It is a retrospective cross-sectional study designed to investigate mutations that are common in patients of acute flaccid paralysis. The permission to undertake this study was obtained from the Ministry of Health, Uganda, the owner of the specimens and the Uganda Virus Research Institute, Research and Ethics Committee approved the study.
Archived poliovirus isolates from AFP patients aged 1 day–180 months were identified through the routine national AFP surveillance system. The Sabin poliovirus 1 and 3 that were investigated were collected between 2010 and 2016 before the switch from trivalent OPV (OPV 1, 2, and 3) to bivalent OPV (OPV1 and 3). The incidence of AFP patients during this period ranged from 2.3 to 8.5% (data not published). The percentage described represents the number of AFP patients who were shedding the poliovirus compared to the total number of AFP patients who were reported annually. The highest rate was observed in 2016 and corresponded to the period of enhanced polio campaigns prior to the switch.
Virus isolation
Poliovirus isolation was primarily characterized through cell culture using the rhabdomyosarcoma (RD) and mouse-derived L cell lines (L20B) [22]. The poliovirus type was then confirmed using real time Reverse Transcriptase Intra-Typic Differentiation and Vaccine-Derived Poliovirus assays (rRTPCR ITD and VDPV assays) [23].
RNA extraction
RNA extraction was performed from archived virus isolates using QIAamp Viral RNA mini kit (Qiagen) according to the manufacturer’s protocol.
Sabin RT-PCR procedure
The procedure was adopted and optimized using the standard protocol for VP1 gene characterization (CDC Laboratory). The Qiagen One step RT-PCR was used according to the manufacturer’s protocol. Briefly the RT-PCR amplifying a 1.1 kb encompassing the VP1 region was performed in a 50 µl reaction that consisted of: 30.5 µl RNase free water; 10.0 µl 5 × Qiagen One Step RT-PCR buffer; 2.0 µl dNTPs (10 mM each); 2.0 µl 5 × Qiagen One Step RT-PCR enzyme mix; 1.0 µl Y7R primer (40 pmol/µl), 5′GGTTTTGTGTCAGCITGYAAYGA-3′; 1.0 µl Q8 primer (10 pmol/µl), 5′AAGAGGTCTCTRTTCCACAT-3′; 0.5 µl RNase Inhibitor (40 U/µl), and 3.0 µl of the template (RNA extract). The thermo-profile condition used for the RT-PCR reactions was as follows: reverse transcription 50 °C for 30 min, RT enzyme inactivation 95 °C for 15 min; amplification: 94 °C for 30 s, 45 °C for 30 s, 72 °C for 1 min × 35 cycles; final extension 72 °C for 10 min; and finally left to hold at 4 °C.
Gel electrophoresis
One gram (1.00gm) of agarose powder was weighed and added to 100mls of 0.5% TBE buffer which was heated and cooled. Four micro-litres (4.0 µl) of ethidium bromide were added to the gel and a comb positioned in the casting tray to create wells for the samples. After the gel had solidified, the PCR products were then loaded onto the wells. A 100 bp molecular DNA ladder marker and a positive control specimen were added for amplicon sizing to ensure that the correct virus fragment was amplified. The PCR products were run at 130 V for 45 min in the electrophoresis tank. After gel electrophoresis, the 1.1 kb DNA products were viewed under ultraviolet light and the images were taken for documentation. The DNA products were stored at 20 °C till further testing.
Cleaning DNA
The Invitrogen Charge Switch PCR Clean-up Kit was used according to the manufacturer’s protocol. Briefly, binding of cDNA was achieved when 50 µl of the purification buffer and 50 µl PCR product together with 10 µl of the Charge Switch magnetic beads were mixed in a microcentrifuge tube and incubated at room temperature for one minute. The mixture was then placed on a Magna rack and the supernatant was removed and discarded. The complementary DNA (cDNA) bound to the beads was washed twice using 150 µl wash buffer for each sample. The sample was again removed from the rack and 50 µl of the elution buffer was added. The beads and buffer were mixed by gentle pipetting. The mixture was placed back on the Magna Rack and incubated for 1 min. The supernatant which contained the purified cDNA was collected, quantified using nanodrop, and stored at − 20 °C.
VP1 region sequencing
The Big Dye Terminator v3.1 cycle sequencing kit was used according to the manufacturer’s protocol. Briefly, a 10.0 µl sequencing reaction was set up consisting of: 5.0 µl RNase free H20, 1.0 µl sequencing buffer × 5, 1.0 µl primer (3.2 pmol) (primarily 246S/249S/249A/Q8 for SL1 or 248S/251S/ 261A/Q8 for SL3), 2.0 µl Big dye, and 1.0 µl [cDNA] (20-40 ng). The sequencing positive control reaction consisted of: 4.5 µl dH20, 1.0 µl Seq. buffer, 1.25 µl primer M13, 2.0 µl Big dye, and 1.25 µl pGEM DNA. The following thermo-profile condition was used for the sequencing reactions: amplification 95 °C for 15 s, 42 °C for 15 s, 60 °C for 4 min for 25 cycles and left to hold at 4 °C. The DNA was then stored at − 20 °C.
Cleaning sequencing products
Sequencing product cleanup was performed using the Agencourt ‘Clean SEQ according to the manufacturer’s protocol with a modification. Briefly, 10 µl of the magnetic beads were added to each specimen. Forty-two (42) µl of 85% ethanol was added to the specimen and mixed to form a homogeneous mixture. To enhance DNA binding the plate was left for 5 min before placing on the magnet (modification). The plate was then placed on the Magnetic Agencourt block for 5 min. The supernatant was removed, and each well was washed twice using 85% ethanol while the plate was on the magnet. The plate was left to dry for 10 min at room temperature. Thereafter, 42 µl of 0.1 mM EDTA (elution buffer) was added, and the plate was incubated at room temperature for 5 min to elute the DNA. Thirty (30) µl of the clear solution that contained the eluted DNA was carefully removed with a pipette ensuring that there was no bead carry over. The DNA product was transferred into the new wells ready for loading onto the detector. The plate was sealed with a septae and stored at − 20 °C until it was run on the 3500 ABI Genetic analyzer.
Data analysis
The full VP1 contigs were assembled using Sequencher software 4.10.1. Alignment of the consensus sequences and phylogenetic inference were performed using Mega 7.0 sequence analysis software [24]. Conservative and non-conservative mutations in VP1 were then identified in each independent alignment. The identification of nucleotide and amino acid mutations was performed by tally when changes in nucleotide and amino acid residues were compared to the reference Sabin poliovirus 1 and 3. Mutations were captured as point nucleotide and amino acid differences in the study VP1 sequence alignments.
Mutations from previous circulating VDPV outbreaks
Poliovirus VP1 sequences from outbreaks linked to circulating VDPVs [25] were downloaded from the National Center for Biotechnology Information (NCBI) and analyzed. Fifty-nine (59) and 24 sequences of SL1 and SL3 were available. The study VP1 mutation sites described were compared with those identified in viruses from the reported outbreaks.
AFP surveillance system
The study specimens investigated were kindly obtained from the routine national AFP surveillance system and the data reported was obtained from the “poliomyelitis/ AFP investigation data collection tool for acute illness”. The clinical presentation, immunization history, together with the preliminary specimen tracking were obtained from the collection tool. Sabin follow-up was performed for the AFP cases in whom the Sabin-like viruses were isolated.