G3 and G9 Rotavirus genotypes in waste water circulation from two major metropolitan cities of Pakistan

Rotavirus A (RVA) is a diarrheal pathogen affecting children under age five, particularly in developing and underdeveloped regions of the world due to malnutrition, poor healthcare and hygienic conditions. Water and food contamination are found to be major sources of diarrheal outbreaks. Pakistan is one of the countries with high RVA related diarrhea burden but with insufficient surveillance system. The aim of this study was to gauge the RVA contamination of major open sewerage collecting streams and household water supplies in two major metropolitan cities of Pakistan. Three concentration methods were compared using RNA purity and concentration as parameters, and detection efficiency of the selected method was estimated. Water samples were collected from 21 sites in Islamabad and Rawalpindi in two phases during the year 2014–2015. Meteorological conditions were recorded for each sampling day and site from Pakistan Meteorological Department (PMD). Nested PCR was used to detect the presence of RVA in samples targeting the VP7 gene. Logistic regression was applied to assess the association of weather conditions with RVA persistence in water bodies. Statistical analysis hinted at a temporal and seasonal pattern of RVA detection in water. Phylogenetic analysis of selected isolates showed a close association of environmental strains with clinical RVA isolates from hospitalized children with acute diarrhea during the same period. This is the first scientific report cataloging the circulating RVA strains in environmental samples from the region. The study highlights the hazards of releasing untreated sewerage containing potentially infectious viral particles into collecting streams, which could become a reservoir of multiple pathogens and a risk to exposed communities. Moreover, routine testing of these water bodies can present an effective surveillance system of circulating viral strains in the population.

RVA prevalence in sewage water. During the first phase of sample collection, thirteen water samples from Islamabad and seven from Rawalpindi were collected. During this phase of sampling, 45% water samples collected from 10 sites were found positive for RVA. Five samples from Nullah Lai, three from its tributaries from Rawalpindi and Islamabad tested positive for RVA, while samples from Rawal dam and Banni gala were also positive for RVA. During the second round of sampling, RVA was not detected in any sample using same methods (Supplementary Table I). Overall detection rate of RVA in water during the whole study period was 23%.
Analysis of climatic parameters associated with RVA detection. Incidence of RVA diarrhea shows seasonal patterns globally. It has a direct and immediate relationship with cold weather in different zones of world 18, 19 . Although in Pakistan RVA diarrheal cases are reported throughout the year; incidence rate shows significant relationship to seasonality 20 . In this study, we investigated the effect of climatic factors on persistence of   RVA in water. Statistical analyses revealed that climate conditions significantly impacted the presence of Rotavirus in water samples. Mann Whitney U test was applied to non-normally distributed data that showed that the environmental factors are significantly different between the two study groups. Relative humidity (p = 0.005) and temperature (p = 0.005), solar radiation (p = 0.005) wind speed (p = 0.02) seemed significantly different among the groups (Table 2). High relative humidity, low temperature, less solar radiation and slower wind speed were found associated with rotavirus presence in water samples. Univariate logistic regression, showed that temperature (OR = 0.72(95%CI = 0.50-0.88), p = 0.02) and solar radiation (OR = 0.70(95%CI = 0.49-0.87), p = 0.009) are negatively associated with RV presence. Moreover, relative humidity had a causal effect (OR = 1.27(CI = 1.08-1.64), p = 0.02) (Table 3). However, precipitation, pH of water and wind speed was not found significantly associated with RVA presence in water. In multivariable logistic regression analyses, temperature was adjusted for humidity and solar radiation, depicting temperature as the only co-dependent variable with RV presence in water (OR = 0.10(95%CI = 0.01-0.54), p = 0.02) ( Table 3).
RVA genotypes in waste water. G3 and G9 genotypes were detected in waste water sample. Maximum likelihood tree was constructed for RVA genotypes based on partial sequencing of gene segment VP7 (Fig. 2

Discussion
RVA diarrhea is considered to be the main cause of childhood mortality particularly in developing countries like Pakistan. According to an estimate in 2013, Pakistan is one among four countries in the world with approximately half (45%) of deaths due to RVA diarrhea 22 . Water and food contamination is the major source of diarrheal spread.
With the support of GAVI, the Vaccine Alliance, UNICEF and WHO the government of Pakistan has recently implemented RVA vaccination in the EPI schedule of each of the four provinces (Punjab, Khyber-Pakhtunkhwa, Sindh and Baluchistan) of the country 23 . The disease burden due to RVA diarrhea is expected to be reduced in the future after vaccine implementation. However, there are many additional challenges involved in improving health facilities and socio-economic conditions of houses in highly populated areas. Moreover, introduction of a vaccine imposes a shift in circulating viral strains 24 . Therefore, continued surveillance of viral genotypes in circulation is necessary for effective control. Present study investigated circulating RVA strains in Nullah Lai and Rawal lake from twin cities (Rawalpindi/ Islamabad) during 2014 and 2015. Although surveillance model is in place for detection of Polio virus in water bodies, not enough evidence is available of RVA circulation in water bodies of Pakistan. The meteorological risk   factors associated with RVA persistence in water were also investigated. The overall prevalence rate of RVA in water during study period was 23%. The RVA detection rate in the present study is lower than the previous study from Karachi, which detected RVA in 35% of water filtration plants and 5% tap water using adsorption-elution virus concentration method with positively charged membranes 25 . On the other hand, an another study from Peshawar detected RVA in 9.47% of drinking and sewage water samples using adsorption-elution virus concentration method with negatively charged membranes 26 . Both studies used concentration methods that are expensive and costly in terms of implementation for routine surveillance purposes. The concentration methods used in this study are relatively inexpensive and less laborious that can be widely employed in a developing country like Pakistan. A number of concentration methods were evaluated for viral RNA yield and quality that can be used for detection of RVA using PCR amplification. Method III using 10% PEG for virus concentration along with TRIzol Method for RNA extraction was able to detect virus particles at 10 -8 dilution. Using this concentration method along with downstream processing, overall 23% of water samples were tested positive for RVA.
A seasonal trend was observed as water samples collected during winter and autumn months tested positive for RVA. It is in contrast to studies that reported persistence of RVA throughout the year in some other parts of the world including tropical countries 27 , but in accordance with studies that reported temporal trends in persistence of RVA in water with more RVA detection in winter and autumn months 28 .
Rotaviruses are a large genetically diverse population which varies in each country 20 . The first Rotavirus outbreak from Pakistan was reported during 1983-1985. Over the years, the RVA incidence and genotype diversity has changed ( Table 4). The most common G and P-genotypes affecting humans are G1, G2, G3, G4, G9, G12 and P [4], P [6] and P [8], respectively 29 . Previous studies from Pakistan have reported high prevalence of G and P genotypes including G1, G2, G3, G9, and P [4], P [6] and P [8], respectively 20 . In the present study two RVA genotypes (G3 and G9) were detected in water sample from Dhoke Naju locality in Rawalpindi. Sequence analysis of these strains indicated a close association (>99% identity) with RVA strains accession number KX 681821,KX681822, KX681823, MH279580, MH279581, MH279583, MH279585 detected in patients suffering from acute diarrheal illness in Rawalpindi during 2014-2016 20,21 , the same period as our study. G3 genotype has not previously been reported in Pakistan (Table 4). It was first detected during the year 2014-2015 and is now second most common genotype in patients suffering from RVA related acute diarrhea 20,21,30 . Similarly, our environmental G9 strains have high similarity with strains accession number KX702196, KX702198, KX702199, MH277395, MH277396 previously reported in Pakistan during 2014-2016 20,21,30 .
Nullah Lai basin is prone to flooding as happened in case of unprecedented rainfalls like in 2001. Muhallah Raja sultan and Dhoke Naju were badly affected by flooding 31 , while Rawal lake is a recreation point and source of water supply to Rawalpindi and Islamabad households. Frequently children take a swim in these waters. RVA is a highly contagious virus and its persistence in the water bodies can pose a serious public health threat in settings with poor sanitation and hygienic conditions and in less efficient water treatment plants 32 . During this study, www.nature.com/scientificreports www.nature.com/scientificreports/ water sample from Dhoke Naju was used for sequencing for RVA strains. Dhoke Naju locality is present along one of major converging points of streams in almost plain area of Nullah Lai basin. Nullah Lai may not be a direct environmental hazard to communities living along banks, but still can pose threat if this water is used for agriculture and bathing animals, as is frequently observed. Also, it could be an excellent source for monitoring changing genotypic diversity of rotavirus strains in twin cities that can advise vaccination campaigns. Although this study has constrains of short study period, small sample size and inclusion of only two sentinel sites, it is promising and can be used for disease surveillance on large scale. The above mentioned limitations should be considered for better understanding of disease surveillance in future studies.
This study concludes the first ever reported prevalence of G3 and G9 RVA genotypes in water bodies in Pakistan. It highlights the close association of environmental RVA strains with disease associated clinical isolates reported from hospitals during the same period. Shedding of viral particles in excreta in sewage water can be significant in determining the circulating genotypes prevalent in a community rather than investing huge funding in population surveillance. Moreover, it is an inert way to get the complete picture of circulating viral types in the community by indirect sampling and without involving patients/population at risk and community etc. These types of studies are highly advisable for enteric viruses' surveillance utilizing waste water bodies.

Methods
Sampling sites. The three main tributaries of Nullah Lai pass through less populated and also residential sectors of Islamabad, and conjoin as main Nullah Lai before entering into densely populated areas of Rawalpindi. The sampling sites were selected based on ease of access, proximity to drainage points and conjoining points of tributaries. For this study, twenty sites along Nullah Lai and its tributaries and one site along Korang river tributary and one from Rawal dam was selected (Fig. 3).
Sample collection. In total, 42 samples were collected. First phase of sampling was conducted from November 2014 to February 2015 and second phase of sampling was undertaken during the months of April 2015 to July 2015. One liter water was collected in duplicate, along the bank of Nullah, in clean, sturdy and clear bottles using Grab sampling method. Samples were transported in cold conditions to Molecular Virology laboratory at COMSATS University Islamabad campus and were stored at 4 °C.
Meteorological conditions. Meteorological data (temperature, relative humidity, precipitation, solar radiation, wind speed) for each sampling day was obtained from Pakistan Metrological Department (PMD) and pH of each sample was measured (Mi 150). Samples were further processed within 48 hours for RVA detection.
Virus concentration. Initially, three concentration methods were taken from literature to adopt a suitable method for this study. For this purpose, a surface water sample collected from Muhalla Raja Sultan locality (Supplementary Table I) was concentrated by these methods and their efficiency was determined by comparing the extracted RNA purity and concentration yield using a Nanodrop spectrophotometer (Implen Nanophotometer-Germany).
Method I. Pellet method: Concentration method of Kargar and coworkers was used 33 , where 100 ml of sewage water was centrifuged at 4000 rpm for 30 minutes at 5 °C. Tubes were kept cold in ice bucket and upper aqueous phase was removed until 4 ml of pellet solution was left. 1 ml of pure chloroform was added to this and pellet was dissolved by vigorous shaking. The tubes were centrifuged first at 200 × g for 20 minutes, then at 2000 × g for 10 minutes. Upper aqueous phase was shifted to a sterile tube and stored at −20 °C.
Method II. 500 ml of water sample was centrifuged at 1000 × g for 30 minutes. Supernatant I was removed and kept at 4 °C, while pellet was dissolved in 10 mL of supernatant I. 1 mL of chloroform was then added and centrifuged at 1000 × g for 5 minutes. Supernatant II was removed and combined with supernatant I. Then 0.2 M  Method III. This was a modified version of method II. Briefly, 500 mL of water sample was centrifuged at 4000 × g for 30 minutes. 0.5 M NaCl (Sigma) and 10% PEG 6000 (Fluka, Germany) was added to supernatant, respectively and stirred for 30 minutes at 4 °C with magnetic stirrer. After overnight incubation at 4 °C, the sample was centrifuged for 35 minutes at 7000 rpm at 4 °C (Backman model J2_21 centrifuge). Supernatant was drained and pellet was resuspended in 10 ml supernatant and mixed with 1 ml of pure chloroform and centrifuged at 2000 × g for 10 minutes at 4 °C. Pellet was discarded and PEG precipitation step (same as above) was repeated with the supernatant. The final pellet containing virus was dissolved in 1 mL of PBS. RNA extraction. Total RNA was extracted from 250 µl of concentrated water sample using 750 µl TRIzol LS (Invitrogen) according to established protocol 36 . RNA was eluted in 20 µl nuclease free water and stored at −20 °C.
Determination of detection limit of method III. The detection limit of the selected method, out of three, was then estimated by preparing suspension of two grams of Rotavirus positive fecal sample in 10 ml autoclaved distilled water. This dilution of original stool sample was considered 10° dilutions in this series and was then used to prepare twofold serial dilutions upto10 −10 dilutions.

Reverse transcriptase polymerase chain reaction (RT-PCR).
Purified RNA (5 µl) was reverse transcribed by using a 20 µl reaction mix of primers Beg 9 and End 9 (10 pMol/µl) (Supplementary Table II), 1 × RT buffer, 1mM dNTPs, 40 U of RNase inhibitor, 200 U of reverse transcriptase enzyme 33,37 . The reaction was first heated at 37 °C for 5 minutes, then 48 °C for 60 minutes and lastly 5 minutes at 95 °C. The concentration of cDNA was measured using Nanodrop spectrophotometer (Implen Nanophotometer-Germany) and reduced to final concentration of 50 ng/µl. VP7 gene amplification. VP7 gene of Rotavirus was amplified through Nested PCR. A reaction volume of 25 µl containing 1-2 µl cDNA, Beg 9 and End 9 primers (10 pMol/ul), 1 × PCR buffer, 0.5 mM dNTPs, 1.2 mM MgCl 2 and 1 U Taq polymerase enzyme was used. Amplification was carried out at following thermal cycling parameters: 95 °C for 5 minutes, 40 cycles of 94 °C for 30 sec, 48 °C for 45 sec, 72 °C for 1 min and final extension at 72 °C for 10 min 37 . Second round of Nested PCR was performed in 25 µl reaction volume containing 1st round PCR product, VP7F and VP7R primers (Supplementary Table II