Molecular characterization of norovirus stains circulating in rural communities of Limpopo Province of South Africa

Abstract

Globally, one in ten child deaths before the age of 5 years is due to diarrheal disease, causing almost 800,000 mortalities worldwide, which mostly occur in Sub-Saharan Africa and South Asia. Eighty-eight percent (88%) of diarrheal deaths worldwide are attributable to unsafe water, inadequate sanitation and poor hygiene. Unsanitary environments and poor hygiene practices allow diarrhea causing pathogens including viruses, bacteria and parasites to spread more easily. Norovirus (NoV) are now considered the most common cause of outbreaks of nonbacterial gastroenteritis. However, the factors which control the genetic diversity, the sources of sporadic NoV infections, the transmission and persistence of infection are poorly understood. Limited data are available for NoVs strains in South Africa, especially in rural and peri-urban areas. Despite the excessive burden of diarrhea disease in developing countries, NoVs outbreaks have been to date mostly reported in developed countries. Given that the contribution of the various pathogens to diarrhea may differ substantially between regions depending on local meteorological, geographic, and socio-economic conditions, there is a need to investigate intensively the role of viral agents associated with diarrhea in different settings in Africa continent. How would poor living conditions in rural setting impact the prevalence and genetic characteristics of Norovirus strains circulating Limpopo province is the research question of this study. ix To determine the prevalence and genetic diversity of NoVs strains circulating in the rural communities in the Limpopo Province, South Africa and investigate the genetic relationship between NoVs strains, a cross-sectional study was performed on human stools collected from rural communities. We used qualitative variables of poor living environmental conditions including type of water used at the household of child’s parent or guardian, use of toilet seat, presence of livestock at the household and parent employment status to assess possible environmental risk factors of NoV infection within the study area. Prior to this prospective study, we conducted a systematic review of the PubMed and EMBASE databases for published articles of Human NoVs in Africa between 1990 and 2013 in order to assess the contribution of Human NoVs to diarrhoeal diseases in Africa. This review provides a picture of Human NoVs studies in Africa and reveals that unreported sporadic gastroenteritis cases of Human NoVs are common in Africa. Most are community-associated infections reported from urban settings. Possible environmental transmission routes have been documented. Combined environmental and clinical studies are required for targeted actions to control transmission of Human NoVs in Africa. Between July 2014 and April 2015, outpatient children under 5 years of age from rural communities of Vhembe district, South Africa, were enrolled for the study. A total of 303 stool specimens were collected from those with diarrhea (n=253) and without (n=50) diarrhea. NoVs were identified using real-time one-step RT-PCR. Nucleotide sequencing methods were performed to genotype the strains. Phylogenetic analyses x were performed to compare identified NoVs genotypes to the worldwide circulating strains. One hundred and four (41.1%) NoVs were detected. NoV detection rates in symptomatic and asymptomatic children (OR = 1.24; 95% CI 0.66 – 2.33) were not significantly different. Comparison of the median CT values for NoV in symptomatic and asymptomatic children revealed significant statistical difference of estimated GII viral load from both groups, with a much higher viral burden in symptomatic children to our knowledge this is the first study reporting on the differences in estimated viral load of GII and GI NoV positive cases and controls. The study findings may have implications for the diagnosis of NoV disease and future vaccine development, which may only need to consider GII as the genogroup associated with diarrhea in the South African population. Sequence analyses demonstrated multiple NoV genotypes identified in rural communities of Vhembe district. The most prevalent NoV genotypes were GII.4 Sydney 2012 variants (n=7) among the capsid genotypes, GII.Pe (n=9) among the polymerase genotypes and GII.Pe/GII.4 Sydney 2012 (n=8) putative recombinants among the RdRp/Capsid genotypes. Two unassigned GII.4 variants and an unusual RdRp genotype GII.P15 were found. With note, the rare GII.P15 identified in this study, has a common ancestor with GII.P15 strain from Japan previously reported as GII / untypeable recombinant strain implicated in a gastroenteritis outbreak. To our knowledge this is the first report of this unusual genotype in the African continent. Though not proven predictive of diarrhea disease in this study, the high detection rate of NoV reflects the substantial exposure of children from rural communities to enteric xi pathogens possibly. However in this study no risk factor has been found between NoV positive and qualitative environmental variables of poor living conditions in rural setting. The results also suggest that the difference between asymptomatic and symptomatic children with NoV may be at the level of the viral load of NoV genogroups involved. The findings highlighted NoV genetic diversity and revealed continuous pandemic spread and predominance of GII.Pe/GII.4 Sydney 2012, indicative of increased NoV activity. An unusual RdRp genotype GII.P15 and two unassigned GII.4 variants were also identified from rural settings of the Vhembe district/South Africa. NoV surveillance