Norovirus-host interaction studies HBGA phenotypic and genotypic profiles of paediatric patients with diarrhoea from rural communities of Limpopo South Africa

Ph.D. (Microbiology)

Department of Biochemistry and Microbiology

Human norovirus (HNoV) are the leading aetiological agents of viral acute gastroenteritis (VAGE) worldwide. Approximately 685 million cases of VAGE and 219 000 deaths associated with norovirus infection are recorded each year. The burden is more severe in developing nations such as those in Africa, and children below the age of five are the most vulnerable. Noroviruses are commonly transmitted from person-to-person via the faecal oral route and contaminated fomite, food and water. Norovirus are extremely prevalent and exhibit great genetic diversity and rapid rates of evolution. Studies have shown that histo-blood group antigens are essential genetic susceptibility factors for HNoV infection. Histo-blood group antigens (HBGAs) are a class of carbohydrates antigens found in the surface of red blood cells and bodily fluids or tissues. HNoVs recognize and bind to HBGAs for attachment to human epithelial cells in the gastrointestinal tract. The synthesis of HBGAs is mediated by fucosyltransferase and glycosyltransferase, which are genetically regulated by FUT2 (Secretor), FUT3 (Lewis) and ABO (H) genes. Individuals with functional FUT2 gene are termed secretors; they express HBGAs in gut epithelial cell surface and body fluids. Natural variability and inability to express HBGAs (due to mutations) plays a role in population genetic susceptibility to norovirus infection and genotype distribution. Approximately 20% of VAGE are caused by norovirus. Recently, global prevalence of norovirus was reported at 16%. In African countries, the range of reported prevalence was between 13% and 20%. However, higher prevalence has been reported, particularly in Ghana and South Africa. Previous data in South Africa have demonstrated the occurrence of norovirus, with higher number of cases reported in rural communities. There are limited or no studies to explain the elevated prevalence of enteric HNoVs in rural communities of Limpopo, South Africa. Various factors including genetic, geographical, meteorological and socio-economic may play a role in the epidemiology of enteric viruses. A potential interaction between human and viral genetic diversity has been demonstrated via association of population secretor profiles and viral genotype susceptibility and distribution. Therefore, this study, aimed to investigate the prevalence and norovirus host genetic susceptibility profiles in paediatric population under five years, within rural communities of South Africa. To achieve the aim of this study, the following objectives were set: 1) To determine molecular characteristics of HNoV strains circulating in the rural communities of Vhembe district, South Africa; 2) To determine host phenotypic profiles of HBGAs (ABO/H and Lewis system) in HNoV infections among children from rural communities of Vhembe district, South Africa; 3) To determine the host genotypic profiles of HBGAs (FUT2/FUT3) in HNoV infections among children from rural communities of Vhembe district, Limpopo Province (South Africa) and 4) To assess the correlation between circulating HNoV genotypes and HBGAs profiles in young children from rural communities of Vhembe district, Limpopo Province (South Africa). To achieve the study objectives, a cross-sectional survey in children with (200) and without (100) diarrhoea, below the age of five was performed. Co-paired stool (300) and saliva (300) samples were collected from October 2019 to September 2021, in South African healthcare institutions (clinics and hospitals) within the Vhembe district of Limpopo province. Objective 1: HNoVs were detected from stool samples using real-time RT-PCR, then amplified by conventional RT-PCR, and further genotyped by Sanger sequencing. Norovirus strains from this investigation were compared to the worldwide circulating strains by phylogenetic analysis using MEGA11. ClustalW software was used to compare nucleotide similarity among strains genotyped in this study and those previously obtained within the same study region. Objective 2: HBGAs phenotypic profiles were determined from saliva samples by ELISA assays. Monoclonal antibodies including A, B, Le-a, and Le-b were used to determine HBGAs phenotypic profiles in saliva samples. In samples where the phenotypic status was inconclusive, the secretor and non-secretor phenotypes were further confirmed using the Ulex europaeus agglutinin (UEA-1), which is specific for Fucα1-2Gal-R present in secretor, but not in non-secretor saliva. Objective 3: Genotypic characterization of HBGAs were determined by Touch-Down-PCR using the Platinum Taq DNA Polymerase High Fidelity enzyme. The primers used to detect SNPs were also used for nucleotide sequencing of both FUT2 and FUT3 partial genes of selected samples (100). Objective 4: Statistical analysis was performed using Graphpad 10.0 for the association of HNoV prevalence and HBGAs profiles. The results indicate that HNoVs were still highly prevalent in children from Vhembe district, with a confirmed significant difference (p < 0.0001) between symptomatic (37%) and asymptomatic (14%). Genogroup II noroviruses predominated children with AGE (80%), whereas genogroup I norovirus were high in children without AGE. Genotype GII.4 Sydney 2012 [P31] were dominant throughout the study period. The detected strains were phylogenetically closely related to the worldwide circulating norovirus strains but showed low nucleotide similarity when compared to other HNoV strains previously characterized in Vhembe region. Elisa assay findings showed diversity of HBGA phenotypic profiles including Lewis and ABO type amongst study participants. Both Lewis negative and positive phenotypes were identified with the latter being the predominant and mostly infected with HNoV. All ABO phenotypes were also identified within the study population. The highest occurring was type O, which also represented the primarily norovirus infected group, followed by type A. Overall, majority of children had secretor phenotypes (81%) over non-secretors (19%), similarly, higher cases on HNoV were detected in children with secretor phenotype (93%). Amongst the selected samples for FUT2 genotyping, only 42 could be amplified and of those, 36 (36%) were successfully genotyped. Majority of FUT2 secretors were homozygous mutations (64%) compared to heterozygous (28%). However, only 8% were determined as non-secretor with homozygous nonsense mutation on the G428A position. FUT3 genes were also associated with norovirus positivity. The G508A SNP was the most common and predominant among positive norovirus cases. Interestingly, 2 mutant SNPs were detected. Although non-secretor was the lowest in HBGAs genotypic profiles and is often related to resistance to infection, norovirus GII.4 Sydney 2012 [P31] were identified in two samples from hospitalized children. Overall, the data revealed correlation between the presence of HNoV and the population HBGAs profiles. Individuals expressing HBGAs of secretor status were highly infected with norovirus, whereas non-secretor individuals demonstrated protection or less susceptibility towards norovirus infection. The GII.4 Sydney 2012 [P31] recombinant strain was dominant, followed by the GII.4 Sydney 2012 variant in all the secretor profiles. This study concludes that the prevalence and susceptibility of human norovirus infection in symptomatic and asymptomatic children from rural communities of Vhembe District is related to their HBGAs genetic profiles. The dominance of GII.4 Sydney 2012 [P31] in AGE children may be explained by the strain’s epidemiological fitness, and genetic susceptibility related to the prevalence of secretor profiles. Population’s genetic profiles may potentially drive human norovirus genetic diversity and evolution. This has implications for vaccine formulation in the current era of vaccine development. Further surveillance on impoverished communities where preventive strategies will most likely have greater impact is needed to monitor distribution of the virus and overall population HBGAs status.

NRF