Virus isolation, evolution and screening of the effective antiviral drugs of feline coronavirus infection in Taiwan
Date Issued
2009
Date
2009
Author(s)
Lin, Chao-Nan
Abstract
Feline infectious peritonitis (FIP) is one of the major infectious causes of mortality in kitten. The causative agent, feline coronavirus (FCoV) is also associated with mild or subclinical enteric infections. Despite the high prevalence of FCoVs in feline populations around the world, only 5-12% of seropositive cats develop FIP. Up to present, there is not a single virological or serolsogical assay that can distinguish virulent from avirulent virus, moreover no effective vaccine or therapy available for FIP. The objectives of this study were to investigate the prevalence of FCoV infection in Taiwan (part I), to further clarify the correlation between FIP, ORF 7b (part II) and different types of FCoV S gene (part II), to isolate the first local FCoV isolate (part IV), to explore the evolutionary insights of type II (part V) and to screen the effective compound for their antiviral activity against a local FCoV strain in fcwf-4 cells. During 2000 to 2009, a total of 1703 cases with a history of FIP-suspected cats from National Taiwan University Animal Hospital were subjected to this study (Part I). FCoV RNA was detected from 651 (38.2%) cats. Among the 67 cases with definite diagnosis of FIP, the highest incidence was found in kittens under 6-months old (50.7%, 34/67). Significantly highest viral load in the effusive fluids of cats from confirmed FIP cases. Eighty-four clinical healthy cats co-hatitated with the FIP cats from various multi-cat households were subjected to further examination. About 67.9% (57/84) of the clinically healthy cats were FCoV positive. These data suggested that the prevalence of FCoV infection is widely in Taiwan and especially in multi-cat population. The open reading frame (ORF) 7b of FCoV has been speculated to play a determining role in virulence as deletions were found to be associated with avirulent viruses. Our part II study revealed that deletions in the ORF7b gene are not constrained to low pathogenicity/enteric biotypes but also associated with pathogenicity/FIP biotypes of FCoV. On the basis of in vitro neutralization tests, FCoVs can be divided into two serotypes. Our results showed that irrespective of the predominance of type I FCoV infection in Taiwan, type II FCoV demonstrated a higher correlation with FIP. Analysis of partial S gene sequences of the local type I and II FCoVs strains revealed that type I viruses were more genetically divergent (6.2-11.7%) than type II viruses (0.6-3.2%) within the 5-year study period (part III). By co-cultivation of pleural effusion from a 4.5-month-old FIP kitten with feline fcwf-4 cells, a local FCoV was isolated. This novel FCoV isolate with distinct growth characteristics is denominated as FCoV/NTU156/P/’07 (NTU156), the first FCoV strain in Taiwan (part IV). We further explore the evolutionary insights of local FCoVs, a complete genome of one local type II FCoV NTU156 and partial genome sequence from one type II virus NTU26-2 were determined. The genome of FCoV NTU156 was found to be 28,897 nucleotides in length, excluding the 3’ polyadenylated tail. Analysis of the sequence identified conserved open reading frames and revealed an overall genome organization similar to known FCoVs. Bearing an in frame deletion of 442 nucleotides in the ORF3c, the genome size of NTU156 was found to be the smallest among subgroup 1a (FCoV Black, FCoV C1Je, and FCoV 79-1146). Bootscan analysis of NTU156 revealed two crossover events took place between type I FCoV and type II CCoV. One of the possible recombination site was located in RNA-dependent RNA polymerase (RdRp) gene and the other in M gene. Analysis of sequence around possible recombination spot of another local type II virus NTU26-2 identified a possible recombination site located in M gene. Comparing the finding from our local viruses with the prototype FCoV 79-1146 strain, of which one possible recombination was located in RdRp (further upstream 900 nucleotides) and the other in ORF 3-E gene, neither of them were identical to our local strains. These data indicated that each type II FCoV strain might has arisen from individual recombination event. Multiple alignments further narrowed the potential hot spot of RNA recombination to a pentanucleotide U(C/U/G/A)U(U/A)A (part V). In our search for agents that may prove clinically effective against FCoV infection, a local FCoV strain was isolated and subsequently used in the screening tests. Our results show that combined use of Galanthus nivalis agglutinin and nelfinavir to FCoV-infected cells, a synergistic antiviral effect with complete blockage of viral multiplication was observed. These results suggest that the combined use of GNA and nelfinavir has therapeutic potential in the prophylaxis and treatment of cats with early-diagnosed FIP (part VI).
Subjects
feline coronavirus
genotype
virus isolation
virus evolution
antiviral agent
SDGs
Type
thesis
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