Computational protocol: Low genetic diversity among historical and contemporary clinical isolates of felid herpesvirus 1

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Protocol publication

[…] Ten historical and fourteen recent Australian FHV-1 isolates, as well as two live attenuated vaccines currently available in Australia (Table ) were selected from our laboratory archive. Historical isolates were obtained from clinical samples submitted to our laboratory as a component of past diagnostic services. Recent samples were collected from shelter-housed cats in 2004 to 2006 with approval from the Faculty of Veterinary Science, Animal Ethics Committee at The University of Melbourne (reference number 0004055.1) and with the consent from the shelter owners. Viruses were propagated in cultures of Crandell Rees feline kidney (CRFK) cells [] for six passages, during which three plaque purifications were performed. CRFK cells are derived from a normal, female, 12 weeks old cat and are commercially available (ATCC CCL-94). Isolates selected for sequencing dating from 2004 to 2006 were all from animals that were negative for feline calicivirus, as determined by RT-PCR []. Viral nucleocapsid genomic DNA was purified and sequenced as described previously []. Libraries were prepared using 50 ng of viral genomic DNA with the Illumina Nextera DNA library preparation kits according to manufacturer’s instructions, and loaded onto an Illumina MiSeq. Sequencing was carried out using a 300 cycle V2 SBS kit (Illumina) in paired-end format. Reads were trimmed to an error probability limit of 0.5 % and mapped against the prototype FHV-1 sequence, strain C-27 [GenBank:NC_013590], using medium-low sensitivity options in the bioinformatics package Geneious V6.1.7 []. For comparison, a de novo assembly of the genome of the F2 strain of FHV-1 from the Feligen vaccine (Virbac) was also performed using the medium-low sensitivity settings in Geneious. The details of the sequencing metrics are provided in Additional file : Table S1. [...] Alignments of the complete genome sequences of the FHV-1 isolates, excluding the terminal repeat regions (TRS), were prepared using the Multiple Alignment with Fast Fourier Transformation (MAFFT) version 7 plugin in Geneious []. The F2 vaccine strain of FHV-1 was used as the reference sequence. The historical C-27 strain of FHV-1 was also included throughout the analyses. Phylogenetic analyses were performed on the complete genome sequences, excluding the TRS and large sequence gaps, using the neighbor-joining method in Geneious with the Jukes Cantor model of nucleotide substitution []. One thousand bootstrap replicates were used to assess the significance of the phylogenetic tree topology.To detect evidence of historical recombination events between the isolates, two programs were used, SplitsTree4 [] and RDP4 [], as previously described []. Short tandem repeat regions were identified in the aligned genomes prior to analysis using the Phobos plugin in Geneious V6.1.7. Default settings and score constraints for satellites with a maximum repeat unit length of 50 nucleotides were used. The identified repeat regions were removed from all viruses in the alignment as the methods used in this study do not allow the length of these regions to be accurately determined, and differences in the length of these regions could give rise to false positive recombination events being detected. The resultant whole genome alignments, as well as the separate UL, US and IRS genomic region alignments, were analysed for evidence of recombination. Splits network trees were generated with an uncorrected P characters transformation model, ignoring gapped sites. Other models were tested but yielded no significant topological differences. Statistical analyses of the recombination networks were performed using the Phi test as implemented in SplitsTree4 []. In RDP4 six different methods were used to assess the sequences for recombination breakpoints, MaxChi, Bootscan, SiScan, Chimaera, GENECONV and RDP, with default RDP4 settings used throughout. […]

Pipeline specifications

Software tools Geneious, MAFFT, SplitsTree
Applications Phylogenetics, WGS analysis
Organisms Felid alphaherpesvirus 1, Felis catus, Viruses
Diseases Conjunctivitis, Respiratory Tract Diseases