Molecular epidemiology of group A Rotaviruses in water sources and selected raw vegetables in Southern Africa

Group A rotaviruses (RVs) are the most important cause of acute viral gastroenteritis in infants and young children. In this study raw and treated drinking water supplies at plants in two geographic areas, as well as selected irrigation water and corresponding raw vegetables in three regions of southern Africa, were screened for the presence of RVs using molecular techniques. Group A RVs were detected in 11.8% of partially treated and 1.7% of finally treated drinking water samples and in 14% of irrigation water samples and 1.7% of corresponding raw vegetable samples. Type-specific reverse transcriptase-PCR and sequence analysis revealed the presence of multiple types (G1, G2, G8, and G9) in irrigation water and single types (G1 or G3) in raw and treated drinking water. Group A RVs detected in all samples consisted of mixed P types (P[4], P[6], P[8], and P[9]), with P[6] predominating. The detection of types G8, G9, and P[6] reflects the emergence of these types in clinical infections. The similarity of environmental types to those in patients with clinical RV infections confirms the value of wastewater screening as a tool for assessing RVs circulating in communities, with the benefit of detecting types that cause both clinical and subclinical infections. The results provide new information on RV types in water and related environments and identify the potential risk of waterborne transmission. In addition, the presence of RVs in drinking water underlines shortcomings in quality specifications. These data provide valuable information regarding the prevalence of RVs in environmental sources, with important implications for vaccine development.This study was supported by grants from the Water Research Commission, the Poliomyelitis Research Foundation, and the Research Committee, School of Medicine, University of Pretoria. A postdoctoral fellowship for W.B.V.Z. from the South African Medical Research Council is also gratefully acknowledged

Prevalence of vaccine-derived polioviruses in sewage and river water in South Africa

Polioviruses (PVs) are not associated with waterborne transmission to the same extent as many other enteric viruses. However, they are typically transmitted by the faecal-oral route, which implies that the risk of infection by exposure to the viruses in water cannot be underestimated. The risk appears particularly high for rural communities, which use sewage-polluted river water for domestic purposes. Thus, the presence in the environment of highly evolved, neurovirulent vaccine-derived poliovirus (VDPV) strains in the absence of polio cases would have important implications for strategies to terminate immunisation with oral poliovirus vaccine (OPV) following global polio eradication. The aim of the current study was to determine the prevalence of VDPVs in selected sewage and river water samples collected from 2001 to 2003, and to construct phylogenetic trees of the partially sequenced 5′untranslated region (5′UTR) and the VP1 region of the genomes to deduce the genetic relatedness between the PV strains. Using the monolayer plaque assay, 703 plaques from sewage and 157 plaques from river water samples were analysed. Application of a RT-multiplex PCR revealed that 176 of these plaques were non-polio enteroviruses, and 49 were PV isolates. The Sabin-specific RT-triplex PCR revealed the presence of 29 Sabin PV type 1, 8 Sabin PV type 2 and 12 Sabin PV type 3 isolates. The 5′UTR and the VP1 region of 13 PV type 1, 7 PV type 3 and 6 PV type 2 isolates were partially sequenced. The majority of the OPV isolates (24 out of 26) displayed close sequence relationships (>99% VP1 sequence identity) to the parental Sabin PV vaccine strains and were classified as “OPV-like viruses”. Two isolates (D1 08/28 and OF1 05/21) were found to be highly divergent and were classified as “suspected” VDPVs. Isolate OF1 05/21 (a “suspected” VDPV type 1) showed more than 0.9% divergence in VP1, whereas isolate D1 08/28 (a “suspected” VDPV type 2) showed 1.4% divergence in VP1 from the parental Sabin PV vaccine strains. As with most of the other OPV-like isolates, these “suspected” VDPVs were carrying mutations, which have previously been associated with reversion of the attenuated Sabin PV strains to increased neurovirulence. It was estimated that the total period of replication for the two “suspected” VDPVs was between 12 and 16 months. In conclusion, this study provided new and relevant information on the prevalence of “suspected” VDPVs in sewage andMedical Virolog

Poliovirus vaccine strains detected in stool specimens of immunodeficient children in South Africa

After exposure to the oral poliovirus vaccine (OPV), immunocompetent persons excrete poliovirus (PV) vaccine strains for a limited period. In contrast, immunodeficient individuals remain sometimes chronically infected, and in some cases, PV excretion times as long as 10 years have been reported. During prolonged replication in the human intestine, the PV vaccine strain almost invariably reverts its attenuated character and acquires neurovirulent properties (vaccine-derived PVs, or VDPVs), which resemble wild-type PV strains. The aim of this study was to determine the occurrence of OPV strains in stools of immunodeficient children from a selected area in South Africa, as a first step toward future research on the prevalence and potential health impact of VDPVs. In a period of 1 year, a total of 164 stool samples of HIV-positive children aged 4 months to 8 years were studied for the excretion of OPV strains. In addition, 23 stool samples from healthy immunocompetent children were analyzed after receiving their OPV immunization. By applying a reverse transcription–polymerase chain reaction in combination with a nested PCR, a total of 54 enteroviruses (EVs) were detected in the stool specimens of the immunodeficient children. Using restriction enzyme analysis, 13 PVs were distinguished from 41 nonpolio EVs (NPEVs). A Sabin-specific RT-triplex PCR confirmed the presence of 7 Sabin PV type 1, 4 Sabin PV type 3, and 2 Sabin PV type 2 isolates. The majority of the NPEV group was made up of 7 coxsackievirus B3 (CBV3), 6 echovirus 11 (ECV11), 5 ECV9, and 3 coxsackievirus A6 (CAV6) isolates. According to the results, two of the immunodeficient patients (P023 and P140) who had received their last OPV immunization more than 15 months before (vaccinated at 14 weeks of age) tested positive for Sabin PVs types 3 and 1, respectively. A 5-year-old immunodeficient patient (P052) who had received her last OPV immunization more than 42 months before (vaccinated at 18 months of age) tested positive for Sabin PV type 1. These results suggested that immunodeficient patients vaccinated with OPV might excrete potentially pathogenic VDPVs for a prolonged period. These VDPVs may circulate in the community, resulting in possible infections in the unvaccinated population. Therefore, the information obtained in this study would be essential for strategies aimed at the protection of both immunodeficient as well as immunocompetent individuals against complications of vaccination with OPV

Prevalence of vaccine-derived polioviruses in stools of immunodeficient children in South Africa

Aims: The aim of the study was to determine the prevalence of vaccine-derived polioviruses (VDPVs) in stool specimens of immunodeficient patients such as HIV-positive children (including those with an AIDS indicator condition, according to the Centres for Disease Control and Prevention classification) by applying various molecular techniques. Methods and Results: A total of 164 stool samples from HIV-positive children and 23 stool samples from healthy immunocompetent children (the control group) were analysed during 2003 and 2004. By applying a reverse transcription polymerase chain reaction (RT-PCR) in combination with a nested PCR, a total of 54 enteroviruses were detected in the stool specimens of the immunodeficient children. The use of restriction enzymes and a Sabin specific RT-triplex PCR confirmed the presence of 13 polioviruses (PVs), such as seven Sabin PV type 1, four Sabin PV type 3 and two Sabin PV type 2 isolates. The 5'untranslated region and the VP1 capsid-encoding protein of the 13 PVs and the three PVs from the stools of the immunocompetent children were partially sequenced and their genetic relatedness was deduced from the constructed phylogenetic trees. The majority of the PVs isolated from the stools of the immunodeficient children (10 of 13 isolates) were classified as 'oral poliovirus vaccine (OPV)-like viruses', as these isolates had close sequence relationships (>99% in VP1 nucleotide sequences) to the original Sabin PV vaccine strains. Three PVs showed ≤99% VP1 sequence identity to the Sabin PV vaccine strains and were classified as 'suspected' immunodeficient VDPVs (iVDPVs). All of the OPV-like isolates and the 'suspected' iVDPVs carried mutations at specific positions in their partially sequenced regions, which have been associated with reversion of the attenuated Sabin PV vaccine strains to increased neurovirulence. Conclusions: Thus, this study adds further evidence to the observation that immunodeficient individuals may excrete OPV strains with potential neurovirulent phenotypes