Genotyping and whole genome classification of group A rotaviruses originating from an urban and rural site in Mozambique

Abstract

English: In 2015, a study by Nhampossa and co-workers reported that rotaviruses are among the leading causes of severe gastroenteritis in children in Mozambique. However, there is not much published information on rotavirus surveillance in this country. In addition, currently there has not been any published information on the genotypes of strains circulating in Mozambique. In 2012, Dr Nilsa de Deus (National Institute of Health, INS) initiated a rotavirus surveillance project at Manhiça Health Research Centre (CISM) where the aim was to study the prevalence as well as the genotypes of strains circulating in two regions of the country i.e Manhiça and Mavalane, rural and urban site, respectively. The work done in the current study is part of a collaborative project on Mozambican rotavirus strain characterisation, molecular epidemiology and rotavirus strain evolution between the research groups of Dr de Deus at INS/CISM and the University of the Free State (UFS). The aims were to use reverse-transcriptase polymerase chain reaction (RT-PCR) and Sanger sequencing to confirm the genotypes obtained by the Mozambican researchers with genotyping PCR and to use next generation sequencing (NGS) to determine genotypes of rotavirus previously typed as mixed genotypes or untypeable with genotyping PCR. The Mozambican reseachers collected stool specimens of children with severe diarrhoea seeking medical attention in hospitals in Mavalane and Manhiça. Positive specimens for the rotavirus antigen were typed by genotyping PCR and were sent to UFS. RNA was successfully extracted from 40/58 specimens received. From the 40 RNA positive specimens, characterisation of strains into P and G genotypes using RT-PCR and Sanger sequencing was performed for 30 specimens. Phylogenetic analysis of strains whose genotypes could be determined by RT-PCR and Sanger sequencing showed that strains circulating in Mozambique were similar to strains circulating in the low income countries in southern Africa and India. In Mozambique, the genotypes detected included G2P[4], G12P[6], G8P[4] and G12P[8]. Whole genome sequencing of nine selected Mozambican samples using NGS was successful and samples which were previously typed as mixed by genotyping PCR appeared as single infections. Interestingly, one specimen that was previously typed with genotyping PCR as G12P[NT], and subsequently as G12P[6] with RT-PCR in combination with Sanger sequencing was typed as a mixed genotype with NGS, revealing a relatively rare P[14] genotype. Phylogenetic analysis of specimens characterised with NGS indicated that Mozambican rotavirus strains are similar to strains circulating in southern Africa, India and Bangladesh. Rotavirus infections are a problem in low-income countries such as Mozambique as their rate of detection is high, with various genotypes circulating and making continual surveillance a necessity before and after the introduction of vaccines.Afrikaans: 'n Studie deur Nhampossa en mede-werkers het in 2015 gerapporteer dat rotavirusse een van die grootste oorsake van ernstige gastro-enteritis by kinders in Mosambiek is. Daar is egter nie veel gepubliseerde inligting oor rotavirus waarneming in hierdie land nie. Daar is ook geen gepubliseerde inligting oor die genotipes van rotavirus stamme wat in Mosambiek voorkom nie. Dr. Nilsa de Deus (Nasionale Instituut van Gesondheid, INS) het ʼn projek in 2012 by die Manhiça Gesondheid Navorsingsentrum (CISM) begin wat die verspreiding en voorkoms van genotipes van rotavirus stamme in die Manhica en Mavalane streke in Mozambiek bestudeer. Hierdie streke verteenwoordig beide landelike en stedelike gebiede van die land. Die werk wat in hierdie studie beskryf word, is deel van 'n samewerkingsprojek op Mosambiek rotavirus stam karakterisering, molekulêre epidemiologie en rotavirus stam evolusie tussen die navorsingsgroepe van Dr. de Deus by INS/CISM en die Universiteit van die Vrystaat (UV) (sien bylaag 1 vir meer inligting). Die eerste doel van hierdie studie was om genotipes van rotavirus stamme wat voorheen deur die Mosambiekse groep met genotipering polimerase kettingreaksie (G-PKR) bepaal is, te bevestig met tru-transkriptase polimerase kettingreaksie (TT-PKR), sowel as Sanger basisopeenvolgorde bepaling. Tweedens is volgende generasie basisopeenvolgorde bepaling (VGB) gebruik om genotipes van rotavirus stamme wat voorheen as gemengde genotipes geïdentifiseer was, te onderskei en verder is monsters waarvan die genotipes nie met G-PKR vasgestel kon word nie, ook deur VGB bepaal. Die Mosambiekse groep het stoelmonsters van kinders met ernstige diarree in hospitale in Mavalane en Manhiça ingesamel. Monsters wat deur G-PKR getipeer is, is na die UV gestuur. RNS is suksesvol vanuit 40 (n=58) monsters geëkstraeer. Dertig van hierdie RNS monsters is met dubbele genotipering van die P en G genotipes deur middel van TT-PKR en Sanger basisopeenvolgorde bepaling suksesvol ontleed. Filogenetiese ontleding gebaseer op die TTPKP en Sanger basis volgorde bepaling data het getoon dat rotavirus stamme in Mosambiek soortgelyk is aan stamme wat hoofsaaklik in die ontwikkelende lande van Suider-Afrika en in Indië voorkom. In Mosambiek het 44% van die monsters ʼn G2P[4] genotipe kombinasie gehad, gevolg deur 37% met G12P[6], G8P[4] met 11.7% en G12P[8] met 7.4%. Heel-genoom basis opeenvolgordes van nege monsters was suksesvol bepaal met VGB. Dit was ook moontlik om genotipes van monsters vas te stel wat voorheen nie met G-PKR geskei kon word nie. Een spesifieke monster wat met G-PKR as G12P [NT] geïdentifiseer was, is met TT-PKR gevolg deur Sanger basis opeenvolgorde bepaling as G12P [8] bepaal. Interessant genoeg het VGB ook 'n relatief seldsame P[14] genotipe in hierdie monster geïdentifiseer. Filogenetiese analise gebaseer op VGB data het ook aangedui dat die Mosambiek stamme soortgelyk is aan rotavirus stamme wat in Suider-Afrika en in ontwikkelende lande in Indië en Bangladesj voorkom. Die resultate van hierdie studie het getoon dat rotavirus infeksies 'n probleem in ontwikkelende lande soos Mosambiek is. Aangesien daar ʼn verskeidenheid rotavirus genotipes in Mosambiek voorkom, is voortdurende waarneming van hierdie stamme noodsaaklik voor en na die bekendstelling van entstowwe.