Molecular detection and genotyping of group A rotavirus in two wastewater treatment plants, Iran

Abstract

In different countries especially developing countries, treatment of urban wastewater might be ineffective removal of pathogens such as group A rotavirus. The objective of this study is to evaluate the efficiency of rotavirus removal in two wastewater treatment plants (WWTPs) in Isfahan, Iran. To meet the study objectives, 96 sewage samples from influent (n = 48) and final effluents (n = 48) were collected by grab sampling. Two different concentration methods included pellet and two-phase used for concentrating sewage samples. The presence of rotavirus antigens in all concentrated sewage samples was screened by enzyme-linked immunosorbent method. To analyze the study samples, real-time PCR technique with SYBR Green I fluorescent dye and nested multiplex PCR for rotavirus genotyping were utilized respectively. Result indicated positive rotavirus percentage in two methods of ELISA and real-time PCR was equal to 61.45% (59 cases) and 44.79% (43 cases). In addition, analyzing seasonal distribution of rotavirus shows different distributions as below: in spring (18.64%), summer (20.33%), autumn (35.60%), and winter (25.42%). Finally, rotaviruses illustrate significantly higher frequency in cold seasons. G10 and G1 types are considered the most, among common genotypes which identified in 11 (25.58%) and 5 (11.62%), out of the 43 positive samples in WWTPs, followed by non-typeable genotypes (13.95%) and mix genotypes (11.62%); and different genotypes including G2, G3, G4, G8, G9, and G12 were equal to 2.33, 9.30, 9.30, 2.33, 7, and 7% in the WWTPs, respectively. Such high prevalence underlines the significance of environmental surveillance. Also, to eliminate potential pathogens especially enteric viruses from sewage, the improvement of treatment systems is essential.

Introduction

Urban wastewater treatment in developing countries is widely used to reduce suspended solids and organic matter, but may be ineffective for the elimination of pathogens such as enteric viruses. Despite advances made in water and wastewater treatment in the last decade, the water-and sewage-borne diseases are globally dangerous for human health. Chemical and biological agents are the main pollutants in urban sewages. Among the biological agents, viruses are the most important and most persistent pathogens in water and sewage [1].

Studies show that various viruses can be detected in wastewater, even when sewage treatment is fully and effectively carried out. As a result, environmental contamination occurs through discharged of untreated and treated sewage from infected persons. Evacuation of untreated sewage into surface water causes circulation of gastroenteritis viruses in water bodies and plays a central role in development and transmission of infectious diseases [1, 2]. The human health can be threatened by enteric viruses evacuated to sewage and surface waters [3]. The stools of infected people contain many enteric viruses, usually 10⁵–10¹¹ virus particles/g stool, meaning the contamination of different types of environmental water samples like river, groundwater, seawater, and drinking water. Several viruses, such as norovirus, rotavirus, adenovirus, astrovirus, and sapovirus, are responsible for gastroenteritis [4].

Acute gastroenteritis, which is predominantly related to the deaths of children under the age of 5 years, is recognized as an important waterborne disease; rotavirus is the main cause of gastroenteritis. Severe pediatric diarrhea among 2-year-old UK patients infected by rotavirus accounts for 20–60% of hospitalizations [5]. According to the World Health Organization (WHO), the rotavirus, among enteric viruses, is more serious in developing countries where an estimated 527,000 (475,000–580,000) rotavirus deaths occur annually [6–9]. This virus is responsible for about 200,000 deaths in children below 5 years of age annually in low-income countries. Rotavirus A (RVA) belongs to the genus Rotavirus and family Reoviridae. RVA has a segmented double-stranded RNA genome surrounded by a non-enveloped three-layered icosahedral capsid [10]. There are eleven double-stranded RNA gene segments in the rotavirus genome, encoding six structural (VP1–VP4, VP6, VP7) and six nonstructural (NSP1–NSP6) proteins in the virus [11]. Several reports have also shown the importance of sewage as a major contributor to rotavirus environmental propagation [12]. It has been reported that a combined sewage overflow has significantly contributed to the concentration of viruses in the receiving aquifers and, in wet weather, is more likely to occur compared with dry weather periods. The infectious enteric viruses can be released to final effluents. One of the sources of human enteric viruses is an inadequate refined wastewater [8]. About 7 log₁₀ units of viral genomic copies per liter can be eliminated by wastewater treatment plants (WWTPs) during treatment process [13]. However, there are treatment-resistant viruses that can be isolated from the final effluent [14]. Many rotavirus particles are released to feces from infected patient, which is transported to wastewater treatment plants. Therefore, the prevalence and genotype diversity of rotavirus among the community can be estimated by calculating the viral load in untreated wastewater [2, 15].

Many studies were performed on the human enteric viruses in different WWTPs around the world. Osuolale and Okoh detected human enteric viruses in five WWTPs in the Eastern Cape, South Africa (2017). They found rotavirus in several effluent samples, but no enterovirus was observed. At WWTP-C, 10⁵ genome copies/L of rotavirus was there in 41.7% of the samples. In recent years, environmental virology studies have shown that there are two main causes of this disease: first, the resistance of enteric viruses to the treatment of sewage and second, no diagnostic relationship between coliforms commonly used as quality standards of bacteria and enteric viruses in environmental water samples [1, 2, 8, 16–22].

In this research, the molecular monitoring of rotavirus was carried out simultaneously in two WWTPs in the south and the north of Isfahan, Iran. Evaluation of the efficiency of WWTPs in eliminating rotavirus is an effective biological index for quality control of water and wastewater. In addition, environmental monitoring of rotavirus can provide valuable regional information about the transmission of human rotavirus genotypes in Isfahan province in order to provide appropriate vaccine for immunization of children.

Materials and methods

Area of study

This study was conducted in two north and south wastewater treatment plants in Isfahan, Iran. These WWTPs are the two main treatment plants in the city, which cover a population over 1,200,000 and 890,000, respectively.

Sample collection

Ninety-six sewage samples were collected from influent and effluent points of the two IWWTPs using grab sampling procedure. In each point, the sewage samples (4 L) were collected and stored in sterile plastic bottles, and kept at 4 °C (cold chain) and transported to the biotechnology laboratory.

Virus concentration

Two different concentration methods included pellet and two-phase for sewage samples [11]. Briefly, 400 mL of sewage samples was centrifuged for 10 min at 5000 g. The pellet precipitated was kept in tubes at 4 °C. In the two-phase method, the supernatant was concentrated with the polyethylene glycol 6000 (30%), dextran from Leuconostoc mesenteroides (20%) (D5376, Sigma), and NaCl 5 M (Merck), followed by incubation at 4 °C overnight. After formation of two phases in the separation funnel, 5 mL was harvested combining bottom phase and the interphase. The resulting suspended pellet in this concentration was extracted by chloroform (10 mL) by centrifuging in 260 rpm at 20 min, followed by clarification of the water phase using centrifugation, as mentioned. The final product was stored at − 80 °C in a microtube [11].

Detection of rotavirus antigen by enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assay (ELISA) method using Kit (EIA) (Rotavirus Ag ELISA, DRG, Germany) was used to screen the rotavirus antigens present in all concentrated sewage samples, according to the manufacturer’s instructions. This method has a high sensitivity to detect rotavirus antigen (group-specific proteins, especially major capsid protein VP6). The optical density (OD) was read by spectrophotometer at a wavelength of 450 nm, with a value greater than 0.15 as positive.

Nucleic acids extraction and cDNA synthesis

Viral RNA was extracted from 140 μL of all antigen-positive sewage samples. First, the samples were suspended in phosphate-buffered saline (PBS), and then centrifuged for 10 min with 1500×g. The supernatant was then used for RNA extraction using a QIAamp viral RNA mini kit (Qiagen, Germany), based on the protocol of the manufacturer. The purified viral RNA was eluted in 60 μL of elution buffer and then, RT reaction was carried out using PrimeScript strand cDNA Synthesis Kit (Takara, Japan) in accordance with the program as follows: 30 °C for 10 min, 42 °C for 10 min, 70 °C for 15 min, and then cooling immediately to 4 °C. The RT product was stored at − 20 °C until further use for qPCR.

Real-time PCR

RT-PCR was conducted for cDNA using SYBR Green I fluorescent dye strategy. Each 20-μL RT-PCR mixture consisted of 1× SYBR Premix (Takara, Japan), 100 nM each primer, and 2 μL DNA template. Primers of rotavirus designed for this study (ACCESSION=LC178569) are as follows: RVA-F: 5′-CAAACTGACGAAGCGAATAAATG-3′, RVA-R: 5′-TGTAGCATCAGTTGTCAAGCATC-3′ [Tm] 61 °C. A detection system (Rotor-Gene 6000) was applied for RT-PCR amplification and detection. The amplification procedures were run in thermal cycler program: 40 cycles including denaturation at 95 °C for 15 s, annealing at 61 °C for 30 s, and extension at 72 °C for 30 s. The emission of each sample was recorded in thermal cycling and the raw fluorescence data were inserted to SDS software (ABI, Tokyo, Japan) to obtain threshold cycle (Ct) values, followed by computing standard curve using the Ct values of the diluted standards. Then, the Ct values were used to extrapolate absolute quantities for the unknown samples.

Nested multiplex PCR for Rotavirus G genotyping

The G-typing was performed by cDNA products as templates for PCR VP7 gene amplification using pair primers according to Barril et al. For genotyping, G1 to G4, G8 to G9, and G10 and G12 specific primers were used in the multiplex nested PCRs, respectively. The technique was performed according to the rotavirus detection and genotyping protocol provided by the World Health Organization. Table 1 shows the primer sequences used [7].

Table 1.

Primers sequence and positions used for genotyping RV in the present study

Primer	Sequence (5′→3′)	Position	Type
aBT1	CAA GTA CTC AAA TCA ATG ATG G	314-335 nt	G1
aCT2	CAA TGA TAT TAA CAC ATT TTC TGT G	411-435 nt	G2
aET3	CGT TTG AAG AAG TTG CAA CAG	689-709 nt	G3
aDT4	CGT TTC TGG TGA GGA GTT G	480-498 nt	G4
aAT8	GTC ACA CCA TTT GTA AAT TCG	178-198 nt	G8
aFT9	CTA GAT GTA ACT ACA ACT AC	757-776 nt	G9
mG10	ATG TCA GAC TAC ATA TAC TGG	666-687 nt	G10
G12	CCG ATC GACGTAACGTTGTA	548-567 nt	G12
Beg9	GGC TTT AAA AGA AAT TTC CGT CTG G	1-28 nt	–
End9	GGT CAC ATC ATA CAA TTC TAA TCT AAG	1062-1036 nt	–
RVG9	GGT CAC ATC ATA CAA TTC T	1062-1044 nt

Statistical analysis

Data were analyzed by statistical software SPSS (version 16.0, USA). Statistical differences between concentrations of enteric viruses in water samples were determined with one-way ANOVA. Chi-square test was performed and differences were considered significant at values of p value < 0.05.

The removal efficiency in WWTPs was computed as follows:

$$\%\mathrm{Eff}=\frac{M_{\text{in}}-M_{\text{out}}}{M_{\text{in}}}\times 100$$

where %Eff is the efficiency percent, M_in is the input parameter, and M_out is the output parameter.

Result

Distribution of rotaviruses

Table 2 illustrates the distribution of rotavirus and positive frequency of the samples in effluent and influent of WWTPs. Total numbers of sample of the research have been evaluated to be 96. EIA showed detection of rotaviruses in 61.45% (59 cases) of the sample. In particular, they contained 42.37% (25 cases) of effluent and 57.62% (34 cases) of raw sewage influent. In general, analyzing the removal efficacy of the wastewater treatment plants showed 26.47% mean removal of rotaviruses in WWTPs systems (21.05% in north WWTP and 33.33% in south WWTP) (Table 2).

Table 2.

Detection of rotavirus using ELISA in sewage samples from two WWTPs (n = 96)

WWTP	Influent system	Effluent system	Total	Removal efficiency†
North	19* (32.7)**	15* (25.2)**	34* (57.9)**	21.05
South	15* (25.2)**	10* (16.9)**	25* (42.1)**	33.33
Total	34* (57.9)**	25* (42.1)**	59* (100)**	26.47

*The number of rotavirus positive samples

**The percent of rotavirus positive samples

^†The removal efficiency = (The number of rotavirus positive in influent samples) − (The number of rotavirus positive in effluent samples) ∕ The number of rotavirus positive in influent samples × 100

Table 3 reports seasonal distribution of rotavirus by real-time PCR in the north and south WWTPs. Results show that rotaviruses were identified with a significantly higher frequency in autumn. The highest prevalence of rotaviruses was detected in autumn (41.9%), followed by winter (20.9%) and spring (20.9%), respectively. Table 4 compares 2 methods’ concentration with higher total viral extracted from two-phase.

Table 3.

Seasonal distribution of rotavirus in WWTPs by qPCR method

Season	WWTP (north)		WWTP (south)		Total
	Influent system	Effluent system	Influent system	Effluent system
	No. (%)	No. (%)	No. (%)	No. (%)	No. (%)
Spring	3 (7)	1 (2.3)	3 (7)	0 (0)	7 (16.3)
Summer	4 (9.3)	2 (4.7)	2 (4.7)	1 (2.3)	9 (20.9)
Autumn	6 (14)	4 (9.3)	4 (9.3)	4 (9.3)	18 (41.9)
Winter	4 (9.3)	2 (4.7)	3 (7)	0 (0)	9 (20.9)
Total	17 (39.5)	9 (20.9)	12 (27.9)	5 (11.6)	43 (100)

Table 4.

Comparison of the two concentrates for virus recovery in WWTPs

Concentration	Methods
	ELISA (%)			Real-time PCR (%)
	Two-phase	Pellet	Total	Two-phase	Pellet	Total
Influent—N	10	9	19 (56%)	9	8	17 (65%)
Effluent—N	8	7	15 (44%)	5	4	9 (35%)
Influent—S	8	7	15 (60%)	6	6	12 (70%)
Effluent—S	5	5	10 (40%)	3	2	5 (30%)
Total positive	18	16	59 (100%)	23	20	43 (100%)

N, north of WTTP; S, south of WWTP

Distribution of rotavirus genotypes

G-typing has been done on rotavirus-positive samples via the nested RT-PCR. Figure 1 a and b depict rotavirus genotype survey’s findings. The commonest genotypes have been G10 and G1 types, which are recognized in 11 (25.58%) and 5 (11.62%) among the 43 positive specimens in WWTPs that are respectively accompanied by non-typeable genotypes (13.95%) and mix genotypes (11.62%). The genotypes G2, G3, G4, G8, G9, and G12 respectively have been 2.33, 9.30, 9.30, 2.33, 7, and 7% in the WWTPs. The commonest rotavirus genotype that has been identified so far is G10 in autumn (54.54%), winter (27.27%), and spring (9.09%). According to the seasonal distribution, it was revealed that rotaviruses were identified with a significantly higher frequency during the cold seasons of the year. A significant relationship was found (Fig. 1) between distribution of the genotype and seasons (P = 0.04).

Fig. 1.

Seasonal distribution of rotavirus genotypes in north WWTP (a) and south WWTP (b)

Discussion

Fecal coliform and total coliform indices have been conventionally used for evaluating micro-biological qualities of urban sewage effluents. However, these bio-indicators do not reflect the risk of other pathogens such as viruses. Since wastewater treatment system has been considered a significant way to transmit waterborne human enteric pathogens, it is of high importance to monitor rotavirus in wastewater for public health, specifically in areas where such viruses are not surveilled. However, there are many concerns about the potential resistance of rotaviruses to treatment processes. There have been few researches conducted on such viruses in wastewater treatment samples in developing countries that are caused by complicated viral concentration techniques to retrieve in aqueous medium and high costs. There is not enough knowledge of the micro-biological supervision, risks evaluation, and epidemiologic findings on public health, particularly in developing countries (for example, Iran). Therefore, it is necessary to investigate rotaviruses in the treated and raw wastewater [23].

In the current study, we evaluated the removal efficiency of rotaviruses in two wastewater treatment plants from Iran. Hovi and colleagues showed that the virus concentration in sewage samples can be increased up to 50-fold by two phases. In our study, pellet and two-phase concentration methods were used for recovery rotaviruses. However, two-phase concentration method using PEG 6000 and dextran is a very efficient recovery method to be applied to viruses from water and sewage [24].

Since results provided by ELISA technique can present incorrect positive samples on the basis of detecting proteins, qPCR procedure has been applied. This research determined group A rotavirus in 61.41% of the samples analyzed from raw and treated sewage by ELISA and 44.79% by qPCR. The requirement for higher free-antigen concentration for generating a positive reaction is a restriction of antibody-based experiments in detection of enteric pathogens. Comparing obtained results to other revealed a prevalence of rotaviruses of 14 to 62% in wastewater samples [11, 12, 21]. Osuolale and colleagues studied human enteric viruses in effluent samples collected from WWTPs in South Africa. They used absorption-elution technique and singleplex RT-PCR to retrieve human viruses. 41.7% of the samples contained rotavirus titer [8].

The seasonal results of the research indicated a considerable relationship between seasonal profile and positive numbers of rotavirus. Seasonal distribution of rotavirus in 2 WWTPs from Isfahan, Iran, has been often observed in autumn and winter. This result is an indicator of a higher risk of transmission during the cold seasons of the year. Other studies have reported prevalence of rotaviruses during the cold seasons. Li and colleagues made a report of seasonal changes in rotaviruses in all effluents that have been greater in winter compared to summer. This suggests an association between seasonal patterns of rotavirus found in wastewater effluent–receiving river. All three WWTPs showed similar patterns of seasonal variation for infectious rotaviruses and rotavirus VP7 gene concentrations in different effluents. According to the results, greater viral concentrations have been seen within fall and winter (November 2007–March 2008) and lower concentrations during summer (June–September 2008) [11, 21, 23, 25].

Analysis of rotavirus genotype in this research showed that genotypes G10 and G1 have been the predominating genotypes in the treated and raw sewage of two WWTPs. The type of genotypes has been different in previous studies conducted on environmental and clinical monitoring. The study conducted by Kargar and colleagues [11] in Shiraz Hospital reported that the predominant genotype is G1.

The non-typeable genotype was detected in 13.95% of the samples collected from the sewage. The non-typeable rotavirus strains for the G genotype could be attributed to the presence of novel strains or the failure of the genotyping due to the presence of other genes that were not investigated in the survey. Seasonal distribution of G genotype indicated greater numbers of G10, G1, and G9 genotypes determined during cold seasons. This is in line with previous reports from Iran [11, 26]. Wastewater treatment plant plays an important role in the removal of human pathogens from wastewater in the world. Hence, one of the effective sewage treatments is necessary to all communities. Although in our study it is indicated that the rotavirus removal is not complete in the effluent of WWTPs, the virus pollution has been significant and revealed a critical public health danger. This result is an agreement with the result of previous studies that display the presence of rotaviruses not only in sewage raw but also in treated wastewater [4, 11, 19, 26]. Studies conducted to evaluate the efficacy of wastewater treatment plants in developing countries are limited to bacterial indicators such as coliforms, while viruses are ignored. Research on the evaluation of the efficiency of the wastewater treatment plants in developing countries is related to bacterial indices, including coliforms, so they did not deal with virus. For this reason, during recent years, more attention has been paid to using viruses as indicators of sewage contamination, the risk of waterborne viral diarrheal disease, and the necessity of routine surveillance of water source for monitoring the viral contamination. The raw sewage is one of the main sources of shedding enteric viruses into water environments, even in the absence of fecal coliforms as standards in microbial water quality assessment [27]. Viruses are resistant to chlorination and ozonation and other treatment processes, such as sedimentation. Thus, viral indices are more accurate indicators with regard to the prevalence of rotavirus infections.

Conclusion

Quality control management is of high importance in wastewater treatment plants because of the increasing worries about water deficiencies, water pollution, and requirement of reusing wastewater in agriculture and drinking water. Reusing the sewage is enhancing in order to be used for industrial, environmental, agricultural, and human purposes. This research results revealed the genotype data of rotaviruses in environment surveillance for the first time in Isfahan, Iran. The high frequency of rotavirus in this research could be justified by higher survival rate of viruses in the wastewater treatment procedures as well as comparative inefficacy of wastewater treatment plants to eliminate such viruses. Hence, regular viral controlling must be regarded as one of the further analyses in the routine testing already performed in order to improve policies of wastewater management by the national water quality monitoring bodies.

Abbreviations

WWTP

Wastewater treatment plant

ELISA

Enzyme-linked immunosorbent assay

rpm

Revolutions per minute

RVA

Rotavirus A

NT

Non-typeable samples

CT

Cycle threshold

CFU

Colony-forming unit

OD

Optical density

NSP

Nonstructural proteins

VP

Viral protein

qPCR

Real-time polymerase chain reaction

Nested RT-PCR

Nested reverse transcription (RT) polymerase chain reaction

RT-PCR

Reverse transcription polymerase chain reaction

WHO/CDC

World Health Organization and Centers for Disease Control and Prevention

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

Not required