G3 and G9 Rotavirus genotypes in waste water circulation from two major metropolitan cities of Pakistan

Abstract

Rotavirus A (RVA) is a diarrheal pathogen affecting children under age five, particularly in developing and underdeveloped regions of the world due to malnutrition, poor healthcare and hygienic conditions. Water and food contamination are found to be major sources of diarrheal outbreaks. Pakistan is one of the countries with high RVA related diarrhea burden but with insufficient surveillance system. The aim of this study was to gauge the RVA contamination of major open sewerage collecting streams and household water supplies in two major metropolitan cities of Pakistan. Three concentration methods were compared using RNA purity and concentration as parameters, and detection efficiency of the selected method was estimated. Water samples were collected from 21 sites in Islamabad and Rawalpindi in two phases during the year 2014-2015. Meteorological conditions were recorded for each sampling day and site from Pakistan Meteorological Department (PMD). Nested PCR was used to detect the presence of RVA in samples targeting the VP7 gene. Logistic regression was applied to assess the association of weather conditions with RVA persistence in water bodies. Statistical analysis hinted at a temporal and seasonal pattern of RVA detection in water. Phylogenetic analysis of selected isolates showed a close association of environmental strains with clinical RVA isolates from hospitalized children with acute diarrhea during the same period. This is the first scientific report cataloging the circulating RVA strains in environmental samples from the region. The study highlights the hazards of releasing untreated sewerage containing potentially infectious viral particles into collecting streams, which could become a reservoir of multiple pathogens and a risk to exposed communities. Moreover, routine testing of these water bodies can present an effective surveillance system of circulating viral strains in the population.

Figure 1

Agarose gel (1.5%) electrophoresis of nested PCR amplified product for VP7 gene of rotavirus (cropped). M (marker), 100 bp DNA ladder; Lane 1 to Lane 6, 10⁻¹⁰ 10⁻⁸ 10⁻⁶ 10⁻⁴ 10⁻² 10⁻⁰ serial dilutions of stool sample containing positive sample (G1P). (Original gel picture is attached in Supplementary Materials).

Figure 2

Maximum Likelihood Phylogenetic Tree of two G9 and one G3 strains of Rotavirus A (RVA) detected in this study, constructed in MEGA program 6.0 with kimura-2-parameter model using 1000 bootstrap replicates. The sequences generated in this study are highlighted in red color.

Figure 3

Map showing sampling sites in twin cities of Islamabad (Capital city of Pakistan) and Rawalpindi. Imagery taken from Google Earth: Image 2018 © DigitalGlobe and annotated using Microsoft © 2018 built in apps.