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Molecular and clinical epidemiology of rotavirus group A, in children less than 5 years of age, pre and post vaccine introduction, in a matched case-control study in Manhiça district, Mozambique, 2008- 2021
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Resumo
Rotavírus é o principal agente etiológico da diarreia em crianças <5 anos, com maior peso de mortes na Ásia e África Subsaariana, incluindo Moçambique, onde ainda existem poucos dados sobre a sua epidemiologia, antes e depois da introdução da vacina. O presente projeto visava avaliar o impacto da vacina contra o rotavírus em crianças <5 anos; Descrever os genótipos circulantes entre crianças com diarreia moderada-a-severa e leve (siglas em inglês MSD e LSD) e sem diarreia (controlos comunitários saudáveis) e realizar a sequenciação do genoma completo de genótipos comuns nestes grupos, para avaliar as suas diferenças ou semelhanças, antes e depois da introdução da vacina no Distrito da Manhiça, Moçambique. Foram analisados casos de diarreia dectados passivamente através do sistema de vigilância de morbidade, e análise laboratorial para a deteção de rotavirus usando o ensaio de imunoabsorção enzimática. Os genótipos foram determinados através de transcrição reversa-reacção em cadeia de polimerase e a sequenciação do genoma completo através da sequenciação de nova geração, usando a plataforma Illumina® MiSeq.
A prevalência de rotavirus reduziu de 22.9% antes para 11.5% depois da introdução da vacina. O genótipo G12P[8] foi mais frequente em casos MSD e controlos e G12P[6] em casos LSD antes da introdução da vacina. Depois da introdução da vacina, G3P[4] e G3P[8] foram mais frequentes em casos MSD e LSD. Foi feita a sequenciação das estirpes G3P[8], G2P[4], G2P[6], G3P[4], G8P[4], G8P[6] e G9P[6] de casos MSD, LSD e controlos. Verificou-se que, G3P[8] apresentava a constelação designada Wa-like (G3P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1) e as outras combinações a constelação DS-1-like (G2/G3/G8-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2); (G2/G8/G9-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2). As análises filogenéticas mostraram similaridades entre as estirpes estudadas e estas com estirpes que circulam em Moçambique e mundialmente.
Os segmentos 9, 6, 1, 7 e 10 que codificam a VP7, VP6, VP1, NSP3 e NSP4 respectivamente, das estirpes Wa-like, agruparam-se com estirpes suínas, equinas e bovinas, com similaridades entre 86.9–99.9% de nucleótidos e 97.2–100% de aminoácidos. O segmento 4 que codifica a VP4 (P[4]) e o segmento 9 que codifica a VP7 (G9, G3), o segmento 10 que codifica a NSP4 nas estirpes G3P[4] e G8P[4] agruparam-se com estirpes porcinas, bovinas e caprinas. O segmento 1 que codifica a VP1 nas estirpes G2P[6] agrupou-se com uma estirpe de camelo, com identidades médias de 89,1%-99,0% de nucleótidos e 89,5%-99,0% de aminoácidos.
A vacina contribuiu para a diminuição de casos de RVA em crianças <5 anos. No entanto, ainda é necessária uma vigilância ativa para avaliar o peso em outras faixas etárias, não elegíveis para a vacinação. Foram observados genótipos semelhantes entre casos e controlos, embora com uma mudança na prevalência e genótipos que circularam antes e depois da introdução da vacina, que pode ser resultado da capacidade de evolução do virus. As estirpes com similaridade com estirpes animais, podem ser resultado de rearranjos genéticos, daí a necessidade de monitorar os genótipos por técnicas avançadas para compreender as alterações evolutivas. Estes resultados reforçam a necessidade de avaliar o impacto dessas estirpes na efectividade da vacina atualmente em uso em Moçambique.
Abstract Rotavirus constitutes a major aetiology of diarrhoea in children under 5, with the highest burden of disease in low and middle-income countries from Asia and sub-Saharan Africa, including Mozambique, where there is still scarce data about the epidemiology of rotavirus, pre and post-vaccine introduction. The present project aimed to evaluate the impact of rotavirus vaccine in children under 5; Describe rotavirus genotypes circulating among children with diarrhoea (MSD and LSD) and without diarrhoea (healthy community controls) and perform whole genome sequencing of common genotypes in these groups, to better understand their differences or similarities, pre and post-vaccine introduction in Manhiça District, Mozambique. There were analysed cases of diarrhoea detected passively through the morbidity surveillance system and rotavirus detection by enzyme-linked immunosorbent assay. Rotavirus genotypes were characterized using conventional reverse transcription-polymerase chain reaction and genotype constellations determined through whole genome sequencing, using the illumina® MiSeq platform. The prevalence of rotavirus reduced from 22.9% pre-vaccine to 11.5% post-vaccine introduction. The genotype combination G12P[8] was more frequently observed in MSD and controls, G12P[6] was frequent in LSD pre-vaccine introduction. Post-vaccine introduction G3P[4] and G3P[8] were more prevalent in MSD and LSD. Sequencing was performed on G3P[8], G2P[4], G2P[6], G3P[4], G8P[4], G8P[6] and G9P[6] strains from children with MSD, LSD and controls. It was found that G3P[8] had the Wa-like (G3P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1) constellation, while other strains had a DS-1-like constellation (G2/G3/G8-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2); (G2/G8/G9-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2). Phylogenetic analysis showed that the study strains were close among them and to strains detected in Mozambique and globally. In addition, the segments 9, 6, 1, 7 and 10 encoding VP7, VP6, VP1, NSP3 and NSP4 respectively, of the Wa-like strains, formed a cluster with swine, equine, and bovine strains, with similarities raging from an average of 86.9–99.9% nucleotide and 97.2–100% amino acid. By the other hand the segment 4 encoding VP4 (P[4]), segment 9 encoding VP7 (G9, G3), segment 10 encoding NSP4 cognate of G3P[4] and G8P[4] strains, were closely related to porcine, bovine and caprine strains. The segment 1 encoding VP1 cognate of G2P[6] strains clustered with a camel strain. The average identities ranged from 89.1%-99.0% of nucleotide and 89.5%-99.0% of amino acid. Although the vaccine contributed for decreasing of rotavirus cases in children under 5, active surveillance is still needed to assess its burden in other age groups not eligible for vaccination. We observed circulation of similar genotypes among cases and controls, however, a switch in dominant circulating strains was observed pre and post vaccine introduction, and this may be related to the capability of rotavirus to evolve. The observation of strains with similarity with animal strains, demonstrates a possible occurrence of reassortment events between human and animal strains, and show the need to implement whole genome sequencing to monitor the evolutionary changes of rotavirus. Additionally, these results reinforce the need to evaluate the impact of these strains on the effectiveness of the currently vaccine in use in Mozambique.
Abstract Rotavirus constitutes a major aetiology of diarrhoea in children under 5, with the highest burden of disease in low and middle-income countries from Asia and sub-Saharan Africa, including Mozambique, where there is still scarce data about the epidemiology of rotavirus, pre and post-vaccine introduction. The present project aimed to evaluate the impact of rotavirus vaccine in children under 5; Describe rotavirus genotypes circulating among children with diarrhoea (MSD and LSD) and without diarrhoea (healthy community controls) and perform whole genome sequencing of common genotypes in these groups, to better understand their differences or similarities, pre and post-vaccine introduction in Manhiça District, Mozambique. There were analysed cases of diarrhoea detected passively through the morbidity surveillance system and rotavirus detection by enzyme-linked immunosorbent assay. Rotavirus genotypes were characterized using conventional reverse transcription-polymerase chain reaction and genotype constellations determined through whole genome sequencing, using the illumina® MiSeq platform. The prevalence of rotavirus reduced from 22.9% pre-vaccine to 11.5% post-vaccine introduction. The genotype combination G12P[8] was more frequently observed in MSD and controls, G12P[6] was frequent in LSD pre-vaccine introduction. Post-vaccine introduction G3P[4] and G3P[8] were more prevalent in MSD and LSD. Sequencing was performed on G3P[8], G2P[4], G2P[6], G3P[4], G8P[4], G8P[6] and G9P[6] strains from children with MSD, LSD and controls. It was found that G3P[8] had the Wa-like (G3P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1) constellation, while other strains had a DS-1-like constellation (G2/G3/G8-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2); (G2/G8/G9-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2). Phylogenetic analysis showed that the study strains were close among them and to strains detected in Mozambique and globally. In addition, the segments 9, 6, 1, 7 and 10 encoding VP7, VP6, VP1, NSP3 and NSP4 respectively, of the Wa-like strains, formed a cluster with swine, equine, and bovine strains, with similarities raging from an average of 86.9–99.9% nucleotide and 97.2–100% amino acid. By the other hand the segment 4 encoding VP4 (P[4]), segment 9 encoding VP7 (G9, G3), segment 10 encoding NSP4 cognate of G3P[4] and G8P[4] strains, were closely related to porcine, bovine and caprine strains. The segment 1 encoding VP1 cognate of G2P[6] strains clustered with a camel strain. The average identities ranged from 89.1%-99.0% of nucleotide and 89.5%-99.0% of amino acid. Although the vaccine contributed for decreasing of rotavirus cases in children under 5, active surveillance is still needed to assess its burden in other age groups not eligible for vaccination. We observed circulation of similar genotypes among cases and controls, however, a switch in dominant circulating strains was observed pre and post vaccine introduction, and this may be related to the capability of rotavirus to evolve. The observation of strains with similarity with animal strains, demonstrates a possible occurrence of reassortment events between human and animal strains, and show the need to implement whole genome sequencing to monitor the evolutionary changes of rotavirus. Additionally, these results reinforce the need to evaluate the impact of these strains on the effectiveness of the currently vaccine in use in Mozambique.
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Palavras-chave
Biologia molecular Rotavírus Crianças Moçambique