Computational protocol: Sequencing of emerging canine distemper virus strain reveals new distinct genetic lineage in the United States associated with disease in wildlife and domestic canine populations

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Protocol publication

[…] Samples were submitted between 2010 and 2014 to the UTCVM Clinical Virology Lab for canine distemper virus detection. Submissions were mainly canine from eastern Tennessee, but samples from Canada, Texas, Washington, Kentucky, West Virginia, Virginia, and South Carolina were also tested. Samples were also submitted for wildlife including raccoons and foxes from Tennessee.RNA was extracted from clinical samples (including nasopharyngeal/conjunctival swabs, urine, and tissues) and cell culture supernatant containing virus with a commercially available extraction kit (QIAamp Viral RNA Mini Kit, Qiagen, Valencia, CA, USA) as previously described []. RNA previously extracted was stored at −80 °C.Samples were tested for CDV by real-time RT-PCR. cDNA from positive samples were genotyped by sequencing the variable M-F intergenic region, and a phylogenetic tree was constructed according to previously described methods [].Samples used for genome sequencing included CDV sample number 13–1941, which was isolated from lung that had been stored at −80 °C from a fox that displayed neurologic signs prior to euthanasia and tested negative for Rabies virus. It was submitted to the pathology department for disposal from the Avian and Zoological Medicine service in the UTCVM. The virus was cultured on Vero SLAM cells (kindly provided by Dr. Edward Dubovi, Cornell University, NY, USA). CDV sample number 13–2262 was collected via urine from a 6 month old puppy from South Carolina with clinical signs consistent with canine distemper, and the sample had a real-time RT-PCR Ct value of 13.11. This strain was not isolated and whole RNA from the urine sample was used directly for genome amplification.Approximately 50 bp overlapping primer sets with ~1000 bp products were designed using Primer 3 [, ] to amplify complete CDV genome sequence (minus the extreme 5’ and 3’ non-coding ends) (Table ). Two μL of RNA per sample were run in 25 μL total volume reactions using a commercially available master mix (SuperScript III Platinum One-Step RT-PCR kit, Invitrogen, Life Technologies, Grand Island, NY, USA) using 300 nM of each primer and one unit of RNAse inhibitor (RNAse Out, Invitrogen, Life Technologies, Grand Island, NY, USA) for RT-PCR. Samples were amplified in a thermal cycler (GenePro, BIOER Technology, China) with a RT step at 50 °C for 30 min., activation step for the hot start Taq polymerase at 94 °C for 2 min., followed by 35 cycles of denaturation at 94 °C for 30 s., annealing at 60 °C for 1 min., and elongation at 72 °C for 3 min., with an additional elongation step at 72 °C for 10 min. The RT-PCR products were electrophoresed on a 1 % TBE agarose gel stained with SYBR Safe®, and visualized by SYBR© Green-filtered UV light with a CCD camera system (UVP, Inc., Upland, CA, USA). Products with a single band were purified using ExoSAP IT (Affymetrix, Santa Clara, CA, USA). Products with more than one band but a single clear product at ~1000 bp (3/15 sets) were excised and gel purified (QIAquick gel extraction kit, Qiagen, Valencia, CA, USA) and all products were capillary sequenced at the UT Molecular Biology Core Facility using the same primers used for the PCR reactions.Chromatograms for capillary DNA sequence were manually edited and assembled using Geneious©, and all positions in the sequence had at least 2x coverage. Available reference genomes and H genes representing the major CDV lineages were downloaded from GenBank and aligned to CDV 13–1941 and CDV 13–2262 using MAFFT v7.017 []. Nucleotide substitution model GTR was selected using ModelGenerator v0.85 [] and phylogenetic trees were constructed using MRBAYES v2.0.9 (genomes) [] with 1,000,000 iterations with subsampling every 1000 trees and a burnin of 20,000 iterations [] or Neighbor-Joining method (H gene). Individual coding sequences (CDS) were predicted using GLIMMER3 v1.4 [], extracted, translated and aligned to representative sequences from GenBank. Phylogenetic trees for individual protein sequences were generated using UPGMA in Geneious© Tree Builder (version 6.1.4, Biomatters http://www.geneious.com) with the Jukes-Cantor distance model.For evaluation of antigenicity, blood was collected from 5 adult dogs that are part of the UTCVM teaching colony, according to Institutional Animal Care and Use Committee guidelines (University of Tennessee Knoxville IACUC protocol 1954). Samples were collected from the animals just prior to receiving a Nobivac vaccine (Merck Animal Health, Summit, NJ, USA) and again 5 days following vaccination. Each dog had been given the same vaccine approximately 1 year prior. Serum was separated from the cells and stored at −20 °C. Neutralization titers were determined by a standard procedure [] using 100 TCID50 per well of the fox isolate (13–1941) or Nobivac isolate. The Nobivac isolate was prepared in the same manner as described for the fox isolate. Samples were run in triplicate and each test was duplicated to evaluate reproducibility. A Mixed ANOVA method (SPSS Software, SPSS Inc, Chicago, IL, USA) was used to compare serum neutralization titers between the viruses and the two time points. In order to determine which pairs of means were significantly different, mean separation tests were run using the Tukey-Kramer method at a significance level of p = 0.05. […]

Pipeline specifications

Software tools GenePro, Geneious, MAFFT, ModelGenerator, MrBayes, Glimmer
Application Phylogenetics
Organisms Canis lupus familiaris, Canine morbillivirus
Diseases HIV Infections