Genetic assessment of infectious hepatitis A virus strains detected in selected water sources in Gauteng, South Africa

Abstract

Hepatitis A is a vaccine preventable liver inflammation caused by the hepatitis A virus (HAV). Hepatitis A virus is the most common cause of acute viral hepatitis worldwide that is transmitted via the faecal-oral route with waterborne transmission recognised as a major public health concern. Hepatitis A virus is classified in the genus Hepatovirus of the family Picornaviridae. The stability of HAV regarding pH, temperature and different treatment systems contributes to the virus’ persistence in the environment. The virion of HAV has a 7.5 kb positive-sense single-stranded RNA genome. Nucleotide sequence analysis of the VP1 region has identified six geographically distinct genotypes infecting humans (genotypes I, II and III) and non-human primates (genotypes IV, V and VI). In South Africa (SA), unique HAV IB strains have been detected in surface and wastewater samples, as well as on fresh produce at the point of retail. However, as the viruses were detected by molecular-based assays it is unknown whether the detected strains were still infectious. Although hepatitis A is a notifiable disease in SA there is gross underreporting, no routine surveillance system and a paucity of epidemiological data on HAV strains in circulation. Recently, the pretreatment of water and food samples with intercalating dyes prior to nucleic acid extraction was successfully applied for the quantification of potentially infectious HAV using molecular assays. Given that analysing sewage, wastewater and surface waters would provide a more accurate estimation of the HAV strains circulating in the country, the present study aimed to detect and characterise infectious HAV strains from selected South African water sources. From April 2015 to March 2016, 118 samples consisting of sewage, treated wastewater discharge and a downstream dam water were collected monthly from five wastewater treatment plants (WWTP 1, 2, 3, 4, 5). High titres of HAV were detected in the sewage (1.34 x e5 and 3.70 x e10 genome copies [gc]/litre [L]) and treated discharge (4.74 x e3 to 3.39 x e7 gc/L) samples. None of the dam water samples tested positive for HAV. Genetic characterisation of the detected strains by Sanger sequencing revealed the circulation of HAV IB strains that carried the R298K amino acid change over the VP1 region or the R63K and R71S change over the VP1/P2B junction or the C70S and M104I change over the VP1/P2B junction. The quasispecies dynamic of HAV has been detected in sewage samples. Hepatitis A virus strains carrying amino acid mutation at the immunodominant and neutralisation epitopes were characterised in both the sewage and treated discharge samples. The virus concentrates of HAV-positive sewage and treated discharge samples were treated with a combination of PMA-water (50 μM) and Tween®20 (0.5%) and the quantification of HAV from the samples was repeated. Potentially infectious HAV was quantified from the wastewater samples, with titres ranging up to e6 and e4 gc/L of sewage and treated discharge samples, respectively. Characterisation of these potentially infectious strains by Sanger sequencing confirmed the circulation of HAV strains carrying the R298K amino acid change over the VP1 region or the R63K and R71S change over the VP1/P2B junction or the C70S and M104I change over the VP1/P2B junction. The complete coding sequence, obtained from sewage and treated discharge samples by NGS, confirmed the circulation of HAV strains carrying the R63K and R71S changes but not the C70S and M104I changes over the VP1/P2B junction. The present study provides a methodology for the quantification and genetic characterisation of potentially infectious HAV from wastewaters.