Diversity of enteroviruses from cerebrospinal fluid specimens collected from hospitalised patients in the private and public sector

Abstract

Enteroviruses (EVs) belong to the family Picornaviridae and are composed of more than 300 genotypes including the types that infect animals. Currently there are 15 species, composed of Enteroviruses (A-L) and Rhinoviruses (A-C), whereby humans are known to be infected only by Enterovirus A-D and Rhinovirus A-C. Enteroviruses are non-enveloped and have a genome ranging between 7 200 and 8 500 nucleotides. Enteroviruses are primarily transmitted via the faecal-oral route, but other routes include eye secretions or respiratory droplets such as mucus or saliva. Depending on the presentation of the enteroviral infection, different diagnostic specimens are required, this can vary from cerebrospinal fluid specimen (CSF), stool, respiratory samples, blood, vesicle fluid and eye swabs. Enteroviruses can be associated with a range of disease syndromes such as hand-foot-and-mouth disease (HFMD), respiratory infections, poliomyelitis, encephalitis, meningitis and can be associated with the development of type-1 diabetes. Currently primers directed at the 5’untranslated region (UTR), viral protein 1 (VP1), VP2 and VP4 regions are being used for the detection and genotyping of EVs, because these methods have been proven to be more rapid and sensitive. The aim of this study was to characterise all EV strains detected in cerebrospinal fluid (CSF) specimens of hospitalised patients in the private and public sector to identify genotypes associated with meningitis and encephalitis. A total of 217 EV-positive RNA extracts (public and private) were included in this study. Overall, 42,9% (24/101 - public and 69/116 - private sector) of the EV strains could be genotyped based on the partial VP1 sequences. From the public sector specimens, species, A, B and C were detected, whereas only species B was detected from the private sector specimens. Enterovirus species B was the predominant species detected from the public and private sector clinical specimens. Echovirus E4 (E4) was the predominant genotype detected during this study (64/92) (69,6%). The E4 genotypes obtained during this study clustered with strains identified in the Western and Eastern Cape, South Africa in 2018 and 2019. The study conducted in the Western and Eastern Cape also noted that the majority of their genotypes were echovirus E4 followed by echovirus E9. All the coxsackieviruses genotyped during this study clustered closely with a study conducted in Kenya pertaining to paediatric patients between 2008 and 2011. In addition to using Sanger sequencing, amplicon-based next generation sequencing targeting the entire capsid coding region (VP1-VP4) and some non-structural proteins (2A to 2C and 3A to 3D) were used to generate more data. However, it was not successful when using the RNA extracts or a cell-culture amplified clinical strain. Next generation sequencing of the cell culture amplified positive control (coxsackievirus B6) was successful. In conclusion this study identified a range of genotypes associated with encephalitis and meningitis in South Africa. It was interesting to find that similar strains of echovirus E4 genotypes were circulating in South Africa between 2018 and 2019. During the 2020 season a clear reduction of non-polio EV cases were observed and this could have been due to the rules and regulations set out by the government during the COVID-19 pandemic. Non-pharmaceutical interventions such as handwashing, social distancing, wearing of a mask and closing of schools could have all attributed to the decline in EV cases in South Africa in 2020 and 2021. Future recommendations would be to accompany the CSF specimen with a stool specimen due to the low viral titres of CSF. Lastly it would be better to perform whole genome shotgun sequencing instead of amplicon-based next generation sequencing to avoid bias.