Feline Coronavirus (FCoV): Development of an ELISA for detection of antibodies against serotype I and II
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The Feline Coronavirus (FCoV) belongs to the family Coronaviridae, order of Nidovirales. (Chang et al., 2012). Antibodies against FCoV are found in 20-60% of pet cats and up to 100% in catteries or multi-cat households. (Sharif et al., 2010) The FCoV contains three proteins in its viral envelope: spike glycoproteins (S), transmembrane proteins (M) and the envelope protein (E). (Le Poder, 2011, Belouzard et al., 2012). The S-protein is the most abundant protein. It consist of a S1 and S2 part of which the S1 part is the most divergent. There are two serotypes (I and II) and 80-90% of FCoV infections are caused by serotype I. Serotype II resulted from a recombination between serotype I FCoV and CCoV. Serotype II obtained the S-protein from the CCoV and the rest of the genome from serotype I FCoV. (Le Poder, 2011, Chang et al., 2012, Woo., et al. 2010). Classification based on pathogenicity are the biotypes: Feline Enteric Coronavirus (FeCV) and feline infectious peritonitis virus (FIPV). (Vogel et al., 2010). Only about 10% of the infections with FCoV results in the fatal systemic disease FIP which is caused by a mutation in the FeCV that enables it to enter macrophages and cause a systemic disease. Serotypes can be distinguished by RT-PCR and a virus neutralization test (VNT) but these tests are expensive and take a lot of time. For this project an ELISA was developed to distinguish serotype I and II infection based on antibody detection, which is fast and cheap. With the ELISA the prevalence of serotype I and II was determined for a population of cats. This is important for the sero-epidemiological features of the FCoV. The VNT was used as a gold standard to compare the ELISA results with. Sera were selected from the serum bank. Some of these sera were already screened in a VNT against type 1 and 2 viruses in a previous study (A. blanken). All the sera with a unknown Immune Fluorescence Assay (IFA) titer were screened for antibodies against the FCoV with the IFA. Samples were considered to be seronegative with a titer of < 1:20. Most of the seronegative samples were excluded from the rest of the project. IFA positive sera were subsequently screened in the VNT. Next an ELISA was developed by coating the plates with the whole S1 protein of serotype I (UU23-S1) and serotype II (SeroII-S1). First the sera with a known VNT titer were tested to validate the ELISA. Next all the sera were screened with this ELISA. Followed testing whether the N-part was more discriminative by coating the ELISA plates with serotype I-N (UU23-N-S1) and serotype II-N (SeroII-N-S1). Based on the results, which showed a high background, it was decided to optimize the ELISA conditions by comparing two different block buffers: BSA1% and NGS10%. The IFA results showed that 36.8% of the tested field samples were considered to be seronegative. The ELISA results showed that it was able to discriminate between serotype I and II by determining the ratio of antibody titers for type 1 and 2. All serotype I samples had a ratio I /II of ≥1 and all the type II samples a ratio I /II of <1. Of 80 field samples tested only one serum sample was positive for serotype 2.The estimated seroprevalence of type II was 1.25% and 98.75% for type I for 80 field samples. There was no strong correlation between VNT titers and ELISA titers. After performing ELISA with N-coated plates of serotype I and II, it appeared that the N-part was not more discriminative than the whole S1 protein of both serotypes. However this was only tested for 11 samples. The overall conclusion is that plates coated with UU23-S1 and SeroII-S1 can distinguish serotype I and II infections. The results of the optimization of the ELISA by comparing two block buffers, showed that there was no significant difference between using BSA1% and NGS10% as a block buffer. In the future more information should be gathered about the tested samples to see whether a correlation can be found between the results of the ELISA, symptoms and the strain that infected the cat. Also the ELISA conditions could be optimized by increase the blocking time of the plates or changing the substrate for instance. For this project, only the N-part of both serotypes was tested. The C-part of both serotypes could also be included. Also more samples should be tested with the N- part of serotype I and II to determine whether the N-part is more discriminative. One sample appeared to be a lion sample, which indicated a negative result on the VNT but positive for serotype II in the ELISA. More lion samples could be tested to see whether more lion samples give the same results.