Preliminary Study to Assess the Performance of Mengovirus Elution from Sludge

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B R I E F C O M M U N I C A T I O N

Preliminary Study to Assess the Performance of Mengovirus Elution from Sludge

Hasna Amdiouni^•Abdelaziz Soukri ^• Jalal Nourlil^•Leena Maunula

Received: 15 July 2012 / Accepted: 13 June 2013 ÓSpringer Science+Business Media New York 2013

Abstract

In the virus detection protocol for sludge, the viral elution step from solids to solution is critical. In this study, mengoviruses were detected in artificially contami- nated sludge with a qRT-PCR assay. The viral yields ran- ged between 19 and 66 % for 60 % sludge. This study demonstrates that mengovirus can be used as a sample process control for analysis of sewage sludge.

Keywords

Mengovirus Real-time RT-PCR Sludge Morocco

In addition to treated wastewater effluent, the processing of sewage generates a large volume of solid material, sludge.

Sludge may contain high levels of pathogens, mostly of human fecal origin. Before the use of sludge as a fertilizer for agricultural land or forests, the pathogens must be inactivated. The sanitary quality of sludge should be suf- ficiently controlled to prevent environmental and public health problems.

Sludge has been studied for the presence of such viruses as rotavirus, enterovirus, avian influenza A/H5N1 virus,

adenovirus, norovirus GII, and hepatitis A virus (Sobsey et al.

1980; Monpoeho et al.2001; Horm et al.2011; Prado

et al.

2013). Thus far, no universal method has existed for

identification of all enteric viruses from sludge, although a standard method is available for enteroviruses in the US (US Environmental Protection Agency

2003). In the liter-

ature, the detection of enteric viruses in sludge has been based on either cell culture or molecular biological meth- ods (Monpoeho et al.

2001; Belguith et al.2006; Houssin

et al.

2007).

Several elution–concentration methods have been applied for detection of enteric viruses in sludge (Monpoeho et al.

2001; Belguith et al. 2006; Houssin et al. 2007). Virus

adhesion to solids depends on both environmental conditions and surface properties of the virus (Langlet et al.

2008). To

elute viruses, all bonds between viral particles and sludge compounds must be broken (Schwartzbrod.

2000). Acidic

pH solutions (pH 3.5) have been used for virus extraction with AlCl

3

(Monpoeho et al.

2001). Otherwise, neutral or

alkaline pH solutions enriched with protein and mineral salts, such as glycine solution, beef extract, and beef extract cou- pled with glycine or borate (Chung et al.

1996; Ahmed and

Sorensen

1995; Belguith et al.2006; Deboosere et al.2012)

that compete with viruses adsorbed to the binding sites, are generally used to elute viruses from sludge. Glycine-based buffers and beef extract are also commonly used to elute viruses from food items and the attached viruses from membranes after water filtration (Costafreda et al.

2006;

Pe´rez-Sautu et al.

2012).

The aim of this preliminary study was to assess the per- formance of mengovirus when eluted with four generally used elution buffers PBS (phosphate-buffered saline, pH 7.5), BE/NaCl (7 % beef extract, pH 9.5, containing 0.3 M NaCl), Gly/NaCl (7 % glycine pH 9.5, containing 0.3 M NaCl), and TGBE (100 mM Tris–50 mM glycine, pH 9.5,

H. AmdiouniJ. Nourlil

Medical Virology & BSL3 Laboratory, Institut Pasteur du Maroc. 1, Place Louis Pasteur, 20360 Casablanca, Morocco H. Amdiouni (&)A. Soukri

Laboratory of Physiology and Molecular Genetics, Faculty of Sciences Aıˆn chock, University Hassan II, km 8 road of Eljadida, P.O. Box 5366 Casablanca, Morocco e-mail: hasna.amdiouni@yahoo.fr

L. Maunula

Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of Helsinki, P.O. Box 66, 00014 Helsinki, Finland

123

Food Environ Virol

DOI 10.1007/s12560-013-9116-2

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1 % beef extract), and detected using real-time reverse transcription (RT)-PCR from sewage sludge. Mengovirus, a cardiovirus serologically related to encephalomyocarditis virus and a non-enveloped RNA virus belonging to the

Picornaviridae

family, was used as a model for enteric viruses. Detection of enteroviruses, also picornaviruses, is included in the American (Environmental Protection Agency

2002) and French (JORF 1998) guidelines for

biosolids.

As material, 200 g of dry sludge collected from a wastewater treatment plant (WWTP), located 70 km from Casablanca, Morocco, was used. The sludge was pumped from wastewater treatment lagoons and was dehydrated in beds at ambient temperature for agricultural reuse in nearby lands. It was collected in a sterilized sampling bag and transported to the laboratory within 2 h, and viral analyses were performed within 24 h. For spiking, 10 RT- PCR U of mengovirus strain MC

₀

(ATCC VR-1957) (kindly provided by A. Bosch, University of Barcelona, Spain), propagated in HeLa cells, per gram of sludge was used. Three sludge concentrations, 100 % (10 g/10 ml), 80 and 60 % (w/v), in water were tested. After incubation of sludge in water seeded with mengovirus for 24 h at room temperature, the water was discarded and elution buffer was added to sludge. After the mixture was stirred for 2 h at 4

°C, it was centrifuged for 10 min at 2,0009g, and the

supernatant was recovered. In parallel, each elution buffer was seeded with mengovirus, without sludge, to assess the presence of PCR inhibitors. All experiments were repeated independently twice. For viral RNA extraction, 500

ll of

supernatant from sludge was processed with Trizol LS reagent (Life Technologies, Gaithersburg, MD), followed by real-time RT-PCR using the Superscript III Platinum One Step qRT-PCR System (Invitrogen, Life Technolo- gies) with an ABI PRISM 7500 Fast Real-Time PCR System (Applied Biosystems). For amplification, the primers Me-110 and Me-209 and the probe Me-147 were used as described by Pinto et al. (2009).

The results obtained when four virus elution buffers (PBS, BE/NaCl, Gly/NaCl, and TGBE) were applied are shown in Fig.

1. All elution buffers tested were able to

extract the mengovirus from sludge. Elution of viruses with BE/NaCl provided generally higher recoveries (range 28.44

±

0.38–66.09

±

0.21 %) than PBS, Gly/NaCl, or TGBE (Fig.

1a). The viral yields varied from 0.4 to 28 %,

2 to 62 %, and 19 to 66 % for 100, 80, and 60 % sludge, respectively. In all experiments, the best recoveries were obtained for the 60 % sludge concentration.

Mengovirus has been used as a surrogate for human hep- atitis A (HAV) and other enteric viruses in several publica- tions and as an external control when virus is detected in food and water samples (Costafreda et al.

2006; Pe´rez-Sautu et al.

2012). Recently, Da Silva reported the use of mengovirus as a

virus extraction control together with internal controls to verify the PCR inhibition levels for noroviruses in wastewater.

The use of mengovirus as a process control in the standard detection of norovirus and hepatitis A virus in shellfish was first proposed by the CEN working group (Lees

2010) and now

it is described for norovirus and hepatitis A virus analyses from food and animal feed in ISO technical specifications (ISO/TS 15216-1

2013; ISO/TS 15216-22013).

In agreement with our results, Mignotte et al. (1999) and Monpoeho et al. (2001) demonstrated that the beef extract in alkaline pH eluted enteroviruses efficiently from sludge.

Sludge may contain various ions and chemicals that interact with compounds of the buffer and/or interfere with the amplification step in PCR. The fact that viral yields increased with decreasing sludge concentration indicated the presence of PCR inhibitors (or limited capacity of RNA extraction) in the more concentrated solutions of sludge.

The differences observed between the results with the four elution buffers when no sludge was present probably reflected the inhibiting or enhancing effect of their chem- ical components in the RNA extraction and RT-PCR procedures (Fig.

1b). Our results suggest that the viral

concentration step was not necessary for the detection of

Fig. 1 Mengovirus recoveries obtained with four elution buffers from sludge

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viruses in sludge, in agreement with Horm et al. (2011), who showed that after artificial contamination of sludge with H5N1, the quantity of RNA detected in the elution step alone was higher than that obtained with the elution–

concentration method.

In conclusion, mengovirus could be successfully recovered with the techniques used in the study, although it became apparent that all the buffers were not equally efficacious in eluting viruses from sludge matrices. Other factors, such as the virus to be tested and the sludge matrix, also affect the elution result. Thus, it is important to test the applicability of the assay for each matrix using adequate process and internal controls. More studies are needed to test various kinds of sludge (primary, digested), to evaluate whether the procedure used here is optimal for other enteric viruses and to determine how comparable the results are with those obtained with infectivity assays. Previous studies have revealed that mengovirus behaves similar to HAV and noroviruses, at least in shellfish, wastewater, and river water (Costafreda et al.

2006; Da Silva et al. 2007;

Pe´rez-Sautu et al.

2012). Based on the results obtained in

this study, we recommend the use of mengovirus as a process control also in sewage sludge. In the future, this rapid and straightforward method using real-time RT-PCR could also be applied for analysis of various other viruses in sludge samples in Morocco and elsewhere.

Acknowledgments This study was funded by the Medical Virology

& BSL3 Laboratory, Institut Pasteur du Maroc, Casablanca, Morocco.

We thank Dr. Nozha Cohen and Dr. Kaoutar Hajjami for supplying us with sludge samples.

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