W16-024 Exploring humoral responses to non-structural proteins of feline coronaviruses. (Winn Funded Study), interim progress report
Principal Investigator: Dr. Magdalena Dunowska, Massey University, New Zealand
This study has been designed to test the following hypothesis: Cats infected with feline coronavirus (FCoV) develop humoral immune responses to selected non-structural proteins of the virus. The targets for such immune responses differ between cats with different disease outcomes.
The following has been achieved up to date (20 Jan 2017):
- Development and validation of diagnostic assays for the use in the study:We have developed a real-time PCR assay to detect FCoV RNA. The sensitivity of the assay was determined to be equivalent to 1 copy of viral cDNA based on serial dilutions of cloned PCR product. We have also compared three different methods of RNA extraction in order to select the best RNA isolation method. Finally, we have selected a suitable serological assay for the use in the study.
- Enrolment of cats with various FCoV infection statusCats with confirmed FIP (n=5), FCoV serologically-positive healthy cats (n=5) and an FCoV serologically-negative cat (n=1) were enrolled into the study. Clinically normal cats were sourced from cats belonging to the Massey University Feline Nutrition Unit. All five selected cats represent surviving siblings of cats that had succumbed to FIP in the past. Samples from FIP cats have been sourced via local veterinary practices.
- Design and production of peptide library for use in the studyThe peptide library was designed in collaboration with scientists from LLC Biosciences. Each library included 28,426 12-mer sequences covering all available variants the entire polyprotein 1ab of FCoV, with 1 amino acid walking distance. Custom peptide chips were commercially synthesised by LLC Biosciences.
- Screening of the peptide chips with feline sera
Each chip was hybridised with the following samples:
- Chip1: Control serum from an FCoV antibody negative cat
- Chip 2: Pooled sera from FCoV antibody positive healthy cats
- Chip 3: Pooled sera from FCoV antibody positive FIP cats
There was minimal binding of the negative control serum to Chip1, with clear binding of feline sera detected on Chips 2 and 3. Sera from FIP cats (Chip 3) appeared to recognise more antigens with stronger binding to selected peptides than sera from healthy cats (Chip 2).
These results, while encouraging, need to be further confirmed. They could represent true differences in the antigen recognition between FCoV antibody positive healthy and diseased cats. However, they could also simply reflect individual differences between cats included in each pool (irrespective of health status) or different levels of FCoV antibodies present in the samples. We are currently in the process of developing ELISA assays to screen serum samples from individual FIP-affected and healthy cats against selected peptides.
Report provided by Dr. Dunowska