Standardization of the reverse transcription - seminested polymerase chain reaction (RT-snPCR) for detection of enterovirus direct from the clinical sample

• Grant number: 19/00936-2
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: May 01, 2019
• End date: December 31, 2019
• Field of knowledge: Health Sciences - Medicine
• Principal Investigator: Rita de Cássia Compagnoli Carmona
• Grantee: Lucas Araujo Leme
• Host Institution: Instituto Adolfo Lutz (IAL). São Paulo , SP, Brazil

Abstract

Enteroviruses (EVs) are associated with a variety of clinical manifestations in humans, including aseptic meningitis, acute flaccid paralysis, undifferentiated rash, myocarditis, neonatal sepsis-like disease, and acute haemorrhagic conjunctivitis. For detection of EVs, viral isolation in cell culture was for many years considered the "gold standard" method. Identification of the virus isolated was performed by the seroneutralization (Nt) assay, using reference polyclonal antisera, with limited distribution and indirect immunofluorescence (IFI), using some monoclonal antibodies. However, these methods are time-consuming and laborious, sensitive to virus aggregation and antigenic variation, and require a large number of antisera to identify all serotypes. In addition, there is no antiserum or reference monoclonal antibody available for all types of EVs described. Recently, a protocol was developed by the CDC-WHO using generic VP1 protein primers, the most useful target region for the molecular typing of EVs and for evolution studies. Amplification of the VP1 gene by reverse transcription - seminested Polymerase Chain Reaction (RT-snPCR) followed by genomic sequencing were used to discriminate all prototype strains of EVs and thus enable the identification of virus isolated directly from human clinical samples, and new potentials EVs. This tool will make it possible to shorten the time to characterize the EVs in genotypes and will provide relevant data on the distribution of types of this virus and genetic variability in the infected human population. (AU)