Molecular detection of human adenovirus and bocavirus in wastewater and drain water in the western Nile Delta of Egypt

Background: Human Adenovirus (HAdV) and Human Bocavirus (HBoV) are frequently found in different water sources throughout the year. Due to their health impact on humans, especially children, continuous monitoring of these viruses is one of the important tools to evaluate their circulation within society. This study aimed to investigate the prevalence of HAdV and HBoV in different environmental samples collected from the western Nile Delta of Egypt. Methods A total of 264 samples were collected from treated and untreated sewage (48), sewage sludge (24), drainage water (96), and drainage sediment (96) from December 2019 to November 2021. Molecular detection by nested PCR was performed to detect both HAdV and HBoV in the collected samples. Results The results indicated that HAdV was more prevalent than HBoV in all types of sewage samples, with the highest detection rate in untreated sewage. Moreover, the detection rates of both viruses were relatively lower in sludge sewage samples. In drainage water and sediment samples, HAdV and HBoV were detected with similar frequencies, although the detection rates were generally lower than those observed in sewage samples.


INTRODUCTION
Waterborne diseases are thought to be responsible for almost 12 million deaths each year (Xagoraraki & O'Brien 2019).Enteric viruses are considered the most important causative agents of many cases and outbreaks resulting from contaminated water.Globally, almost 80% of wastewater is dumped into the environment without receiving enough treatment (UN 2017) and nearly 2 billion individuals consume drinking water contaminated with feces (WHO 2019).
Monitoring of sewerage systems provides the valuable community health status and the required epidemiological information regarding serotypes circulating as well as for early detection of many emerging viruses (Mao et al. 2020).For people with viral gastroenteritis infection, the virus can be detected in their feces after a short period of infection, whether or not they have symptoms.Therefore, we can evaluate the virus load and monitor its levels by collecting and testing wastewater samples.Furthermore, direct sampling of wastewater is the ideal way since it contains more pathogens than the receiving environments where wastewater is released.
In the environment, enteric viruses are more stable and resistant to the treatment processes that are applied by the current wastewater treatment plants systems (Plummer & Long 2007).The presence of viral genome in a water sample does not mean it`s belong to an infectious virus particle.However, the infectious viral particles found to be detected in environmental samples for up to 100, 120, 130 days in soil, fresh and sewage water, and seawater respectively (US-EPA 1993).Despite modern water treatments, enteric virus remains present in water at high levels, and for this reason, it is suggested to be used as a viral contamination indicator (Bosch 1998).Adenovirus and Bocavirus are considered the only DNA viral pathogens that cause gastroenteritis and hence tend to outlast in environmental waters for a long time (Jiang & Chu 2004).
Human Adenovirus (HAdV) is the most common of enteric viruses in aquatic environments and considered one of the major causes of gastroenteritis, conjunctivitis, and respiratory diseases (Pina et al. 1998).HAdV serotypes 40 and 41 are the second causative agent for gastroenteritis in children after rotavirus (Fong et al. 2010), with the possibility of infection occurring throughout the year with little or no seasonal variation in shedding (Allard et al. 1992).
Therefore, the Environmental Protection Agency (EPA) recommended monitoring viruses in all water utilities that serve more than 100,000 households (US-EPA 2009).On the other side, adenovirus has unique clinical importance since it is used as a vector for delivering target antigens to mammalian hosts (Ginn et al.

2018)
. Therefore, many studies have been carried out to study its relationship with the host cell (Guissoni et al. 2018;Badr et al. 2019), and on the other hand evaluate its prevalence within the community (Öner et al. 2022) or in the environment (Quintão et al. 2021).
Human Bocavirus (HBoV) was identi ed for the rst time in children with respiratory tract infections in Sweden (Allander et al. 2005).After this, HBoV was identi ed in stool samples from children with diarrhea in Spain (Vicente et al. 2007).Various studies demonstrated the prevalence and the circulation of HBoV among children suffering from gastroenteritis, both with and without respiratory tract infection symptoms (Sousa et al. 2017).Since its discovery, there are four species of HBoV (HBoV1-4) that are mainly associated with respiratory tract infections and gastroenteritis.However, some studies indicated that species 2, 3 and 4 are mainly associated with gastroenteritis infection (Lee et al. 2016).
In Egypt, different studies have been conducted to assess the prevalence of HAdV and HBoV.Some studies showed the clinical assessment of HAdV regarding respiratory infections (Zaki et al. 2020) (Wierzba et al. 2006) from clinical samples of hospitalized children at Abu Homos General Hospital.The data obtained from these studies showed that the selected enteric viruses were the main cause of severe diarrhea symptoms among children in this region.However, the current study aimed to evaluate the prevalence of human adenovirus and human bocavirus in sewage and drainage water from Abu Homos city to gure out the environmental assessment situation of wastewater in this region for the rst time.

Study sites
The samples were collected from Abu Homos wastewater treatment plant (WWTP) located in Abu Homos city, Beheira governorate in the western Nile Delta region of Egypt.Abu Homos is an urban agricultural community with an area of 15.400 km and an estimated population of around 455,000 inhabitants.The city includes approximately 90% of urban households that have municipal water and wastewater disposal as compared with 55% of rural residents receiving municipal water and don't have a sewage disposal system (Wierzba et al. 2006).The WWTP usually receives wastewater from the city and treats the raw sewage to primary treated wastewater then discharges it directly into Al-Mahata drain, which in turn passes through several other drains until it ends in the general drain (Al-Imoum) heading to Lake Mariout in Alexandria governorate.

Collection and concentration of wastewater samples
One liter of each tested sample was collected once monthly from December 2019 to November 2021.A total of 48 sewage samples were collected from untreated raw sewage (24 samples) and treated sewage (24 samples) in addition to 24 sludge samples.Furthermore, 192 samples were collected during the same period from drainage water (96 samples) and drain sediment (96 samples) at four sites.S1 (Sharaan village) and S2 (Al-Twala village) are located on Al-Mahata drain and S3 (Kom Swan village) and S4 (Al-Serafy village) are located on Al-Imoum drain (Fig. 1).

Concentration of viral particles from water samples
All water samples were concentrated by the adsorption-elution method as described by Katayama et al. (2002), using a negatively charged membrane with an acid (H 2 SO 4 solution) rinse procedure.Brie y, water samples were processed in a vacuum system (Nihon Millipore®, Tokyo, Japan) using a negatively charged membrane with a pore size of 0.45 µm and a diameter of 142 mm, and a ow rate of 200 mL/min.After ltration, the membrane was washed with a 200 mL solution of H 2 SO 4 (0.5 mM) at pH 3.0 to ensure that all retained viral particles are eliminated in the biosolids.Virus particles have been eluted from the membrane using 11 mL of NaOH (1 mM) at pH 10.5 in sterile glassware with continuous stirring for 10 min.The obtained eluate was neutralized with 50 µl of H 2 SO 4 solution (50 mM) and 50 µl Tris-EDTA buffer solution (100×) 1 mM pH 8.0.All the concentrated samples were stored at − 20°C until used.

Concentration of viral particles from Sediment and Sludge samples
For Sewage sludge and sediment samples, 100 g of each sample was concentrated using the method described by the EPA (US-EPA, 1993) with a modi cation as described previously by Schlindwein et al. (2009) followed by the PEG 6000 precipitation method as described by Lewis & Metcalf (1988).Brie y, an equal volume of phosphate buffer (PBS) and AlCl 3 (0.05 M) solution was applied to 100 g of wet sediment or sludge, then the pH was adjusted to 3.5 using HCl (5 M).Samples were subjected to ice sonication three times each 30 s to dislodge viruses, followed by centrifugation at 2.422×g for 15 min at 4°C.After this, the pellet was suspended in a 10 mL glycine buffer (0.25 M) at pH 9.5 with stirring for 1 h, followed by further centrifugation at 7.656×g for 30 min at 4°C.The supernatant was transferred to another tube containing 8% PEG 6000 as a nal concentration at pH 7.2 and the tube was stirred for about 2 h at 4°C.A nal centrifugation step was done at 7.656×g for 90 min at 4°C, then the pellet was suspended in 10 mL of PBS (0.1 M) at pH 7.2.The decontamination stage was performed by adding chloroform (1:3) followed by another centrifugation at 7.656×g for 15 min.The upper (aqueous) phase was recovered, aliquoted, and stored at − 20°C until further analysis.

Viral genomic Extraction
The genetic materials (DNA) of both Adenovirus and Bocavirus were extracted from 240 µl of the concentrated samples using the QIAamp Viral RNA and DNA kits (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's instructions.For quality assurance, human adenovirus type 2 (AdV-2) was used as an internal control to examine polymerase chain reaction (PCR) inhibition (data not shown).Puri ed viral DNA was eluted in a nal volume of 60 µl of DNase-free water.

Detection of HAdV DNA by nested PCR
First PCR targeted the conserved hexon region followed by nested PCR was performed as described by Allard et al. (1992) and Pugi et al. (1994), respectively.
The rst PCR reaction was performed using hexAA1885 and hexAA1913 primers to amplify 300 bp for 40 cycles under the following condition: 94°C for 1 minute, 56°C for 1 minute, and 72°C for 45 seconds; with a nal extension step at 72°C for 10 min.Nested PCR was performed to amplify 142 bp using the following primers: nexAA1893 and nexAA1905 under the same cycling conditions of the rst PCR (Table 1).1).

RESULTS AND DISCUSSION
The regular microbiological survey of any pathogen with a health impact on humans is one of the powerful tools that can be used to assess the health status within a community and predict newly emerging pathogens, especially these days after the COVID pandemic (Shaheen et al. 2022).Many studies have reported different prevalence rates of HAdV and HBoV in the environment in many countries around the world.However, both viruses have been detected as the causative agent of respiratory tract infection and/or gastroenteritis in several studies conducted in Egypt.Other studies focused on monitoring the circulation of the virus in the aquatic Egyptian environment, especially in Greater Cairo.Therefore, the current study aimed to evaluate the prevalence of HAdV and HBoV in sewage samples from the western region of the Nile Delta in Egypt.
Previous studies showed that the currently used treatments by WWTPs worldwide have been shown to be ineffective at totally eliminating or inactivating viruses from the sewage (Okoh et al. 2010).Our results showed that HAdV and HBoV genomes were detected in all water samples; untreated and treated sewage, drain water as well as sewage sludge and drain sediment.All samples in this study are environmental specimens that were collected gradually in an equal condition from all selected sites.As it's known that most environmental samples especially sewage and sludge specimens contain different organic and inorganic compounds that might integrate with the nucleic acid and may inhibit the detection by PCR (Schlindwein et al. 2010).Therefore, to increase the speci city of the detection as well as to avoid any false-positive results, we used the nested PCR method and internal controls in our study.
Our results showed that HAdV was detected in 33.33% (8/24) of untreated sewage, 20.8% (5/24) of treated sewage, and 12.5% (3/24) of sewage sludge, in addition to 28.1% (27/96) of drainage water and 13.5% (13/96) of drainage sediment (Tables 2 and 3).The presence of HAdV in some water samples was expected due to their DNA genome composition, which gives it stability with higher frequency and abundance in water in comparison to other RNA enteric viruses (Verani et   Nested PCR was used to detect viral genome and the "+"symbol refers to the detected samples and "-"symbol to not detected samples.On the other hand, HBoV was detected according to our results in 25% (6/24) of untreated sewage, 16.7% (4/24) of treated sewage, and 8.33% (2/24) of sewage sludge, while in drainage water was 20.8% (20/96) and in drainage sediment was 11.5% (11/96) (Tables 2 and 3).Worldwide, HBoV was reported with various detection rates ranging from 60 to 93% in sewage and from 37 to 40% in surface water (La Rosa et al. 2017; Booranathawornsom et al. 2022).The results in this study are considered lower than the rst study conducted in Egypt, which detected HBoV-1, 2, and 3 in untreated (72-100%) and treated (34-100%) sewage samples (Hamza et al. 2017).The second study also mentioned high prevalence rates in raw sewage (41.6%), treated sewage (25%), sewage sludge (16.6%), drainage water (48.6%) and drain sediment (29%) (Shaheen et al. 2020), while a recent study reported 95.8% of HBoV in e uents of wastewater (Rizk & Hamza 2021).These differences in HAdV and HBoV detection rates can be attributed to the variations in population numbers, geographic areas, detection methods, and techniques used.
According to some reports, human enteric viruses may be able to survive in sediments before being released into the aquatic environment (Salvo & Fabiano 2007).In our study, HAdV and HBoV were detected in high numbers in wastewater and surface water more than in sludge or drain sediments which is consistent with other studies (Elmahdy et al. 2016;Shaheen et al. 2020).Moreover, we observed that the number of positive samples for both viruses started to increase at site 3 and continue to site 4 in comparison to sites 1 and 2 (Table 3).This increase can be explained by the fact that site 3 is considered the meeting point where Al-Mahata drain joins the main Al-Imoum drain in addition to the agriculture and urban wastewater which can discharge directly into both drains without treatment.
The results showed that HAdV and HBoV genomes were detected in all samples and throughout the year.Egypt has four seasons divided throughout the year, where winter represents the period from December to February, spring from March to May, summer from June to August, and autumn from September to November.Each season, 66 samples were collected from all sources including water samples from untreated, treated, and drainage water samples in addition to sludge sewage and drain sediment.The Heat map of all samples showed that there are seasonal variations for both viruses, especially in the winter and summer seasons (Figure 2).HAdV was found to be more detected in the winter than in the summer season, in contrast to HBoV which was detected in higher numbers in the summer than in the winter.Some studies noted the same observation for both viruses (Jiang et al. 2016;Elmahdy et al. 2019).In contrast, other studies mentioned that there are no seasonal prevalence rates of these viruses (Fong et al. 2010;Shaheen et al. 2020).Based on our results, the treatment processes in the WWTP were effective at removing both viruses.From the raw sewage in the inlet point to the treated sewage in the outlet point, the percentage of positive samples decreased from 25% to 16.67% for HAdV and from 33.33% to 25% for HBoV.
To our knowledge, this study is the rst documentation of enteric virus monitoring in the aquatic environment (treated wastewater and drains water) in the western region of the Nile Delta in Egypt.The results highlighted the presence of HAdV and HBoV in wastewater that may play a role in diarrheal cases among the population in this region.The present study is limited due to the absence of clinical data, which would have explained the general situation more clearly in addition to the infectivity test for positive viruses.On the other hand, the signi cant incidence of both viruses in untreated raw sewage may point to widespread population circulation of both viruses.The presence of both viruses in the treated wastewater could be one of the sources of contamination of some other sources such as surface and recreational water as well as agricultural crop water.Therefore, conducting continuous monitoring of viruses in the environment, especially surface water, wastewater, and agricultural crops, is considered one of the most important methods of environmental assessment, which is bene cial to the health of society in general.

CONCLUSION
In conclusion, HAdV and HBoV can be found in different water sources, and the continuous monitoring of these viruses in environmental samples can help assess the quality of the water supply and identify potential sources of contamination.HAdV was more prevalent than HBoV in all types of sewage samples tested, especially in untreated sewage.Both viruses were less commonly detected in sludge sewage samples.HAdV and HBoV were detected at similar rates in drainage water and sediment samples, but overall detection rates were lower compared to sewage samples.These ndings can be used to improve water treatment and prevent the spread of infection through contaminated water.The existence of enteric viruses in the treated e uent may be one of the reasons for its transmission to surface water as well as to agricultural crops.More studies are still needed to continuous monitor the circulation of enteric viruses in sewage, water, and agriculture products.

Declarations
Author Contributions: All authors contributed to the study conception and design.Material preparation, data collection and analysis were performed by

[
Kareem Rady Badr],[Elmahdy Mohamed Elmahdy]  and [Mohamed NasrFathy Shaheen].The rst draft of the manuscript was written by [Kareem Rady Badr] and all authors commented on previous versions of the manuscript.All authors read and approved the nal manuscript.Data availability: All data generated or analyzed during this study are included in this published article.COMPLIANCE WITH ETHICAL STANDARDS Funding: The authors declare that this work was supported under the grant numbers: 7357 funded by the Academy of Scienti c Research and Technology (ASRT) in Egypt.Disclosure of potential con icts of interest: The authors have no nancial or non-nancial interests to disclose.Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors.
or gastroenteritis infection (Elmahdy et al. 2019).Also, other studies aimed to detect HBoV in respiratory samples (Roshdy et al. 2020) or from cancer specimens (Abdel-Moneim et al. 2016), in addition to patients with gastroenteritis (Mahmoud et al. 2022).On another hand, different studies focused on the Egyptian aquatic environment in different regions, basically in Greater Cairo to demonstrate the presence of HAdV (Elmahdy et al. 2020; Rashed et al. 2022) and HBoV (Shaheen et al. 2020; Rizk & Hamza, 2021).In the western region of the Nile Delta, two clinical studies have been conducted in Abu Homos city, Beheira governorate, located southeast of Alexandria, Egypt.These studies aimed to evaluate the prevalence of enteric viruses including Adenovirus, Astrovirus, Norovirus (El-Mohammady et al. 2012), and Rotavirus

Table 1
Primer sequences used for the detection of human Adenovirus and human Bocavirus.Detection of HBoV DNA by nested PCRNested PCR was carried out to detect HBoV-2/3/4 species targeting the conserved VP1/VP2 region as described by La Rosa et al.(2016).The extracted viral genome was applied for rst round PCR using primer 234F1 and 234R1 to amplify 543 bp under the following conditions: 95°C for 10 min followed by 40 cycles of 94°C for 30 seconds, 55°C for 3 seconds, and 72°C for 60 seconds; with a nal step at 72°C for 10 minutes.The next round was a Nested PCR to amplify 382 bp using primer 234F2 and 234R2 under the same condition as the rst PCR with a modi ed annealing temperature at 50°C (Table

Table 2 :
(Elmahdy et al. 2020)revious four studies have mentioned the prevalence rates of HAdV in water samples collected from Greater Cairo.HAdV was detected in 91.7%(22/24)of raw sewage collected from Zenin WWTP and 66.7% (16/24) of Nile water samples between 2009 and 2011 (EL-Senousy et al. 2013).However, another study reported the same rates in 2017 from the same WWTP when HAdV genome was detected in 84.4%(27/32)in raw sewage, 50% (16/32) in treated sewage, and 78% (25/32) in sludge sewage samples(Elmahdy et al. 2019).In 2020, another study showed that the median incidence of HAdV in wastewater was 3.06 × 10 5 GC/L and in sludge was 4.52 × 10 4 GC/L, while in surface water was 1.5 × 10 7 GC/L and in sediment was 3.7 × 10 7 GC/L of samples collected between 2017-2018 from Abu-Rawash WWTP and El-Rahawy drainage(Elmahdy et al. 2020).A recent study reported a high prevalence rate of HAdV in samples collected from different water sources from 2015 to 2017(Rashed et al. 2022).In this study, HAdV genome was detected in 85-100% of sewage water with a 75%-100% infectivity rate, while in river water was 100% with more than 65% infectivity in addition to almost 30% without any infectivity rate in drinking water.Detection of HAdV and HBoV in untreated, treated, and sludge sewage samples collected from December 2019 to November 2021