Recovery Comparison of Two Virus Concentration Methods from Wastewater Using Cell Culture and Real-Time PCR

Abstract

Enteric viruses are shed in the feces and may be present in environmental waters. Their detection in wastewater, even at low concentration, is a major challenge. In this study, recoveries of Echovirus 7 (EV7), virions and RNA in wastewater, using virus concentration methods were determined to evaluate the detection of infectious viruses and the possibility of recovering viral genomes. Two virus concentration methods, PEG precipitation method and two-phase separation method, were applied to recovery experiments of EV7-virions from wastewater, in parallel with recovery experiments of EV7 RNA. The titration of EV7 virions was carried out by cell culture using human rhabdomyosarcoma tumor tissue and the EV7 RNA quantification was performed by real-time PCR. The mean recovery yields of EV7 virions using the PEG precipitation method and the two-phase separation method were 78.5 ± 10.99 and 83.1 ± 0.28 %, respectively. Besides, EV7 RNA recoveries obtained using the PEG precipitation method were four times higher than those using the two-phase separation method. According to our results, the two methods enable to concentrate both infectious viruses and viral genomes. Moreover, considering the protocol time and cost together with the ratio of the EV7 virion recovery to the EV7 RNA recovery, the two-phase separation method (83.1/2.71 %, or 30.6) seems to be more appropriate for selective concentration of viral virions than the PEG precipitation method (78.5/10.33 %, or 7.6).

Comparison of Two Virus Concentration Methods for Enteric Viruses Detection in Moroccan Wastewater and Treated Effluent

“…Some of them advised EV (37,50) and others advised HAdV (38,51) . Current study results are in agreement with those obtained in previous study when the same methods were compared using Echovirus 7 as a model of enteroviruses (22) .…”

“…The obtained results showed that Hep2 cells were more suitable for adenovirus detection as reported in previous study (22) . The use of both cell lines was useful for us to isolate the most of HAdV and EV in samples.…”

“…To explain the simultaneously negative and positive results of the CC and PCR, This could be due to that during the transport and treatment of wastewater, some viral particles undergo alterations that reduce their infectivity in vitro which decrease the positive samples by CC (22) . The inactivation of viruses could be caused by virus contact with wastewater treatment lagoons sides or natural inactivation by sunshine (26) .…”

“…The first one uses bags with sorbent macroporous glass to trap viruses and the second uses the two-phase separation, which is applied in this study. Regarding the comparison of virus concentration methods in water, several studies have been conducted using polymerase chain reaction (PCR) and cell culture (13,22) . In the present study, a one-year survey (January -December 2009) was conducted in order to compare two virus concentration methods, the PEG precipitation and the twophase separation using three statistical tests, to analyze the correlation between the cell culture and PCR for both EV and HAdV and to assess the presence of viral genomes of EV, HAV, HEV, HAdV, HuNoV, RV and HAstV by each method.…”

Comparison of Two Concentration Methods for the Molecular Detection of Enteroviruses in Raw and Treated Sewage

“…An effective concentration of samples is necessary to evaluate virological quality of wastewaters, since matrices are highly contaminated with inhibitory substances that will interfere with viral detection [11]. Numerous methods have been described for concentrating and extracting enteric viruses from sewage [14][15][16]. All of them studied on spiked viral particles in decontaminated samples.…”