Sewage sample collection
Sewage sample (approx one liter) was collected and stored in sterile glass bottle from the collection site by directly dipping into the sewage effluent. The outer surface of the bottle was rinsed with 2% sodium hypo chloride solution, placed in a cool box, and transported to the laboratory. The bottle was frozen at − 20° c, until the process of virus isolation. This procedure is already reported in our previously published article (11).
Sewage sample processing
Sewage sample was concentrated by two-phase concentration method described previously (11). In brief, the pH of the sample was adjusted to 7.2 and the sample was centrifuged at 5000 g for 30 min at 4 °C to pellet the solids. 500 ml of clarified sewage was mixed with defined amounts of two polymers, dextran and 15% polyethylene glycol (15% PEG6000). The homogenous mixture obtained by vigorous shaking is left to stand overnight at 4° C in a separating funnel. This allows the polymers to separate in two distinct layers (phases) in the funnel. Enterovirus accumulates in the smaller bottom layer and/or at the boundary between the layers (interphase). The bottom layer and interphase were collected drop-wise. The pellet from the initial centrifugation was suspended in this concentration, and treated with chloroform, then centrifuged at 5000g for 30 min at 4°C and assayed for the presence of virus. The concentrated virus suspension was stored at − 20°C until used for virus isolation (19). This method was published in a study which compared three different methods, including direct isolation, centrifugation and two phase separation, and results of their study suggest that the two phase separation method is the best for maximum virus yield (20).
Inoculation of sewage samples
The extracted concentrate was inoculated on fresh monolayer cultures of L20B and RD cells in 50 ml (25 cm2) flasks. The cultured flasks were incubated at 37 °C and examined at every 24 h for cytopathic effect (CPE). Samples which showed CPE were frozen and thawed two times, then re-passaged on new cells. Samples were observed for CPE up to 7 days before being considered negative. Samples showed CPE were stored for confirmation through serotyping.
Human rhabdomyosarcoma (RD) and L20B (transgenic) cells were obtained from the Center for Disease Control and Prevention, Atlanta, GA, USA. Minimum essential media (MEM) of Earle’s salt solution and fetal bovine serum (FBS) were purchased from (Sigma Aldrich, USA). All cell culture media contained HEPES buffer, L-glutamine, sodium bicarbonate penicillin, streptomycin, amphotericin B from GIBCO, USA. Cell cultures were grown at 37 °C in incubators with supply of 5% CO2. Cell cultures for virus isolation were grown in 25cm2 plastic flasks (Costar, Corning, N.Y.) with 10% FBS (MEM) and maintained at 2% FBS (MEM) containing 7 ml MEM, and tissue culture tube with 1.5 ml (2% FBS).
Virus culture and Serotyping
E30 used in this study was isolated from environmental specimens. Virus stock was prepared by infecting RD cells in 25 cm2 flasks (Corning Inc. USA). Virus infected and mock infected cells were incubated at 36.5 ºC with 2% MEM. After 48 h of infection, the culture was aliquoted and kept at-80 ºC for further use. The virus was serotyped according to World Health Organization’s protocol (21) and confirmed by RT-PCR with specific primers (1).
Isolates grown in the RD cells were freeze-thawed three times and centrifuged at 12000 rpm for 10 minutes. The 5µl isolate supernatant was diluted with 10µl of distilled water. Primer E-1 and E-2 were used 50 pmol concentration as described previously (21, 1), the buffer containing 100 mM Tris-HCl, 15 mM MgCl2, 500 mM KCl (pH 8.3), 1 µL of diluted samples were added to the appropriate tube in the Bio-safety cabinet. The tubes were incubated at 95 ºC in the thermocycler for 5 min. and immediately chilled on ice followed by addition of 5 µl enzyme buffer containing 0.7 µl of 1M DTT (dithiothreitol), 6.9 µL of 40 U µl-1 Protector RNase inhibitor, 4.5 µl of 20 U µl-1 of the Avian Myeloblastosis Virus (AMV) Transcriptase, 13.7 µl of 5 U µl-1 Taq polymerase and 1.0 µl of 10 mM dNTPs. PCR tubes were placed in the thermocycler in the RT reaction at 42 ºC for 20 min. and for inactivation at 95 ºC for 3 min. For PCR amplification, 30 cycles of denaturation at 95 ºC for 45 sec, annealing at 55 ºC for 45 sec, and extension at 70 ºC for 45 sec, followed by cooling at 4 ºC has been used.
RT-PCR product purification
DNA was eluted from gel and has been purified by the QIAGEN gel extraction columns (QIAGEN, Chatsworth, CA, USA). PCR products were examined and randomly five E30 isolates selected for sequencing.
Sequencing reaction mix was contained, 4 μl of big dye terminator ready reaction mix, 2 μl of each primer (10 pmol λ-1), 3 μl Milli-Q water and 1μl of template (100 ng μl-1). PCR conditions followed (25 cycles) initial denaturation at 96 °C for 1min, denaturation at 96° C for 10 sec., hybridization at 50 °C for 5 sec. and elongation at 60 °C for 4 min. The ABI 3130 genetic analyzer and chemistry big dye terminator version 3.1 cycle sequencing kit were used for sequencing. Polymer and capillary array used POP_7 polymer 50 cm capillary array and BDTv3-KB-Denovo_v 5.2 protocol were examined, whereas data were analyzed by software Seq Scape_v 5.2 and reaction were examined in the Applied biosystem micro Amp optical 96-well reaction plate.
Construction of phylogenetic trees
Nucleotide BLASTn analysis (http://www.ncbi.nlm.nih.gov/BLAST) was used to identify related genes of the viruses and construct the phylogenetic tree. The reference sequences were obtained from the GenBank. The ClustalX version 2.0.12 (22) was used to perform multiple nucleotide alignment and tree was constructed (Njplot Version 2.3) (23), the neighbor-joining method according to the distances between all pairs of sequences in a multiple alignment. The confidence of sequence clustering was evaluated by bootstrapping (1000 replicates) (24, 11) in fig.1.