Waled M.

El-Senousy* PREVALENCE OF ROTAVIRUSES AND

NOROVIRUSES IN GROUND WATER
Nagwa M. Sidkey**
OF SOME RURAL AREAS IN EL-GIZA
Amel S.M. Abu Senna** GOVERNORATE, EGYPT
Nermine N. Abed**
Seham F. Hasan**
Rotaviruses and noroviruses the causative agents of
gastroenteritis were investigated in some well water which
*Corresponding author used in drinking and irrigation in some Egyptian rural areas
*Water Pollution Research Department, (Nahya and Saft Al laban) using RT-PCR. Through one year
National Research Centre survey, the results showed that rotaviruses were detected in
(NRC), El-Bohouth st., Dokki, 4/12 (33.3%) samples of well water in Nahya village while
Giza, Egypt, they were detected in 3/12 (25%) samples of well water in
Saft Al laban village. Neither noroviruses genogroup I (GGI)
**Botany and Microbiology Dept.,
nor noroviruses genogroup II (GGII) were detected in the well
Faculty of Science, Al-Azhar
water of both sites all over the year. Rotaviruses are more
University (for girls),
frequent than noroviruses in ground water of rural areas of
Nasr City, Cairo
El-Giza Governorate.

INTRODUCTION human and animal populations (Yates et al.,

Viruses transmitted via the faecal–oral 1985; Metcalf et al., 1995; Muscillo et al.,
route are generally nonenveloped and thus 1997; Koopmans et al., 2002 & La Rosa
very stable in the environment (Rzezutka et al., 2007). Human exposure to even low
and Cook, 2004). These viruses cannot levels of these pathogenic viruses in the
always be effectively eliminated by current environment, such as rotavirus (RV) and
methods of sewage treatment (Vantarakis norovirus (NoV), can cause infection and
and Papapetropoulou, 1999; Thompson et disease (Lindesmith et al., 2003 & Teunis et
al., 2003; Van Heerden et al., 2003; Van al., 2008).
den Berg et al., 2005; & El-Senousy et Group A rotaviruses are the most important
al., 2013a & Béji-Hamza et al., 2014) and viral agents that cause severe dehydrating
consequently cause viral contamination gastroenteritis in children under the age of
of the environment from treated as well five globally (Parashar et al., 2009). The
as untreated wastewater. Other examples World Health Organization (WHO) global
of indirect routes are run-off from manure rotavirus surveillance network estimates that
used in agriculture. There is also direct the annual rotavirus-associated mortality is
faecal contamination of the environment approximately 453,000 worldwide, of which
from humans and animals, for example by 95% occur in children younger than five
bathers or by defecation of free-range or years (Tate et al., 2012). Noroviruses are
wild animals onto soil or surface waters. the second most common cause of severe
The resulting viral contamination of sea and gastroenteritis in children less than five years
coastal water, rivers and other surface water, of age in both developing and industrialized
groundwater, and irrigated vegetables and nations, preceded only by rotaviruses.
fruit is associated with subsequent risks of NoVs are responsible for ~12 % children
reintroduction of the viral pathogens into less than 5 years of age hospitalized for

The New Egyptian Journal of Medicine Vol.:48; No.: 3 Supplement 1st March 2013 18
severe gastroenteritis worldwide. Each year, polyethylene glycol (PEG) was performed
NoVs cause approximately 900,000 cases according to El-Senousy et al. (2013b).
of pediatric gastroenteritis in industrialized Viral nucleic acid extraction
nations and at least 1.1 million episodes and It was done using BIOZOL Total RNA
218,000 deaths in developing nations (Patel Extraction reagent (BioFlux) according to
et al., 2008). The objective of this study the manufacturer’s instructions.
is to compare the prevalence of group A
rotaviruses and noroviruses GGI and GGII Semi-nested RT- PCR and gel
in some well water of rural areas in El-Giza electrophoresis for the detection of
Governorate in Egypt. noroviruses

Materials and Methods Viral RNA of the capsid gene was

amplified with RT-PCR method. Extracted
Collection of Samples: RNA samples were heated to 99οC for 5
From March 2012 to February 2013, a total min and immediately placed on ice. 5μl of
of 24 ground water samples were collected the heated RNA extract was mixed with 5μl
from two villages in El Giza Governorate, of the reaction mixture containing 1x RT-
Nahya and Saft Al Laban. Twenty liters of buffer (Fermentas, EU), 0.2 mM dNTP’s
each sample were collected monthly from (Fermentus, EU), 1 μm of primer , 100 U
one well in Nahya and other well in Saft Al of Reverse Transcriptase (Fermentas, EU).
Laban. The primers used in the RT were GI-SKR
for Norovirus GI and G2-SKR for Norovirus
Concentration of ground water samples
GII according to kojima et al., (2002) &
Adsorption/elution technique (APHA, kageyama et al., (2003). The RT profile
1998) was used for concentration of large was run with 25˚C for 10 min., 42˚C for 1
volumes of water. Virus particles are hr., 99C for 5min, and 5˚C for 5min. Ten
negatively charged at pH 7.0, and for the μl of the cDNA sample was mixed with
water passing through an electronegatively fifteen μl of the reaction mixture containing
charged filter nitrocellulose membranes 1x PCR-buffer, 2.5 U of DNA polymerase
(Shleicher and Schuell, 0.45 μm pore size (Fermentas, EU), 0.2 mM dNTP’s, 1 μm of
and 142 mm diameter filter series). The water primers forward and reverse. the primers
samples were acidified (approximately 3.5) added in the first PCR were COG1F for NoV
to alter the charge of the viral particle before GI and COG2F for NoV GII (kojima, et al.,
filtration so that the virus will be adsorbed to 2002 & kageyama et al., 2003). The PCR
the filter and to enhancing the viral adsorption profile was run as followes, 1 cycle of 94˚C
the AlCl3 was added before acidification and for 3 min, 40 cycles of 94˚C for 1 min., 50˚C
filtration of the samples. An eluate 3% beef for 1 min., and 72˚C for 2 min., and 1 cycle
extract (Lab-Limco powder, Oxoid, UK) of 72˚C for 15 min.
at alkaline pH ~9.5 is left in contact with
The PCR products were amplified with
the filter to allow the viruses to return into
seminested-PCR, in which 2 μl from the
solution (Smith and Gerba, 1982 & Rose
first PCR product were mixed with 1x
et al, 1984). After primary concentration
PCR-buffer, 2.5 U of DNA polymerase,
the secondary step concentration using
The New Egyptian Journal of Medicine Vol.:48; No.: 3 Supplement 1st March 2013 19
0.2 mM dNTP’s, 1 μm of primers and were incubated for 60 min. at 50οC for the
the two primers added in the nested-PCR RT reaction. Five μl of the RT product were
were G1SKF and GI-SKR for NV GI, and added to a final volume of 50 μl of the PCR
G2SKF and G2-SKR for NV GII according reaction mix containing 5 μl of the PCR
to kojima et al., (2002) & kageyama et al., buffer (Fermentas-EU), 2 mM MgCl2, 0.2
(2003). The length of products from the mM of each dNTP,s, 1 μm of each primer and
seminested-PCR was 330 bp for NV GI and 2.5 U of the Taq DNA polymerase enzyme
344 bp for NV GII. The seminested-PCR (Fermentas-EU). After a denaturation step of
profile was the same as for the first PCR 95°C for 3 min, 40 cycles of amplification at
protocol. Ten μl from the seminested-PCR 94°C for 1 min, 50°C for 1 min, and 72°C for
product were analyzed by 3% agarose gel 1 min. were performed with a final extension
electrophoresis and visualized by ethidium of 72°C for 10 min.
bromide staining. The nested PCR involved adding 2 μl
Nested RT-PCR and gel electrophoresis of first-round PCR product to a 48μl PCR
for the detection of rotavirus group A mix containing 10 mM Tris (pH 8.0), 50
The primers used for RT-PCR , to amplify mM HCl, 2.5 mM MgCl2, 0.2 mM of each
a 379-bp region (nucleotides [nt] 747 to deoxynucleoside triphosphate (Fermentas-
1126, coding for amino acid 241 to 367) of EU), 1 μM of VP6NF and VP6NR primers,
the VP6 fragment; were the forward VP6- and 2.5 U of Taq DNA polymerase
F primer (sense) (nt 747 to 766) and the (Fermentas-EU). Cycling conditions for
reverse primer, VP6-R (antisense) (nt 1126 VP6NF/VP6NR were 35 cycles of 94°C for
to 1106) according to Iturriza-Gómara et al. 1 min, 42°C for 1 min, and 72°C for 1 min.
(2002). Nested PCR primers, to amplify 155 Nested-PCR products (10 μl) were analyzed
bp fragment of the target rotavirus VP6 gene, by electrophoresis on 3% agarose gels
were the forward primer, VP6-NF and the (Panreac-spain).
reverse primer, according to Gallimore et al. RESULTS
(2006). Extracted samples (5 μl) were heated Prevalence of Noroviruses GGI and GGII
to 99οC for 5 min and immediately placed and Rotavirus group A in ground water
on ice. Salts, nucleotides, primers and 100 samples collected from Nahia village.
U of reverse transcriptase (Fermentas-EU)
were added in 10 μl final volume to give a The frequency of norovirus GGI, norovirus
working concentration of 50mM Tris-HCl GGII and rotavirus group A in ground water
pH 8.3, 40 mM KCl, 5 mM MgCl2, 5 mM samples, which collected monthly from the
DTT, 0.5 mM Tween 20, 0.2 mM of each well water of Nahya village was 0%, 0%
dNTP,s (Fermentas-EU) and 1 μM of both and 33.33% (4/12), respectively (Table 1).
VP6-F and VP6-R primers. The samples

The New Egyptian Journal of Medicine Vol.:48; No.: 3 Supplement 1st March 2013 20
 Table (1): Frequency of Noroviruses GGI and GGII and Rotavirus group A in well
water samples from Nahya village (March 2012- February 2013)
Noroviruses
Sampling date Rotavirus group A
GGI GGII
Mar. 2012 - - +
Apr. 2012 - - -
May 2012 - - -
Jun. 2012 - - -
Jul. 2012 - - -
Aug. 2012 - - -
Sep. 2012 - - +
Oct. 2012 - - +
Nov. 2012 - - +
Dec. 2012 - - -
Jan. 2013 - - -
Feb. 2013 - - -
Number of positive samples 0/12 0/12 4/12
% of positivity 0% 0% 33.33%

Prevalence of Noroviruses GGI and GGII and Rotavirus group A in ground water samples
collected from Saft Al Laban village.
The frequency of human norovirus GI, norovirus GII and rotavirus group A in ground
water samples, which collected monthly from the well in Saft Al Laban, was 0%, 0% and
25% (3/12), respectively (Table 2).
Table (2): Frequency of Noroviruses GGI and GGII and Rotavirus group A in well
water samples from Saft Al Laban village (March 2012- February 2013).
Noroviruses
Sampling date Rotavirus group A
GGI GGII
Mar. 2012 - - +
Apr. 2012 - - -
May 2012 - - -
Jun. 2012 - - -
Jul. 2012 - - -
Aug. 2012 - - +
Sep. 2012 - - -
Oct. 2012 - - +
Nov. 2012 - - -
Dec. 2012 - - -
Jan. 2013 - - -
Feb. 2013 - - -
Number of positive
samples 0/12 0/12 3/12
% of positivity 0 0 25%

The New Egyptian Journal of Medicine Vol.:48; No.: 3 Supplement 1st March 2013 21
 DISCUSSION followed by HAV 50%, enterovirus 41.7%,
It is well documented that food and water astrovirus 8.3% and caliciviruses 0% in Nile
may act as vehicles for the transmission water samples collected from Cairo, Egypt
of human enteric viruses (Bosch, 2007; (1998-1999). In recent studies, rotavirus
Koopmans et al., 2008 & El-Senousy et al., still has high prevalence in Egyptian Delta
2013b). Large outbreaks of hepatitis and and Cairo (El-Senousy and El-Mahdy, 2009
gastroenteritis of suspected foodborne origin & El-Senousy et al., 2013a) while norovirus
have been reported in the literature (Halliday still has low prevalence in Egyptian naturally
et al., 1991; Oishi et al., 1994; Ponka et contaminated irrigation water and fresh
al., 1999; Berg et al., 2000; Sánchez et al., produce (El-Senousy et al., 2013b).
2002 & Hall et al., 2012). Contaminated The peak of rotavirus was observed in
water employed for irrigation, fertilization the study which rotavirus was detected in
or washing, has been considered a major March, September, October, and November
vehicle for crop contamination (Doyle and (autumn and winter months) in the ground
Erickson, 2008 & Lynch et al., 2009). water samples collected from Nahya village,
To study the prevalence of rotavirus while it was detected in March and October
group A and noroviruses GGI and GGII in months (autumn and winter) in the ground
ground water of some Egyptian rural areas water samples collected from Saft Al Laban
from El-Giza Governorate, Twenty four village. Rotavirus was detected in one sample
water samples were collected from March collected in August from Saft Al Laban
2012 to February 2013 from one well in village. These results agree with the peak of
Nahya village and other well from Saft Al rotavirus in different types of environmental
Laban village. One sample from each well samples (sewage, surface water, drinking
was collected monthly. Nested/semi-nested water) all over the world. In Egypt the peak
RT-PCR assays were used for detection of of rotavirus in environmental samples was
noroviruses and rotaviruses in the samples. observed in several studies (Villena et al.,
Rotaviruses were detected in 33.33% of the 2003; El-Senousy et al, 2004 & El-Senousy
ground water samples collected from Nahya et al, 2013a).
village where they were detected in 25% of Detection of rotaviruses which cause
the ground water samples collected from severe gastroenteritis especially in young
Saft Al Laban village. Complete absence of children (less than 5 years) in ground water
noroviruses GGI and GGII was observed in used in drinking and irrigation of crops
all the samples from the two villages and all represents a high risk for consumers. The
over the year. contamination of well water with enteric
These results revealed that Rotaviruses viruses may result from the contamination
was more prevalent than noroviruses GGI of well water with human and animal feces
and GGII in ground water of Egyptian directly or through contaminated surface
rural areas. This agreed with El-Senousy et water and runoff. More studies are needed to
al. (2004), which Rotavirus was the most investigate the prevalence of enteric viruses
frequent RNA enteric viruses (66.7%) in Egyptian ground water.

The New Egyptian Journal of Medicine Vol.:48; No.: 3 Supplement 1st March 2013 22
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