Computational protocol: Mutation in Spike Protein Cleavage Site and Pathogenesis of Feline Coronavirus

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Protocol publication

[…] Clinical and demographic data are reported in ). Fecal samples from asymptomatic infected domestic cats were solicited from shelters and veterinarians throughout the United States. RNA was extracted by using QIAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA, USA). FCoV primers that detect most circulating strains were used to screen all fecal samples (). RNA extracted from FIPV-TN406 (Black) laboratory-adapted strain was used as a positive control.We analyzed 22 FIPV-positive tissue samples (Veterinary Pathology Archives, Cornell University, Ithacan, NY, USA) from 11 cats with FIP. Diagnosis of FIP was based on the standard method of immunohistochemical evaluation by board-certified pathologists. Each sample was retrieved from formalin-fixed, paraffin-embedded tissue blocks from which sections were stained by using FIPV 3–70 antibody (Custom Monoclonals, Sacramento, CA, USA). Positively stained regions were thinly sectioned and RNA was extracted by using RecoverAll (Ambion, Foster City, CA, USA).Fecal samples collected from FCoV-positive housemates, cats 234 and 304, were processed as previously described in this section. After the referring veterinarian made a diagnosis of FIP in cat 234, the owner elected to euthanize the animal. Fresh tissue was harvested and RNA extracted by using MagMAX Express (Life Technologies, Grand Island, NY, USA).For all samples, 50 μL reverse transcription PCRs (RT-PCRs) were performed with One-Step RT-PCR (QIAGEN) by using gene-specific S primers, encompassing S1/S2. The PCR primers sequences are found in Table 2. PCR conditions were 30 min at 50°C, 15 min at 95°C, and 39 or 35 cycles of 1 min at 94°C, 1 min at 55°C, 1 or 1.5 min at 72°C, and 10 min at 72°C. PCR products were purified by using a QIAquick Gel Extraction Kit (QIAGEN). Sanger sequencing was performed at the Life Sciences Core Laboratories (Cornell University). Nucleotide archive accession numbers are shown in Table 4. DNA sequences were translated into protein sequences and alignments were performed by using Geneious 5.4 (Biomatters Ltd., Auckland, New Zealand). Sequence logos were generated by using Weblogo 3.1 (http://weblogo.threeplusone.com/). Statistical analysis was performed by using 2-tailed Fischer exact test. In the test, the numbers of FIPV-infected and FECV-infected cats were counted. For each category of FIPV or FECV infection, cats harboring viruses with or without mutations at the S1/S2 site were counted. […]

Pipeline specifications

Software tools Geneious, WebLogo
Application Genome data visualization
Organisms Feline coronavirus
Diseases Feline Infectious Peritonitis, Mastocytosis, Systemic