are covered with at least 500 reads per base space. Furthermore, R-SPA covers both the 5′ and 3′ ends of each reovirus genes. In summary, this study presents a universal and fast amplification protocol that yields sufficient double-stranded cDNA and facilitates and expedites the whole-genome sequencing of reoviruses. Protocolo universal de amplificación con un iniciador único para realizar la secuenciación del genoma completo de orthoreovirus aviares con ARN de doble cadena y segmentados La familia Reoviridae representa la familia más grande de virus de ARN de doble cadena y se han aislado miembros de una amplia variedad de mamíferos, aves, reptiles, peces, insectos y plantas. El género Orthoreovirus, uno de los 15 géneros reconocidos en la familia Reoviridae, pueden infectar a humanos y a casi todos los mamíferos y aves. La caracterización genómica de los reovirus no se ha adoptado a gran escala debido a la complejidad de obtener secuencias para los 10 segmentos. En este estudio, desarrollamos un método práctico y eficiente para enriquecer las lecturas de secuenciación de reovirus a partir de aislamientos que permite la recuperación del genoma completo utilizando un método de amplificación con un iniciador único junto con la secuenciación de próxima generación. Nos referimos a este protocolo como amplificación de un solo iniciador de reovirus (R-SPA). Estos resultados demuestran que la mayoría de los genes están cubiertos con al menos 500 lecturas por espacio base. Además, el método R-SPA cubre los extremos 5′ y 3′ de cada gene de reovirus. En resumen, este estudio presenta un protocolo de amplificación rápido y universal que produce suficiente ADN complementario de doble cadena y facilita y acelera la secuenciación del genoma completo de los reovirus. [ABSTRACT FROM AUTHOR]

Mycoplasma pulmonis is a naturally occurring respiratory pathogen in rodents. To date, this pathogen was not isolated from humans. This study aimed to evaluate the prevalence and seropositivity to M. pulmonis in humans who have had direct contact with rats. Moreover, the prevalence of M. pulmonis in pet and laboratory rats was assessed. Overall, 131 and 235 oropharyngeal swab samples were collected from human individuals and rats, respectively. In humans, M. pulmonis was detected by PCR in 21 of 86 pet rat keepers (24.42%), 10 of 13 technicians (76.32%), and 8 of 32 (25.0%) veterinarians. In rats, M. pulmonis was identified by PCR in 86 of 122 pet rats (70.49%) and 56 of 113 (49.56%) laboratory rats. Seroprevalence in humans was examined by screening sera from 44 individuals for M. pulmonis-specific IgG using ELISA. In total, 26 out of 44 (59.09%) humans were seropositive to M. pulmonis (4 out of 9 technicians, 8 of 12 veterinarians, and 15 of 23 pet rat keepers).The high antibody titer was found in 4 individuals (2 pet rat keepers and 2 veterinarians), whereas the moderate and low antibody titers were found in 8 and 14 individuals, respectively. The high antibody titer found in humans might indicate an active infection. However, it is unknown whether the presence of M. pulmonis in humans might be associated with disease and whether the foreign Mycoplasma can survive for long in its new environment. [ABSTRACT FROM AUTHOR]