Computational protocol: Universal primers that amplify RNA from all three flavivirus subgroups

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Protocol publication

[…] Primers were designed using a strategy similar to that used by Vercruysse et al []. All available full-length flavivirus sequences were retrieved from NCBI in March 2005. Sequences were sorted using Bioedit [,] and aligned using ClustalX []. Several divergent sequences were identified by examining neighbour joining trees found using ClustalX and removed from the alignment. Conserved regions were identified by calculating redundancy scores [], and the average dominant base counts using an early version of the NCSF program and a window length of 20 bases []unpublished software, 2005; P. Wayper and M.J. Gibbs]. Average dominant base counts were calculated by summing the number of occurrences of the most common base at each position in the window and averaging those counts across all positions in the window. The distance between conserved regions was taken into account when selecting conserved sites as was the potential for using mixed bases or deoxyinosines, to enhance bonding at variable positions []. Standard nucleotides were favoured close to the 3' termini of the oligonucleotides. The primer sequences and their positions relative to the genome of Yellow fever virus (NC_002031) are shown in Table . Primers were synthesised by Geneworks (Hindmarsh SA, Australia). The primers target the end of the region encoding the methyltransferase and the start of the region encoding the RNA-dependent RNA-polymerase in the flavivirus NS5 gene.Virus stocks produced in the molecular virology laboratory at the University of Queensland, Australia were prepared from the supernatant medium of infected PS-EK cell cultures (Table ). PS-EK cells were grown in Dulbecco's Modified Eagle Medium with 10% Foetal Bovine Serum (Gibco, Carlsbad, California), 50U/mL Penicillin and 50 μg/mL Streptomycin (Invitrogen, Carlsbad, California). To remove cellular debris, the supernatant medium was centrifuged at 1,500 rpm for 5 min at 4°C. To increase the concentration, the virus particles were precipitated using a 40% Polyethylene Glycol (PEG) 8000 NTE solution (0.5 M NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The virus-PEG solution was stirred for 16–24 hours at 4°C, centrifuged at 10,000 rpm for 1 hour at 4°C then resuspended in NTE. Viruses tested at Oxford were prepared from the supernatant medium of infected 10% suckling mouse brain suspensions in PBS []. Viral RNA was isolated from both sources using RNAqueous kit according to the manufacturer's protocol (Ambion, Austin, Texas).One-step RT-PCR was performed using Superscript III in a 50 μL volume (Invitrogen, Carlsbad, California) with touch-down cycling conditions []. The final primer concentration in the RT-PCR was 1pmol per μL. A 40-min reverse transcription step was performed with incubations for 10 minutes at each of 46°C, 50°C, 55°C and 60°C. Enzyme activation at 94°C for 15 minutes was followed by the touch down PCR. During cycling, denaturation and extension were performed at 94°C for 15 seconds and 68°C for 60 seconds respectively. Annealing occurred for 30 seconds during each cycle, with one cycle at each of the following temperatures of 56°C, 54°C, 52°C, 50°C, 48°C, 46°C, 44°C and 42°C. After the touch down stage, 36 cycles with a 40°C annealing temperature, and then a final extension for 10 minutes at 68°C completed the programme. The reaction was held at 11°C until processing then stored at -20°C.The specificity of the primers was investigated by attempting amplification from cultures infected with viruses that are not flaviviruses, including Barmah Forest virus, Ross River virus, Influenza A virus, Human coronavirus NL, Human coronavirus OC43, Human adenovirus, Human bocavirus, Human rhinovirus 1, 2 or 3 and RNA from virus free cell cultures.RT-PCR products were cloned into the pGEM-T easy vector (Promega, Madison, Wisconsin) according to the manufacturer's protocol. Colonies were PCR screened for the presence of an insert. Positive colonies were grown overnight in LB with 1 μg mL-1 ampicillin. The plasmid was purified using a spin column kit (Qiagen, Eppendorf or Invitrogen) according to the manufacturer's protocol. Colony PCRs were performed using a step down protocol as described above although the extension temperature was 72°C (Invitrogen, Carlsbad, California). RT-PCR and PCR products were analysed on a 1% agarose gel containing ethidium bromide, and visualised using a UV transilluminator.Purified plasmid was sequenced using ABI BigDye Terminator Version 3.1 chemistry, on the AB3730xl sequencing platform. SP6 and T7 promoter primers were used for sequencing. Each virus clone was sequenced twice or more in the forward and reverse directions.Sequence data were assembled using Contig Express (Invitrogen, Carlsbad, California). Sequences were then compared to the GenBank non-redundant nucleotide database using BLASTN []; the programme identified the most closely matching sequences and produced alignments. Species and strain names were matched between the GenBank records and the virus isolates from which template RNA was extracted. RT-PCR reactions were considered to have been successful if the highest scoring alignment was made with a sequence from the expected flavivirus and the correct region of the genome. Publications were traced from the Genbank files to confirm that the sequences had been correctly named. Virus strain names were only used for those isolates where the strain had been identified by the International Committee on Taxonomy of Viruses (ICTV) []. If there was no relevant sequence information available in the GenBank database then the identification was based on phylogenetic analysis.Sequences of known species and strains, identified by the ICTV using their Genbank accession codes, were compiled with the sequences from the amplified products; sequences were then aligned using the default single step progressive method of the program MAFFT version 6.0 [,]. Maximum likelihood phylogenetic trees were found for the aligned sequences using the program PhyML []; a general time reversible model was used, nucleotide frequencies and the proportion invariant nucleotides were estimated from the data, and variable rates were allowed at different positions with four rate categories. Bootstrap analyses were done using the program PAUP version 4 [] using the maximum parsimony and neighbour-joining methods. […]

Pipeline specifications

Software tools BioEdit, Clustal W, BLASTN, MAFFT, PhyML, PAUP*
Databases ICTV
Application Phylogenetics
Organisms Japanese encephalitis virus, West Nile virus
Diseases Dengue, Encephalitis, Yellow Fever, Flavivirus Infections