Development and optimisation of a group-specific real-time RT-PCR assay for the broad detection of the Simbu serogroup orthobunyaviruses

• Autor(a) principal: Camarão, António Alexandre Riachos
• Data de Publicação: 2018
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.5/15823
• topic: Peribunyaviridae; Molecular diagnosis; TaqMan assay; Wide spectrum; Phylogenetic clades; Diagnóstico molecular; Ensaio TaqMan; Largo espectro; Clades filogenéticas

The Simbu serogroup within the genus Orthobunyavirus belongs to the family Peribunyaviridae and comprises 32 recognised three-segmented negative-sense single-stranded RNA viruses, divided into two phylogenetic clades. Some members of this group of arthropod-borne viruses, cosmopolitan distributed, cause neurologic disease in humans as well as reproductive and neurologic disease in domestic animals, however, definitive diagnosis always requires laboratorial confirmation. Few real-time RT-PCR assays have been developed for the molecular diagnosis of Simbu serogroup orthobunyaviruses. There are two published methods with broad detection capacity, utilising either a SYBR Green based chemistry able to recognise viruses from both clades, which is not absolutely specific, or a TaqMan based chemistry that recognises only clade B viruses. A novel group-specific TaqMan-based real-time RT-PCR assay was developed, optimised and laboratory validated for the broad detection of the Simbu serogroup orthobunyaviruses. The published genomic data of the Simbu serogroup members were evaluated, and a conserved region, situated in the segment encoding the nucleocapsid protein, was selected to design a universal primer set and a pair of differently labelled hydrolysis probes, which allowed for the distinction between the two phylogenetic clades of the Simbu serogroup. Seven prototype Simbu serogroup isolates were used for the development of the assay, namely Akabane orthobunyavirus, Simbu orthobunyavirus, Shuni orthobunyavirus, Sathuperi orthobunyavirus, Shamonda orthobunyavirus, Ingwavuma virus and Sabo virus. The primer and probe concentrations in the reaction were optimised. Amplification efficiency was determined for each one of the viruses: AKAV (99%), SIMV (96%), SHUV (96%), SATV (97%), SHAV (84%), INGV (93%) and SABOV (110%). A panel constituted of genetically related, causative agents of abortion in ruminants and arthropod-borne viruses was selected for in vitro specificity analysis, and in silico analysis was also performed. The assay was shown to be specific, as no cross-reactions were observed either in vitro or in silico, and sensitive, with a 95% limit of detection ranging from 10-0,39 to 10-3,61 TCID50/reaction, for the detection of Simbu serogroup viruses. The repeatability of the assay was evaluated for both probes detection, using the intra- and inter-run standard deviations and coefficient of variation. This work resulted in a manuscript in submission process to a peer-reviewed journal. In addition, a comprehensive review of the viruses of the Simbu serogroup was carried out, including sites of viral isolation and seroconversion, and a map was generated using a geographic information system tool.