Molecular Quantification of Viruses in Surface Water: Development of Indicator of Viral

Contamination in Water

Dissertation to obtain the degree Doctor Philosophiae (Doctor of Philosophy, PhD)

at the Faculty of Biology and Biotechnology Ruhr-University Bochum

International Graduate School of Biosciences Ruhr-University Bochum

Department of Hygiene, Social and Environmental Medicine Faculty of Medicine

Submitted by

Ibrahim Hamza Ewess

from

Giza, Egypt

Bochum2010

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Molekulare Quantifizierung von Viren in Oberflchenwasser: Entwicklung eines Indikators zur Bestimmung der viralen

Kontamination in Wasser

Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften

der Fakultt fr Biologie und Biotechnologie an der Internationalen Graduiertenschule Biowissenschaften (IGB)

der Ruhr-Universitt Bochum

angefertigt in der

Abteilung fr Hygiene, Sozial- und Umweltmedizin

vorgelegt von

Ibrahim Hamza Ewess

aus

Giza, gypten

Bochum2010

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This work was supervised by: Prof. Dr. Michael Wilhelm 1st Supervisor

Prof. Dr. Franz Narberhaus 2nd Supervisor -------------------------------------------------------------------------------------

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Dedication

Dedication

To My

Parents

Brothers& Sisters

Wife

Kids

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Acknowledgments

Acknowledgements

I am deeply indebted to Prof. Michael Wilhelm for giving me the opportunity to conduct this work at his laboratory and who was an excellent supervisor, supporting me throughout the entire time of my thesis, read this manuscript

and taught me how to publish my data.

I would like to express my deepest gratitude to Prof. Franz Narberhaus for accepting to be my second supervisor.

I am heartily thankful to Prof. Klaus berla, whose guidance, support and critical comments from the initial to the final level were invaluable.

My thanks to Dr. Lars Jurzik for his bacteriological analysis of river water samples and review the German translating of the summary. I extend my

thanks to Mr. Andre Schtze for translating the summary to German.

My special thanks to Dr. Alexander Stang for his cooperation to develop PCR random amplification approach to examine viruses in aquatic environment.

My regards and pleasing to my colleagues in department of hygiene, social- and environmental medicine and department of molecular and medical

virology for creating an inspiring environment and providing constant support. I would like to mention specially Messrs. Klaus Sure, Rosemarie Bohr, Heike Seidenstcker, Andreia Fernandes, Ulrike Hofmann, and Elke Benedens for

their support during my study.

I would like to thank the ministry of higher education, Egypt for the financial support during my PhD study here in Germany.

Lastly, my parents, brothers, sisters, wife and kids, there are no words that

say thank you enough for everything you have done for me.

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Table of Contents

i

Table of Contents

Table of contents ...................................................................................................i

Abbreviations ......................................................................................................iii

I. Introduction ...................................................................................................... 1

1. Viruses associated with water contamination

1.1. Human viruses ..........................................................................................................................................

1.2. Pepper mildmottle virus ...................................................................................................................9

2. Commonly usedmethod for virus examination in aquatic environments

2.1. Concentration of enteric viruses................................................................................................

2.2. Commonmethod for virus detection in water..................................................................

3. Indicators of waterborne enteric viruses

3.1. Conventional bacterial indicators.............................................................................................

3.2. Viral indicator.........................................................................................................................................

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Table of Contents

ii

3.3. Chemical parameters as indicator ............................................................................................

II. Objectives of thiswork.....21

III. Original articles...........................................................................................22

1. Determination of the viral load in rivers of a densly populated area in

Germany using a virus adsorption elution method optimized for PCR

analyses....

2. Detection and quantification of human bocavirus in riverwater.......

3. Chemical andmicrobiological parameters as possible indicators for human

enteric viruses in surfacewater....

4. Evaluation of pepper mild mottle virus, human picobirnavirus and Torque

teno virus as indicators of fecal contamination in river water......

IV. Discussion .....................................................................................................27

V. Summary .......................................................................................................43

VI. Zusammenfassung .....................................................................................46

VII. References.......49

VIII. Attachment.................................................................................................58

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Abbreviations

iii

Abbreviations

% Percent (percentage)

C Degree Celcius

A549 Human Lung Adenocarcinoma Cell Line

APHA The American Public Health Association

B. fragilis Bacteroides fragilis

BGM Green Monkey Kidney Cells

bp Base Pair

C. perfringens Clostridium perfringens

CaCo-2 Human Epithelial Colorectal Adenocarcinoma Cells

cDNA Complementary Deoxyribonucleic Acid

DNA Deoxyribonucleic Acid

E. coli Escherichia coli

EC European Commission

EV Enteroviruses

FRNA Male-Specific RNA Coliphages

gen.equ./l. Genome Equivalents per Liter

GG Genogroup

HAdV Human Adenovirus

HAV Hepatitis A Virus

HBoV Human Bocavirus

Hep2 Human Epithelial Type 2

HEV Human Enterovirus

hPBV Human Picobirnaviruses

HPyV Human Polyomaviruses

ICC-PCR Integrated Cell Culture PCR

Kb Kilobase

kDa Kilodalton

l Liter

MA104 Rhesus monkey epithelial cell line

mg Milligram

MID Minimum Infectious Dose

ml Mililiter

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Abbreviations

iv

mM Millimolar

nm Nanometre

NoV Human Noroviruses

PBS Phosphate Buffer Saline

PCR Polymerase Chain Reaction

PEG Polyethylene Glycol

PMMoV Pepper Mild Mottle Virus

qPCR Quantitative Polymerase Chain Reaction

RD Human Rhabdomyosarcoma Cells

RNA Ribonucleic Acid

RoV Rotavirus

RoV A Rotaviruses Group A

RT Reverse Transcriptase

SOMCPH Somatic Coliphages

TC Total Coliform

TCPP Tris-(2-chloro-1-methyl-ethyl) phosphate

TFF Tangential Flow Filtration

TTV Torque teno virus

USEPA United States Environmental Protection Agency

UV Ultraviolet Light

v/v Volume per Volume

VIRADEL Virus Adsorption and Elution

VP Viral Proteins

w/v Weight per Volume

WHO World Health Organization

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Introduction

1

I. Introduction

Despite the progress in water and wastewater treatment technology, waterborne

diseases keep having far reaching public health and socio-economic implications

in both the developed and developing world. Communities obtain their potable

water from surface or underground sources. Surface waters include lakes, rivers,

and streams, their quality rapidly changes as a response to changes in the

surrounding watershed. However, water can become contaminated by biological

and chemical pollutants originati