World Applied Sciences Journal 27 (10): 1235-1247, 2013ISSN 1818-4952© IDOSI Publications, 2013DOI: 10.5829/idosi.wasj.2013.27.10.81200

Corresponding Author: Waled M. El-Senousy, Department of Water Pollution Research, Environmental Research Division,National Research Center (NRC), Dokki, Cairo, Egypt.

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Molecular Epidemiology of Human Adenoviruses and Rotaviruses asCandidate Viral Indicators in the Egyptian Sewage and Water Samples

Waled M. El-Senousy, Ahmed B. Barakat, Hossam E. Ghanem and Marwa A. Kamel1 2 2 1

Department of Water Pollution Research, Environmental Research Division,1

National Research Centre (NRC), Dokki, Cairo, EgyptDepartment of Microbiology, Faculty of Science, Ain Shams University, Cairo, Egypt2

Abstract: The prevalence of human rotavirus and adenovirus in sewage and water samples before and aftertreatment steps and also in clinical samples was compared in this study to determine the most prevalent virusand the more resistant one to treatment processes. The prevalence percentage of rotaviruses in sewage samplescollected from raw sewage of Zenin wastewater treatment plant (WWTP) from July 2009 to June 2011 was 16.7%(4/24) while the prevalence percentage of adenoviruses in the same raw sewage samples was 91.7% (22/24). Theprevalence percentage of rotaviruses in Nile water samples collected from El- Giza water treatment plant (WTP)from July 2009 to June 2011 was 29.2% (7/24) while the prevalence percentage of adenoviruses in the same Nilewater samples was 66.7% (16/24). Rotavirus could be only detected and quantified as genome copies andinfectious units in the peak months of the virus in autumn and winter. Adenovirus genome copies andinfectious units were higher in winter than in the other months but considerable numbers of both of them werequantified all the year. The means of adenovirus genome and infectious units removals were 3.5±1.1 log and10

2.8±0.8 log respectively while the means of rotavirus genome and infectious units removals were 3.75±0.5 log10 10

and 3±1.2 log respectively after treatment processes of Zenin WWTP. The means of adenovirus genome and10

infectious units removals were 2.3±0.9 log and 2.1±0.9 log respectively while the means of rotavirus genome10 10

and infectious units removals were 2.86±1.1 log and 2.57±1.1 log respectively after treatment processes of10 10

El-Giza WTP. Rotavirus was the most prevalent in diarrheal samples 31.8% (35/110), while adenovirus wasdetected in 18.1% (20/110) of the samples. Sequence analysis of positive adenovirus samples indicated that19 samples (6 raw sewage, 5 Nile water and 8 clinical samples) showed similar sequences clustered with humanadenovirus 41 strain 503804/KOL/2009 hexon gene with 94% homology and 5 samples (2 raw sewage and 3 Nilewater samples) showed similar sequences clustered with human adenovirus 2 strain N3773/TW/05-2 hexon genewith 98% homology. As a conclusion, human adenovirus may be a suitable candidate viral indicator of humanviral contamination in Egyptian water and sewage samples.

Key words: Adenovirus Rotavirus Viral Indicator Wastewater Treatment Plants Water Treatment Plants

INTRODUCTION thus neglected. Bacterial indicators are able to detect fecal

Urban wastewaters discharged into surface waters the viral health risk, but they are not correlated with thecan be sources of environmental viral contamination. presence of enteric viruses or their abatement [1-3].Reuse of wastewaters for agriculture or industrial Enteric viruses are excreted with human feces and havepurposes may also contaminate the environment. been found not only in wastewater but also in rivers,Although controls of the microbial pollution of treated recreational water and seawater as well as in ground waterwastewater are currently required, biological monitoring and even treated drinking water [4]. In the USA, 10% ofis limited to bacterial parameters (fecal coliform, waterborne outbreaks are reportedly associated with viralEscherichia coli, enterococci and Salmonella); viruses are agents [5].

contamination and are therefore an indirect indicator of

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The choice of the most useful viral parameter to be In Cairo, Egypt, rotavirus (Group A) is the mosttested is complicated by the large number of viral species frequent RNA enteric viruses in raw sewage and Nilepotentially present, the variability of strains circulating in water and also the most resistant RNA enteric viruses tothe population, the different significance of qualitative treatment processes in water and wastewater treatmentand quantitative assays (respectively, presence/absence plants followed by HAV, astrovirus and enterovirus andand counts) and difficulties in interpreting the results finally, noroviruses respectively [19, 20]. In this study weobtained from culture and biomolecular tests [4]. A better compared the molecular epidemiology of humanunderstanding of viral contamination and resistance to rotaviruses and human adenoviruses as candidate viraltreatments therefore seems imperative in order to be able indicators in Egyptian water and wastewater and into assess and control the true risks related to wastewater diarrheal samples.recycling and discharge. The enteric viruses mostcommonly found in human stool belong to more than MATERIALS AND METHODS140 types, of which adenovirus, norovirus genotype I andII, rotavirus, astrovirus, enterovirus and hepatitis A virus Sewage Samples: A total of 96 sewage samples were(HAV) are those most often detected in the environment. collected for 2 years from July 2009 to June 2011 fromAmong the diseases caused by enteric viruses are Zenin wastewater treatment plant which uses an activatedgastroenteritis, meningitis, myocarditis, conjunctivitis and sludge treatment with a capacity of 330000 m /day.hepatitis [6]. The samples were collected from raw wastewater, after

Although, adenoviruses are more often associated to primary sedimentation, after secondary sedimentation andrespiratory infections, some serotypes, denominated treated effluents. Four liter of raw sewage and of the otherenteric adenoviruses are related to diarrheal disease. treated sewage samples were collected and transferred inEnteric adenoviruses are classified into the species F of clean sterile bottles and transported to the laboratorythe Mastadenovirus genus of the Adenoviridae family within 3 hrs after collection for examination.and comprise serotypes 40 and 41 [7]. Adenoviruses arevery commonly found in water environments [8] and have Water Samples: A total of 96 water samples werealready been proposed as viral indicators [9], since collected from July 2009 to June 2011 from El-Giza waterseveral studies have reported that human adenovirus treatment plant. The treatment facilities in the treatment(HAdV) exhibits high stability under conditions of plant are pre-oxidation with Cl , coagulation-settling,environmental stress, such as UV radiation, temperature, sedimentation, filtration (rapid sand filters) and final Clchlorine concentration and pH variation, including addition. 20 liters of water sample were collected includingsewage treatment procedures [10, 11]. Rotaviruses are inlet water (Nile water), after sedimentation, after sandresponsible for severe gastroenteritis in both humans and filtration and outlet water (drinking water).animals and have been implicated in waterbornegastroenteritis outbreaks in many countries [12]. The Clinical Samples: 110 stool samples were collectedpresence of enteroviruses has been frequently detected in during the period from May 2011 to April 2012 from Aboboth raw and treated wastewater [13], as well as in sludge El-Reech hospital in Greater Cairo. Stool samples were[14]. Owing to its frequent detection and easy cultivation, collected from children (< 5 years old) suffering from acutethey have been considered as viral references in Italian diarrhea. Samples were collected in clean containers anddrinking water regulations (D.Lgs 31/2001) [15] although transferred to the laboratory within 3 hrs after collectionthey are not a mandatory parameter. HAV is the main for examination.cause of acute hepatitis worldwide and its waterbornetransmission has been thoroughly documented [12]. Concentration of Sewage and Water Samples: SewageThe WHO regards HAV as reference pathogens for and water samples were concentrated by filtration throughdrinking water risk analysis [16]. Finally, Torque Teno negatively charged nitrocellulose membranes (ALBET,Virus (TTV) is prevalent in general populations Spain 0.45 µm pore size and 142 mm diameter filter series)worldwide, but at present is not related with any specific after addition of AlCl to a final concentration of 0.5 mMpathology. Its presence in feces and its remarkable and acidification to pH 3.5 and after passing throughenvironmental stability suggest the possibility of using it Whatmann no. 1 filter paper. The viruses adsorbed to theas an indicator of fecal contamination in the environment membrane were eluted with 75 ml of 0.05 M glycine buffer,[17, 18]. pH 9.5 containing 3% beef extract

3

2

2

3

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(Lab-Limco powder, OXOID, UK) [21, 22]. All samples culture RT-PCR (CC-RT-PCR) assay was performed onwere reconcentrated using an organic flocculation method suspensions of infected MA104 cells. Set of primers[23]. Samples were neutralized and kept at -70°C until VP6-F and VP6-R was used. The RT-PCR method was theused. same as described previously. The detection limit in

Concentration of Clinical Samples: Approximately 0.1 g CC-RT-PCR units/ml (u/ml), where CC-RT-PCR u is theof stool samples was weighed, diluted 1:10 in nuclease- reciprocal end point dilution detectable by CC-RT-PCR.free H O and vortexed for 30 sec. Samples were clarified2

by centrifugation at 7.000 rpm for 10 min at room Extraction of DNA: It was done as described previouslytemperature. Viral RNA was extracted from 140 µl of the by Kapperud et al. [30] and modified by Estrada et al.supernatant. [31]. 50µL of sample concentrate were added to 50µL of

Viral Nucleic Acid Extraction: It was done using being incubated at 37°C for 1h, the suspension wasBIOZOL Total RNA Extraction reagent (BIOFLUX, Japan) boiled for 10 min and then centrifuged at 12500 rpm forand according to the manufacturer’s instructions. 5 min at 4°C. The supernatant was used for performing the

RT-PCR of a Fragment of the VP6-Coding Gene ofRotaviruses (Group A): The primers used for RT-PCR Detection of Adenoviruses Using Nested PCR: It waswere the forward VP6-F primer 5'- done according to Puig et al. [32] using the specificGACGGNGCNACTACATGGT-3' and the reverse primer, primers hex AA 1885, hex AA 1913 for the first round PCRVP6-R 5'-GTCCAATTCATNCCTGGTGG-3' according to and nehex AA 1893 and nehex AA 1905 for the secondIturriza-Gomara et al. [24]. Nested PCR amplification of round PCR for detection of human adenovirus and werethe target rotavirus VP6 fragment was performed using selected from the DNA sequence of the open readingthe forward primer, VP6-NF 5'-GCTAGAA frame of hexon gene. PCR products (10 µl) were analyzedATTTTGATACA-3' and the reverse primer, VP6-NR 5'- by electrophoresis on 3% agarose gels (Panreac-spain).TCTGCAGTTTGTGAATC-3', according to Gallimore et al.[25] to amplify 155 bp fragment. PCR products (10 µl) Real Time PCR for Quantification of Adenoviruses:were analyzed by electrophoresis on 3% agarose gels Real time PCR was done using power SYBR green PCR(Panreac-spain). master mix (Applied Biosystem, UK) with 0.5 µl of both

Quantitation of Rotavirus Using Real time RT-PCR 1913 in a total volume of 25µl. Amplification wasMethod: Real time RT-PCR was done for positive samples performed as described previously [33], in a Real timein previous RT-PCR screening. The RT was done PCR thermal cycler (OneStep, Applied Biosystem). Theaccording to Iturriza-Gomara et al. [24] using primers specificity of the reactions was determined by melting(VP6-F and VP6-R). Real time PCR was done using power curve analysis of the amplicons. The adenovirus genomeSYBR green PCR master mix (Applied Biosystem, UK) copy number was determined by comparison with awith 0.5 µl of both forward primer VP6-F and reverse standard curve generated with serial dilutions of aprimer VP6-R in a total volume of 25µl. Amplification was positive control plasmid, constructed by TOPO TAperformed as described previously [26] in a Real time PCR cloning (Invitrogen) of the PCR product from anthermal cycler (OneStep, Applied Biosystem). The adenovirus positive sample.specificity of the reactions was determined by meltingcurve analysis of the amplicons. The rotavirus genome Cell Culture-PCR (CC-PCR) Technique forcopy number was determined by comparison with a Quantification of Adenovirus Infectious Units: It wasstandard curve generated with serial dilutions (10 to done according to Esawy et al. [34], Abdo et al. [35].1

10 ) of a positive control plasmid (pCR2.1-TOPO, Adenovirus cell culture-PCR (CC-PCR) assay was6

Invetrogen, USA) containing full length of cDNA of performed on suspensions of infected Hep 2 cell line.rotavirus VP-6 genome. Set of primers, hex AA 1885and hex AA 1913 was used.

CC-RT-PCR for Quantification of Infectious Rotavirus 100 µl of inoculum is 1x10 CC- PCR units/ml (u/ml), AnParticles: It was done according to Abad et al. [27], adenovirus CC-PCR unit is defined as the reciprocalEl-Senousy et al. [28] and Ghazy et al. [29]. Rotavirus cell endpoint dilution detectable by CC- PCR.

this tissue culture assay using 100 µl of inoculum is 1x101

1X PCR buffer containing 0.2mg of Proteinase K/mL. After

PCR.

forward primer hex AA 1885and reverse primer hex AA

The detection limit in this tissue culture assay using1

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Confirmation of the RT-PCR and PCR Positivity by were 91.7% (22/24), 83.3% (20/24), 75% (18/24) and 45.8%Amplimer Sequencing: The RT-PCR products of selected (11/24) in raw sewage, after primary sedimentation,positive samples for rotavirus (4 raw sewage, 4 Nile water after secondary sedimentation and after final chlorinationand 8 clinical samples) and PCR products for adenovirus respectively (Tables 3 and 4).(8 raw sewage, 8 Nile water and 8 clinical samples)covering all seasons were sequenced. Fifty to one Prevalence of Human Adenoviruses in Water Samples:hundred µl of the RT-PCR or PCR products were purified The Prevalence of human adenoviruses in waterusing a high pure PCR products purification kit (Qiagen) samples collected from El-Giza water treatment plantfollowing the manufacturer's instructions. Cycle after different treatment steps from July 2009 to June 2011sequencing was performed on 1 to 7 ml of the purified were 66.7% (16/24), 58.3% (14/24), 37.5% (9/24) and 25%products with an ABI prism Big dye termination cycle (6/24) in raw Nile water, after sedimentation, after sandsequencing ready reaction kit (applied biosystem) using filtration and after final chlorination, respectivelythe same primers as in the PCR and following the (Table 5).manufacturer's instructions. The DNA was sequencedwith an ABI prism 310 automated DNA sequencer. Prevalence of Human Rotaviruses and Adenoviruses inSequence data from both strands of the PCR products Diarrheal Samples: 35 samples out of 110 samples werewere aligned and compared by using the clustalw and positive for rotaviruses, the highest prevalence ofblast programs (European bioinformatics institute). rotaviruses was detected during the winter season, while

Statistical Analysis: Mean and standard deviation were and the summer seasons. 24 samples out of 110 samplescalculated to determine the removal efficiency of treatment were positive for Adenoviruses. They were distributedprocesses in Zenin WWTP and El-Giza WTP and also to through the whole year (Fig. 1).determine the difference between the number of genomecopies and infectious units for both adenovirus and Number of Human Adenovirus Genome and Infectiousrotavirus. Units in Positive PCR Samples of Zenin WWTP: The

RESULTS to 6x10 DNA copies/l while, the number of adenovirus

Prevalence of Human Rotaviruses in Sewage Samples: in raw sewage samples. 0 to 1 log reduction wasThe prevalence percentages of human rotaviruses in observed after primary sedimentation, 0 to 2 logsewage samples collected from Zenin wastewater reduction was observed after secondary sedimentationtreatment plant after different treatment steps from July and 1 to 2 log reduction was observed after final chlorine2009 to June 2011 were 16.7% (4/ 24), 20.8% (5/24), 16.7% for adenovirus genome. 0 to 1 log reduction was(4/24) and 16.7% (4/24) in raw sewage, after primary observed after primary sedimentation, 0 to 2 logsedimentation, after secondary sedimentation and after reduction was observed after secondary sedimentationfinal chlorination respectively (Table 1). and 1 to 2 log reduction was observed after final chlorine

Prevalence of Human Rotaviruses in Water Samples: were observed between adenovirus genome andThe prevalence percentages of human rotaviruses in infectious units (Tables 6 and 7).water samples collected from El-Giza water treatment plantafter different treatment steps from July 2009 to June 2011 Number of Adenovirus Genome and Infectious Units inwere 29.2% (7/ 24), 25% (6/24), 20.8% (5/24) and 12.5% Positive PCR Samples of El-Giza WTP: The number of(3/24) in raw Nile water, after sedimentation, after human adenovirus genome ranged from 9x10 to 8x10sand filtration and after final chlorination, respectively DNA copies/l while, the number of adenovirus infectious(Table 2). units ranged from 8x10 to 9x10 CC-PCR unit/l in Nile water

Prevalence of Human Adenoviruses in Sewage Samples: sedimentation, 0 to 1 log reduction was observed afterThe Prevalence of human adenoviruses in sewage sand filtration and 0 to 2 log reduction was observedsamples collected from Zenin wastewater treatment plant after final chlorine for adenovirus genome. 0 to 1 logafter different treatment steps from July 2009 to June 2011 reduction was observed after sedimentation, 0 to 1 log

the lowest prevalence was detected during the spring

number of human adenovirus genome ranged from 9x107

infectious units ranged from 1x10 to 4x10 CC-PCR unit/l5

10

10

10

10

10

10

for adenovirus infectious units. 0 to 3 log difference10

5

4

samples. 0 to 1 log reduction was observed after10

10

10

10

10

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Table 1: Prevalence of human rotaviruses after different treatment steps of Zenin wastewater treatment plant (2009 - 2011)

Sampling date Raw sewage After primary sedimentation After Secondary sedimentation Chlorinated effluents

November 2009 +Ve +Ve -Ve +Ve

December 2009 - Ve - Ve +Ve +Ve

February 2010 - Ve +Ve +Ve - Ve

January 2011 +Ve +Ve -Ve -Ve

February 2011 +Ve +Ve +Ve +Ve

March 2011 +Ve +Ve +Ve +Ve

*All samples in other months were negative

Table 2: Prevalence of human rotaviruses after different treatment steps of El-Giza water treatment plant (2009 - 2011)

Sampling date Raw water After Sedimentation After Sand filtration Chlorinated effluents

November 2009 +Ve -Ve -Ve -Ve

December 2009 +Ve +Ve +Ve +Ve

January 2010 +Ve +Ve +Ve +Ve

February 2010 +Ve +Ve -Ve -Ve

January 2011 +Ve +Ve +Ve +Ve

February 2011 +Ve +Ve +Ve -Ve

March 2011 +Ve +Ve +Ve -Ve

*All samples in other months were negative

Table 3: Prevalence of human adenoviruses after different treatment steps of Zenin wastewater treatment plant (2009 - 2010)

Sampling date Raw sewage After primary sedimentation After Secondary sedimentation Chlorinated effluents

July 2010 +Ve -Ve -Ve -Ve

August 2010 -Ve -Ve -Ve -Ve

September 2010 +Ve +Ve -Ve -Ve

October 2010 +Ve +Ve +Ve -Ve

November 2010 +Ve +Ve +Ve +Ve

December 2010 +Ve +Ve +Ve +Ve

January 2011 +Ve +Ve +Ve +Ve

February 2011 +Ve +Ve +Ve +Ve

March 2011 +Ve +Ve +Ve +Ve

April 2011 +Ve +Ve +Ve -Ve

May 2011 +Ve +Ve +Ve +Ve

June 2011 +Ve +Ve +Ve +Ve

Table 4: Prevalence of human adenoviruses after different treatment steps of Zenin wastewater treatment plant (2010 - 2011)

Sampling date Raw sewage After primary sedimentation After Secondary sedimentation Chlorinated effluents

July 2009 +Ve +Ve +Ve -Ve

August 2009 -Ve -Ve -Ve -Ve

September 2009 +Ve +Ve +Ve -Ve

October 2009 +Ve +Ve Ve+ Ve+

November 2009 +Ve Ve+ Ve+ Ve-

December 2009 +Ve +Ve +Ve +Ve

January 2010 +Ve +Ve -Ve -Ve

February 2010 +Ve +Ve +Ve +Ve

March 2010 +Ve +Ve +Ve +Ve

April 2010 +Ve +Ve +Ve -Ve

May 2010 +Ve +Ve +Ve -Ve

June 2010 +Ve -Ve -Ve -Ve

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Table 5: Prevalence of human adenoviruses after different treatment steps of El-Giza water treatment plant (2009 - 2011)

Sampling date Raw water After Sedimentation After Sand filtration Chlorinated effluents

October 2009 +Ve +Ve +Ve - VeNovember 2009 + Ve +Ve +Ve - VeDecember 2009 + Ve + Ve + Ve + VeJanuary 2010 + Ve + Ve + Ve + VeFebruary 2010 + Ve + Ve + Ve + VeApril 2010 + Ve - Ve - Ve - VeMay 2010 + Ve + Ve - Ve - VeJune 2010 + Ve - Ve - Ve - VeAugust 2010 + Ve + Ve - Ve - VeOctober 2010 + Ve + Ve - Ve - VeNovember 2010 + Ve + Ve - Ve - VeDecember 2010 + Ve + Ve + Ve + VeJanuary 2011 + Ve + Ve + Ve + VeFebruary 2011 + Ve + Ve + Ve + VeMarch 2011 + Ve + Ve - Ve - VeApril 2011 + Ve + Ve + Ve - Ve

*All samples in other months were negative

Table 6: Number of DNA copies (DNA copies/l) and infectious units (CC-PCR unit/l) in samples positive for human adenovirus genome in (Zenin) wastewatertreatment plant (Jul.2009 - Jun.2010)

Raw sewage After primary sedimentation After Secondary sedimentation Chlorinated effluents----------------------------------------- ---------------------------------------- --------------------------------------- --------------------------------------

Sampling date* DNA copies/l CC-PCR unit/l DNA copies/l CC-PCR unit/l DNA copies/l CC-PCR unit/l DNA copies/l CC-PCR unit/l

July 2009 3x10 6x10 1x10 2x10 2x10 7x10 0 04 2 3 2 2

September 2009 1x10 5x10 2x10 4x10 4x10 7x10 0 04 2 3 2 2

October 2009 5x10 6x10 2x10 4x10 8x10 7x10 8x10 9x105 3 5 3 4 2 2

November 2009 4x10 3x10 8x10 1x10 9x10 7x10 0 05 3 4 2 2

December 2009 7x10 6x10 1x10 3x10 6x10 8x10 8x10 1x106 4 6 4 5 3 3 2

January 2010 3x10 2x10 2x10 1x10 6x10 4x10 5x10 5x106 4 6 4 6 3 4 2

February 2010 6x10 4x10 3x10 4x10 8x10 7x10 4x10 9x105 4 5 4 4 3 2

March 2010 8x10 4x10 2x10 8x10 5x10 4x10 1x10 05 4 5 3 4 2 2

April 2010 5x10 9x10 8x10 6x10 2x10 4x10 0 05 3 4 2 2

May 2010 6x10 9x10 2x10 3x10 2x10 2x10 0 05 3 4 2 2

June 2010 1x10 4x10 2x10 1x10 6x10 7x10 3x10 05 3 4 2 3 2

*All samples in other months were negative

Table 7: Number of DNA copies (DNA copies/l) and infectious units (CC-PCR unit/l) in samples positive for human adenovirus genome in (Zenin)wastewater treatment plant (Jul.2010 - Jun.2011)

Raw sewage After primary sedimentation After Secondary sedimentation Chlorinated effluents----------------------------------------- ---------------------------------------- --------------------------------------- --------------------------------------

Sampling date* DNA copies/l CC-PCR unit/l DNA copies/l CC-PCR unit/l DNA copies/l CC-PCR unit/l DNA copies/l CC-PCR unit/l

July 2010 9x10 1x10 0 0 0 0 0 0September 2010 8x10 5x10 2x10 1x10 0 0 0 03 2 2

October 2010 6x10 8x10 6x10 2x10 2x10 1x10 0 05 3 4 2 2 2

November 2010 6x10 2x10 6x10 2x10 9x10 8x10 4x10 04 4 3 3 2 2

December 2010 6x10 4x10 3x10 2x10 1x10 6x10 2x10 9x107 5 6 4 5 3 3

January 2011 5x10 7x10 3x10 3x10 4x10 1x10 4x10 1x106 4 5 3 4 3 2

February 2011 8x10 6x10 9x10 5x10 8x10 9x10 4x10 05 3 4 3 3 2

March 2011 5x10 9x10 8x10 6x10 6x10 7x10 4x10 05 3 4 3 3 2 2

April 2011 9x10 8x10 8x10 7x10 6x10 2x10 0 05 3 4 2 2

May 2011 5x10 6x10 3x10 2x10 2x10 1x10 2x10 1x105 4 4 3 3 2 2

June 2011 8x10 2x10 6x10 2x10 6x10 1x10 4x10 05 4 4 3 4 2 2

*All samples in other months were negative

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Fig. 1: Prevalence of human rotaviruses and adenoviruses in stool samples of children (< 5 years) with acute diarrheafrom May 2011 to April 2012

reduction was observed after sand filtration and 1 to 2 to 5x10 RNA copies/l while, the number of rotaviruslog reduction was observed after final chlorine for infectious units ranged from 2x10 to 1x10 CC-RT-PCR10

adenovirus infectious units. 0 to 3 log differences were unit/l in Nile water samples. 1 log reduction was10

observed between adenovirus genome and infectious observed after sedimentation, 0 to 1 log reduction wasunits (Tables 8 and 9). observed after sand filtration and 1 to 3 log reduction

Number of Human Rotavirus Genome and Infectious 0 to 1 log reduction was observed after sedimentation,Units in Positive PCR Samples of Zenin WWTP: The 0 to 1 log reduction was observed after sand filtrationnumber of human rotavirus genome ranged from 7x10 to and 1 to 3 log reduction was observed after final chlorine3

2x10 RNA copies/l while, the number of rotavirus for rotavirus infectious units. 0 to 2 log differences were7

infectious units ranged from 1x10 to 5x10 CC-RT-PCR observed between rotavirus genome and infectious units2 5

unit/l in raw sewage samples. 1 log reduction was (Table 11).10

observed after primary sedimentation, 1 to 2 log10

reduction was observed after secondary sedimentation Sequence Analysis: Sequence analysis of 24 PCRand 2 to 3 log reduction was observed after final chlorine positive adenovirus samples indicated that 19 samples10

for rotavirus genome. 0 to 1 log reduction was observed (6 raw sewage, 5 Nile water and 8 clinical samples) showed10

after primary sedimentation, 1 to 2 log reduction was similar sequences clustered with Human adenovirus 4110

observed after secondary sedimentation and 1 to 3 log strain 503804/KOL/2009 hexon gene with 94% homology.10

reduction was observed after final chlorine for It indicated also that 5 PCR positive adenovirus samplesrotavirus infectious units. 0 to 3 log differences were (2 raw sewage and 3 Nile water samples) showed similar10

observed between rotavirus genome and infectious units sequences clustered with Human adenovirus 2 strain(Table 10). N3773/TW/05-2 hexon gene with 98% homology.

Number of Human Rotavirus Genome and Infectious samples (4 raw sewage, 4 Nile water and 8 clinicalUnits in Positive PCR Samples of El-Giza WTP: The samples) showed that all the sequences clustered withnumber of human rotavirus genome ranged from 9x10 rotavirus VP6 with 98% homology.

5

4

10

10

10

was observed after final chlorine for rotavirus genome.10

10

10

10

Sequence analysis of the 16 RT-PCR positive rotavirus

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Table 8: Number of DNA copies (DNA copies/l) and infectious units (CC-PCR unit/l) in samples positive for human adenovirus genome in El-Giza watertreatment plant (October 2009 - September 2010)

Raw Nile water After sedimentation After Sand filtration After final Chlorine----------------------------------------- ---------------------------------------- --------------------------------------- --------------------------------------

Sampling date* DNA copies/l CC-PCR unit/l DNA copies/l CC-PCR unit/l DNA copies/l CC-PCR unit/l DNA copies/l CC-PCR unit/l

October 2009 7x10 1x10 8x10 1x10 6x10 1x10 0 04 2 3 2

November 2009 5x10 2x10 6x10 6x10 4x10 1x10 0 04 2 3 2

December 2009 6x10 9x10 8x10 8x10 9x10 1x10 6x10 05 4 4 3 3 2 2

January 2010 5x10 2x10 4x10 6x10 9x10 3x10 2x10 05 3 4 2 3 2 2

February 2010 4x10 2x10 1x10 1x10 3x10 9x10 1x10 04 2 4 2 3 3

April 2010 9x10 0 0 0 0 0 0 0May 2010 2x10 9x10 2x10 0 0 0 0 02

June 2010 8x10 0 0 0 0 0 0 0August 2010 9x10 8x10 8x10 6x10 0 0 0 02

*All samples in other months were negative

Table 9: Number of DNA copies (DNA copies/l) and infectious units (CC-PCR unit/l) in samples positive for human adenovirus genome in El-Giza watertreatment plant (October 2010 – September 2011)

Raw Nile water After sedimentation After Sand filtration After final Chlorine----------------------------------------- ---------------------------------------- --------------------------------------- --------------------------------------

Sampling date* DNA copies/l CC-PCR unit/l DNA copies/l CC-PCR unit/l DNA copies/l CC-PCR unit/l DNA copies/l CC-PCR unit/l

October 2010 9x10 1x10 8x10 5x10 0 0 0 02 2

November 2010 5x10 2x10 7x10 6x10 0 0 0 02 2

December 2010 8x10 8x10 5x10 9x10 6x10 9x10 7x10 05 3 4 2 3 2

January 2011 4x10 7x10 1x10 9x10 9x10 8x10 7x10 04 3 3 2 2

February 2011 7x10 5x10 8x10 8x10 2x10 6x10 3x10 04 2 3 3 2

March 2011 6x10 7x10 9x10 2x10 0 0 0 02 2

April 2011 8x10 9x10 8x10 1x10 0 0 0 02

*All samples in other months were negative

Table 10: Number of RNA copies (RNA copies/l) and infectious units (CC-RT-PCR unit/l) in samples positive for human rotavirus genome in (Zenin) wastewater treatment plant (Jul.2009 -

Jun.2011)

Raw sewage After primary sedimentation After Secondary sedimentation Chlorinated effluents

-------------------------------------------- ------------------------------------------ ------------------------------------------- ----------------------------------------------

Sampling date * RNA copies/l CC-RT-PCR unit/l RNA copies/l CC-RT-PCR unit/l RNA copies/l CC-RT-PCR unit/l RNA copies/l CC-RT-PCR unit/l

November 2009 2x10 5x10 9x10 6x10 0 0 2x10 6x107 5 6 4 3

December 2009 0 0 0 0 8x10 1x10 9x10 1x105 3 3

February 2010 0 0 8x10 7x10 9x10 1x10 0 05 4 3 3

January 2011 7x10 1x10 8x10 1x10 0 0 0 03 2 2

February 2011 3x10 2x10 5x10 1x10 3x10 3x10 9x10 3x106 5 5 4 4 2 2

March 2011 8x10 9x10 4x10 7x10 4x10 8x10 9x10 05 2 4 2 3

*All samples in other months were negative

Table 11: Number of RNA copies (RNA copies/l) and infectious units (CC-RT-PCR unit/l) in samples positive for human rotavirus genome in El-Giza water treatment plant (October 2009 –

September 2011)

Raw Nile water After sedimentation After Sand filtration After final Chlorine

-------------------------------------------- ------------------------------------------ ------------------------------------------- ----------------------------------------------

Sampling date* RNA copies/l CC-RT-PCR unit/l RNA copies/l CC-RT-PCR unit/l RNA copies/l CC-RT-PCR unit/l RNA copies/l CC-RT-PCR unit/l

November 2009 9x10 2x10 0 0 0 0 0 0

December 2009 5x10 1x10 3x10 2x10 2x10 1x10 8x10 05 4 4 3 4 3

January 2010 2x10 7x10 9x10 1x10 6x10 9x10 5x10 05 3 4 3 3 2 2

February 2010 8x10 1x10 9x10 1x10 0 0 0 02 2

January 2011 4x10 1x10 6x10 1x10 9x10 4x10 8x10 05 4 4 3 3 2 2

February 2011 9x10 1x10 3x10 4x10 4x10 1x10 0 04 2 3 2

March 2011 2x10 1x10 9x10 5x10 1x10 1x10 0 03 2 2 2

*All samples in other months were negative

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DISCUSSION Rotavirus (Group A) was the most prevalent RNA

The prevalence of human rotaviruses in sewage and drinking water in Cairo, Egypt, followed by HAV,samples collected from raw sewage of Zenin wastewater astrovirus and enterovirus and finally, norovirusestreatment plant from July 2009 to June 2011 was 16.7% respectively [19, 20]. In recent studies, rotavirus still has(4/ 24). The prevalence of human adenoviruses in the high prevalence in Egyptian Delta and Cairo [39, 40] whilesame raw sewage samples was 91.7% (22/24). The higher norovirus still has low prevalence in Egyptian naturallyprevalence of adenovirus in raw sewage samples is contaminated irrigation water and fresh produce [41]. Inobvious. It may return to the peak of rotavirus in winter our study, Adenovirus was detected in all samplesand autumn months which is clear in our results. The positive for rotavirus except in treated effluent of Zeninprevalence of adenovirus was all the year without any WWTP in November 2009. Rotavirus was not detected inobserved peak. The number of adenovirus genome ranged 18 samples raw sewage of Zenin WWTP and 8 samples offrom 9x10 to 6x10 DNA copies/l while, the number of its treated effluents, 10 samples of Nile water of El-Giza7

adenovirus infectious units ranged from 1x10 to 4x10 WTP and 3 samples of its drinking water.5

CC-PCR unit/l in raw sewage samples. The number of Zenin wastewater treatment plant which uses anrotavirus genome ranged from 7x10 to 2x10 RNA copies/l activated sludge treatment technology, the most prevalent3 7

while, the number of rotavirus infectious units ranged treatment technology in Egypt was chosen as a model forfrom 1x10 to 5x10 CC-RT-PCR unit/l in raw sewage Egyptian wastewater treatment plants using an activated2 5

samples. Rotavirus could be only detected and quantified sludge treatment technology. In Zenin WWTP, 0 to 1as genome copies and infectious units in the peak months log reduction was observed after primary sedimentation,of the virus in autumn and winter. Adenovirus genome 0 to 2 log reduction was observed after secondarycopies and infectious units were higher in winter than in sedimentation and 1 to 2 log reduction was observedthe other months but considerable numbers of both of after final chlorine for adenovirus genome. 0 to 1 logthem were quantified all the year (except August). On the reduction was observed after primary sedimentation, 0 toother hand, again this observation was confirmed by the 2 log reduction was observed after secondarydata of water samples of El-Giza water treatment plant. sedimentation and 1 to 2 log reduction was observedThe prevalence of rotaviruses in Nile water samples after final chlorine for adenovirus infectious units. 1 logcollected from July 2009 to June 2011 was 29.2% (7/ 24). reduction was observed after primary sedimentation, 1 toThe prevalence of adenoviruses in the same Nile water 2 log reduction was observed after secondarysamples was 66.7% (16/24). Rotavirus was detected only sedimentation and 2 to 3 log reduction was observedin autumn and winter months while adenovirus was after final chlorine for rotavirus genome. 0 to 1 logdetected all the year. The number of adenovirus genome reduction was observed after primary sedimentation, 1 toranged from 9x10 to 8x10 DNA copies/l while, the number 2 log reduction was observed after secondary5

of adenovirus infectious units ranged from 8x10 to 9x10 sedimentation and 1 to 3 log reduction was observed4

CC-PCR unit/l in Nile water samples. The number of after final chlorine for rotavirus infectious units. In El-Giza,rotavirus genome ranged from 9x10 to 5x10 RNA copies/l water treatment plant, the treatment facilities are5

while, the number of rotavirus infectious units ranged pre-oxidation with Cl , coagulation-settling,from 2x10 to 1x10 CC-RT-PCR unit/l in Nile water samples. sedimentation, filtration (rapid sand filters) and final Cl4

Again the higher rotavirus genome copies and infectious addition which is the most prevalent water treatmentunits were observed in autumn and winter without any facilities in Egypt was chosen as a model of Egyptianpresence for rotavirus in spring and summer while water treatment plants. In El-Giza WTP, 0 to 1 logadenovirus genome copies and infectious units could be reduction was observed after sedimentation, 0 to 1 logquantified all the year (except July). Our results agree reduction was observed after sand filtration and 0 to 2with the study results of Villena et al. [19] and log reduction was observed after final chlorine forEl-Senousy et al. [20], who reported that the peak of adenovirus genome. 0 to 1 log reduction was observedrotavirus incidence in Cairo raw sewage and river Nile after sedimentation, 0 to 1 log reduction was observedwater samples is in autumn and winter months. On the after sand filtration and 1 to 2 log reduction wasother hand in our results, adenoviruses showed no peak observed after final chlorine for adenovirus infectiousmonths for prevalence in the two years survey. This was units. 1 log reduction was observed after sedimentation,also confirmed by several reports [8, 10, 36-38]. 0 to 1 log reduction was observed after sand filtration

enteric viruses in raw sewage, treated effluents, Nile water

10

10

10

10

10

10

10

10

10

10

10

10

2

2

10

10

10

10

10

10

10

10

World Appl. Sci. J., 27 (10): 1235-1247, 2013

1244

and 1 to 3 log reduction was observed after final chlorine process and then after the whole treatment processes in10

for rotavirus genome. 0 to 1 log reduction was observed10

after sedimentation, 0 to 1 log reduction was observed10

after sand filtration and 1 to 3 log reduction was10

observed after final chlorine for rotavirus infectious units.Quantitatively, The means of adenovirus genome andinfectious units removals were 0.77±0.4 log and 0.6±0.510

log respectively while the means of rotavirus genome10

and infectious units removals were 1±0 log and 0.75±0.510

log respectively after the primary sedimentation of Zenin10

WWTP. The means of adenovirus genome and infectiousunits removals were 1.2±0.6 log and 0.95±0.4 log ,10 10

respectively while the means of rotavirus genome andinfectious units removals were 1.5±0.6 log and 1.25±0.510

log respectively after the secondary sedimentation of10

Zenin WWTP. The means of adenovirus genome andinfectious units removals were 1.65±0.6 log and 1.4±0.510

log respectively while the means of rotavirus genome10

and infectious units removals were 2.25±0.5 log and10

1.75±1 log respectively after the final chlorine of Zenin10

WWTP. The means of adenovirus genome and infectiousunits removals were 3.5±1.1 log and 2.8±0.8 log10 10

respectively while the means of rotavirus genome andinfectious units removals were 3.75±0.5 log and 3±1.210

log respectively after the whole treatment processes of10

Zenin WWTP.The means of adenovirus genome and infectious

units removals were 0.9±0.3 log and 0.86±0.4 log10 10

respectively while the means of rotavirus genome andinfectious units removals were 1±0 log and 0.86±0.4 log10 10

respectively after the sedimentation of El-Giza WTP. Themeans of adenovirus genome and infectious unitsremovals were 0.9±0.3 log and 0.69±0.5 log ,10 10

respectively while the means of rotavirus genome andinfectious units removals were 0.67±0.5 log and 0.5±0.510

log respectively after the sand filtration of El-Giza WTP.10

The means of adenovirus genome and infectious unitsremovals were 1.1±0.6 log and 1.3±0.5 log respectively10 10

while the means of rotavirus genome and infectious unitsremovals were 1.8±0.8 log and 1.8±0.8 log respectively10 10

after the final chlorine of El-Giza WTP. The means ofadenovirus genome and infectious units removals were2.3±0.9 log and 2.1±0.9 log respectively while the10 10

means of rotavirus genome and infectious units removalswere 2.86±1.1 log and 2.57±1.1 log respectively after10 10

the whole treatment processes of El-Giza WTP.The means of adenovirus genome and infectious

units removals were less than the means of rotavirusgenome and infectious units removals after each treatment

Zenin WWTP and in El-Giza WTP (except sand filtration).So, both, adenovirus and rotavirus are resistant totreatment processes in water and wastewater treatmentplants with higher removal for rotavirus genome andinfectious units and higher resistance for adenovirusgenome and infectious units in water and wastewatertreatment plants. Detection of rotavirus and adenovirusgenome or infectious units in treated effluents anddrinking water samples indicates the resistance ofrotavirus and adenovirus to treatment processes(especially chlorine as a disinfectant) in the water andwastewater treatment plants. The high stability ofrotavirus in front of disinfectants (free chlorine, copper,silver) was previously studied at laboratory scale [42, 43]and in field scale [20]. This may explain the high positivityof rotavirus genome in treated sewage effluents and indrinking water samples. El Senousy et al. [20] reportedthat rotavirus is the most resistant enteric virus totreatment processes in 3 Egyptian WWTPs and 3Egyptian WTPs among all RNA enteric viruses studied(HAV, astrovirus, enterovirus and norovirus). In ourstudy the resistance of adenovirus to water andwastewater treatment processes had been proved. Otherreports confirmed the high resistance of adenovirus totreatment processes in water and wastewater treatmentplants [10, 44-46]. The resistant of both adenovirus androtavirus to treatment processes in both water andwastewater treatment plants represents a human healthrisk. In our results the presence of genome copies andinfectious units of both adenovirus and rotavirus in thewastewater chlorinated effluents represents a badperformance of the WTP in the removal of viruses.However adenovirus genome copies and infectious unitswere detected and quantified all the year. They maycontaminate irrigation water, Nile water or even drinkingwater. Also, the presence of genome copies of bothadenovirus and rotavirus in drinking water even withoutinfectivity represents a bad performance of the WTP inthe removal of viruses.

In Zenin WWTP, 0 to 3 log differences were10

observed between genome and infectious units foradenovirus and rotavirus which the means of differenceswere 1.5±0.6 log and 1.5±0.7 log for adenovirus and10 10

rotavirus respectively. Also, in El-Giza WTP 0 to 3 log10

(with mean 1.3±0.8 log ) differences were observed10

between genome and infectious units for adenoviruswhile 0 to 2 log (with mean 1.1±0.6 log ) differences10 10

were observed between genome and infectious units forrotavirus. It indicates comparable survival of the genomeafter the loss of infectivity.

World Appl. Sci. J., 27 (10): 1235-1247, 2013

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In our result rotavirus was the most prevalent 31.8% 2. Carducci, A., R. Battistini, E. Rovini and M. Verani,(35/110) in diarrheal samples while adenoviruses weredetected in 18.1% (20/110) of the samples. Adenovirusesare second as leading causes of childhood gastroenteritisafter rotaviruses worldwide. Rotavirus is the mostcommon cause of acute diarrhea in infants and youngchildren, which causes approximately 111 millionepisodes of gastroenteritis and 352,000–592,000 deathsin children of less than 5-years-old worldwide each year [47]. In our results, the peak of rotavirus indiarrheal samples was very obvious in autumn and wintermonths. Adenoviruses showed no peak for prevalence indiarrheal samples.

Sequence analysis indicated that 19 samples(6 raw sewage, 5 Nile water and 8 clinical samples) showedsimilar sequences clustered with Human adenovirus 41strain 503804/KOL/2009 hexon gene with 94% homologyand 5 samples (2 raw sewage and 3 Nile water samples)showed similar sequences clustered with Humanadenovirus 2 strain N3773/TW/05-2 hexon gene with 98% 6. Fong, T. and E. Lipp, 2005. Enteric viruses of humanshomology. It indicated that, among adenoviruses, humanenteric adenovirus 41 were the most frequent type inEgyptian diarrheal, sewage and water samples.Epidemiological and environmental studies usingmolecular tools reported that HAdV-41 is clearly the mostprevalent enteric strain [47]. Our results also indicatedthat some sewage and water samples had only nonenteric adenovirus (Human adenovirus 2). These resultsmay indicate that human adenoviruses and not onlyhuman enteric adenoviruses could be suggested as acandidate viral indicator of human viral contaminationin Egyptian sewage and water samples due to its highprevalence, stability in the environment and its humanorigin. Due to their prevalence and their survivalcharacteristics, HAdVs are part of the 12 microorganismslisted on the Candidate Contaminant List (CCL3)established by the US Environmental Protection Agency[48].

More studies are still needed to confirm the best viral N.A. Gimenez and R. Girones, 2006. Identification ofindicator candidate in Egyptian sewage, water and foodsamples.

Human adenoviruses and not only human entericadenoviruses could be suggested as a candidate viralindicator of human viral contamination in Egyptiansewage and water samples.

REFERENCES

1. Leclerc, H., D.A.A. Mossel, S.C. Edberg andC.B. Struijk, 2001. Advances in the bacteriology ofthe coliform group: their suitability as markers ofmicrobial water safety. Annu. Rev. Microbiol.,55: 201-234.

2009. Viral Removal by Wastewater Treatment:Monitoring of Indicators and Pathogens. FoodEnviron. Virol., 1: 85-91.

3. Carducci, A., I. Federigi and M. Verani, 2013. VirusOccupational Exposure in Solid Waste ProcessingFacilities. Ann Occup Hyg doi: 10.1093/annhyg/met043.

4. Bosch, A., 1998. Human enteric viruses in thewater environment: a minireview.nternatl. Microbiol.,1: 191-196.

5. Yoder, J.S., M.C. Hlavsa, G.F. Craun, V. Hill, P.A. Tu,L.A. Hicks, N.T. Alexander, R.L. Calderno, S.L. Royand M.J. Beach, 2008. Surveillance for waterbornedisease and outbreaks associated with recreationalwater use and other aquatic facility – associatedhealth events-United States, 2005-2006. MMWRSurveill. Summ., 57: 1-29.

and animals in aquatic environments: health risks,detection and potential water quality assessmenttools. Microbiol. Mol. Biol. Rev., 69: 357-371.

7. Benkõ, M., B. Harrach and W.C. Russell, 2000. FamilyAdenoviridae. In Virus Taxonomy. Seventh Report ofthe International Committee on Taxonomy of Viruses,Eds, Van Regenmortel, M., C.M. Fauquet, D.H.L.Bishop, E.B. Carstens, M.K. Estes, S.M. Lemon,J. Maniloff, M.A. Mayo, D.J. McGeoch, C.R. Pringleand R.B. Wickner. San Diego: Academic Press,pp: 227-238.

8. Fong, T., M. Phanikumar, I. Xagoraraki and J. Rose,2010. Quantitative Detection of Human Adenovirusesin Wastewater and Combined Sewer OverflowsInfluencing a Michigan River. Appl. Environ.Microbiol., 76: 715-723.

9. Hundesa, A., C. Maluquer, S. Bofill Mas,

Human and Animal Adenoviruses andPolyomaviruses for Determination of Sources ofFecal Contamination in the Environment. Appl.Environ. Microbiol., 72: 7886-7893.

10. Pina, S., M. Puig, F. Lucena, J. Jofre and R. Girones,1998. Viral pollution in the environment and inshellfish: human adenovirus detection by PCR as anindex of human viruses. Appl. Environ. Microbiol.,64: 3376-3382.

11. Carter, M.J., 2005. Enterically infecting viruses:pathogenicity, transmission and significance forfood and waterborne infection. J. Appl. Microbiol.,6: 1354-1380.

World Appl. Sci. J., 27 (10): 1235-1247, 2013

1246

12. Leclerc, H., L. Schwartzbrod and E. Dei-Cas, 2002. 24. Iturriza Gomara, M., C. Wong, S. Blome, U.Microbial agents associated with waterborne. CriticalReviews in Microbiology, 28: 371-409.

13. Payment, P., R. Plante and P. Cejka, 2001. Removal ofindicator bacteria, human enteric viruses, Giardiacysts and Cryptosporidium oocysts at largewastewater primary treatment facility. Can. J.Microbiol., 47: 188-193.

14. Guzmán, C., J. Jofre, A.R. Blanch and F. Lucena, 2007.Development of a feasible method to extract somaticcoliphages from sludge, soil and treated biowaste.J. Virol Methods, 144: 41-48.

15. Decreto Legislativo n. 31/2001. Attuazionedelladirettiva 98/83/CE relativa alla qualità delle acquedestinate al consumo umano. Gazz Uff, 52, 3.03.2001-Suppl Ordinario, 41.

16. Fewtrell, L. and J. Bartram, 2001. Water Quality:Guidelines Standards Health. Assessment of RiskManagement for Water Related Infection Disease.World Health Organization Series, IWA Publishing,London, pp: 61-88.

17. Verani, M., B. Casini, R. Battistini, F. Pizzi, E. Roviniand A. Carducci, 2006. One-year monthly monitoringof Torque teno virus (TTV) in river water in Italy.Water Sci. Technol., 54: 191-195.

18. Griffin, J.S., J.D. Plummer and S.C. Long, 2008. Torqueteno virus: An improved indicator for viral pathogensin drinking waters. Virology Journal, 5: 112-117.

19. Villena, C., W.M. El-Senousy, F.X. Abad, R.M. Pintóand A. Bosch, 2003. Group A rotavirus in sewagesamples from Barcelona and Cairo: Emergence ofunusual genotypes. Appl. Environ. Microbiol.,69: 3883-3891.

20. El-Senousy, W., R. Pintó and A. Bosch, 2004.Epidemiology of human enteric viruses in the Cairowater environment. In the Proceedings of the 1st

International Conference of Environmental ResearchDivision on Sustainable Development EnvironmentalChallenges Facing Egypt, pp: 72-73.

21. Smith, E.M. and C.P. Gerba, 1982. Development of amethod for detection of human rotavirus in water andsewage. Appl. Environ. Microbiol., 43: 1440-1450.

22. Rose, J.B., S.N. Singh, C.P. Gerba and L.M. Kelley,1984. Comparison of microporous filters forconcentration of viruses from wastewater. Appl.Environ. Microbiol., 47: 989-992.

23. Katzenelson, E., B. Fattal and T. Hostovesky, 1976.Organic flocculation: an efficient second-stepconcentration method for the detection of viruses intap water. Appl. Environ. Microbiol., 32: 838-839.

Desselberger and J. Gray, 2002. Molecularcharacterization of VP6 genes of human rotavirusisolates: Correlation of genogroups with subgroupsand evidence of independent segregation. J. Virol.,76: 6596-6601.

25. Gallimore, C.I., C. Taylor, A.R. Genney, A.J. Cant,A. Galloway, M. Iturriza-Gomara and J.J. Gray, 2006.Journal of Clinical Microbiology, 44: 395-399.

26. Kang, G., M. Iturriza-Gomara, J.G. Wheeler, P. Crystal,B. Monica, S. Ramani, B. Primrose, P.D. Moses,C.I. Gallimore, D.W. Brown and J. Gray, 2004.Quantitation of Group A Rotavirus by Real-TimeReverse-Transcription-Polymerase Chain Reaction.J. med. Virol., 73: 118-122.

27. Abad, F.X., R.M. Pintó, C. Villena, R. Gajardo andA. Bosch, 1997. Astrovirus survival in drinkingwater. Appl. Environ. Microbiol., 63: 3119-3122.

28. El-Senousy, W.M., S. Guix, I. Abid, R.M. Pintó andA. Bosch, 2007. Removal of Astrovirus from Waterand Sewage Treatment Plants, Evaluated by aCompetitive Reverse Transcription-PCR. Appl.Environ. Microbiol., 73: 164-167.

29. Ghazy, M.M.E., W.M. El-Senousy, A.M. Abdel-Aatty, B. Hegazy and M. Kamel, 2008. Performanceevaluation of a waste stabilization pond in a ruralarea in Egypt. American Journal of EnvironmentalSciences, 4: 316-326.

30. Kapperud, G., T. Vardnund, E. Skjerve, E, Hornes andT.E. Michaelsen, 1993. Detection of pathogenicYersinia enterocolitica in foods and water byimmunomagnetic separation, nested polymerasechain reaction and colorimetric detection of amplifiedDNA. Appl. Environ. Microbiol., 59: 2938-2944.

31. Estrada, C.S.M.L., L.D.C. Velasquez, S.D. Genaro andA.M.S.D. Guzman, 2007. Comparison of DNAextraction methods for pathogenic Yersiniaenterocolitica detection from meat food by nestedPCR. Food Research International, 40: 637-642.

32. Puig, M., J. Jofre, F. Lucena, A. Allard, G. Wadelland R. Girones, 1994. Detection of adenovirusesand enteroviruses in polluted waters by nestedPCR amplification. Appl. Environ. Microbiol.,60: 2963-2970.

33. Kohdera, U., M. Kino and M. Ito, 2006. Detection ofAdenovirus DNA in Throat Swabs and Blood bySYBR Green Real-Time PCR Assay in Patients withAdenovirus-Associated Tonsillitis. Jpn. J. Infect.Dis., 59: 394-396.

World Appl. Sci. J., 27 (10): 1235-1247, 2013

1247

34. Esawy, M.A., E.F. Ahmed, W.A. Helmy, 42. Abad, F.X., R.M. Pintó, J.M. Diez and A. Bosch,N.M. Mansour, W.M. El-Senousy and M.M. El-Safty, 1994. Disinfection of human enteric viruses in water2011. Production of a halophilic levansucrase from by copper and silver in combination with low levelsnovel honey Bacillus subtilis isolates capable of of chlorine. Appl. Environ. Microbiol., 60: 2377-2383.producing antiviral levans. Carbohydrate Polymers, 43. Bosch, A., J.M. Diez and F.X. Abad, 1993.86: 823-830. Disinfection of human enteric viruses in water by

35. Abdo, S.M., M.H. Hetta, W.M. El-Senousy, copper:silver and reduced levels of chlorine. Wat.F.A. Samhan, R.A. Salah El Din and G.H. Ali, 2012. Sci. Tech., 27: 351-356.Antiviral activity of freshwater algae. Journal of 44. Muniain-Mujika, I., M. Calvo, R. Girones andApplied Pharmaceutical Science, 2: 21-25. F. Lucena, 2000. Viral contamination of shellfish:

36. Griffin, D.W., E.K. Lipp, M.R. McLaughlin and evaluation of methods and analysis ofJ.B. Rose, 2001. Marine recreation and public health bacteriophages and human viruses. J. Virol. Meth.,microbiology: quest for the ideal indicator. 89: 109-118.Bioscience, 51: 817-825. 45. Muniain-Mujika, I., M. Calvo, F. Lucena and

37. Jiang, S., H. Dezfulian and W. Chu, 2005. Real-time R. Girones, 2003. Comparative analysis of viralquantitative PCR for enteric adenovirus serotype pathogens and potential indicators in shellfish.40 in environmental waters. Can. J. Microbiol., International Journal of Food Microbiology,51: 393-398. 83: 75-85.

38. Katayama, H., E. Haramoto, K. Oguma, H. Yamashita, 46. Serracca, L., M. Verani, R. Battistini, I. Rossini,A. Tajima, H. Nakajima and S. Ohgaki, 2008. One-year A. Carducci and C. Ercolini, 2010. Evaluation ofmonthly quantitative survey of noroviruses, Adenovirus and E. Coli as indicators for humanenteroviruses and adenoviruses in wastewater enteric viruses presence in mussels produced in Lacollected from six plants in Japan. Water Res., Spezia Gulf (Italy). Letters in Applied Microbiology,42: 1441-1448. 50: 462-467.

39. El-Senousy, W.M. and E.M. El-Mahdy, 2009. 47. Ogorzaly, L., S. Bonot, B. El Moualij, W. Zorzi andDetection and genotyping of rotaviruses in water H. Cauchie, 2013. Development of a quantitativetreatment plants of El-Dakahlia Governorate. immunocapture real-time PCR assay for detectingEgyptian Journal of Biotechnology, 31: 25-34. structurally intact adenoviral particles in water. J.

40. El-Senousy, W.M., Y.E. Shaheen, A.B. Barakat, Virol. Meth., 194: 235-241.H.E. Ghanem, A.E. El-Hakim and S.M. Ameen, 2013. 48. United States Environmental Protection AgencyMolecular cloning and immunogenicity evaluation of (EPA), 2009. Final Contaminant Candidate List 3rotavirus structural proteins as candidate vaccine. Microbes: PCCL to CCL Process, pp: 1-110.International Journal of Biological Macromolecules,59: 67-71.

41. El-Senousy, W.M., M.I. Costafreda, R.M. Pinto andA. Bosch, 2013. Method validation for norovirusdetection in naturally contaminated irrigation waterand fresh produce. International Journal of FoodMicrobiology, 167: 74-79.