Infection Of Cells With Fipv Essay

1308 words - 6 pages

Feline coronavirus (FCoV) is known to be prevalent and common infection in cat populations, with particularly high prevalence in catteries and multiple-cat households [1, 2, 3]. The genome is characterized as an RNA virus under the family Coronaviridae, order Nidovirales [3, 4]. It was first recognized in 1950s as a specific disease of cats [5] and its first occurrence in Malaysia in 1981 [6]. Two pathotypes of coronaviruses are described in cats; feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV). These FCoVs are spread world-wide and infect cats and other members of the family Felidae. FECV is the common form of FCoV, which causes from ...view middle of the document...

Feline coronavirus serotype I strain are difficult to grow in vitro; hence studies on these viruses have been limited in view of their fastidious growth in cell culture, whereas serotype II FCoV propagated more readily than serotype I in cell cultures. Growth of feline coronavirus in a continuous feline cell line of felis catus whole fetus-4 (Fcwf-4) which possess the properties of macrophages [15, 16, 17] was characterized by cytopathic changes and giant cell formation. After binding itself to specific receptor of infected cell, the virus enters the cells by fusion either with the plasma membrane or the endosome. The viral nucleocapsid is released into the cytoplasm of infected cell to become available for translation and transcription [13, 18].
Feline coronaviruses have been described as large plemorphic particles with numerous spike-like projections extending from their envelope. Particle measurements ranged from 75-150 nm in diameter. Surface projections have been observed to vary in size and shape reported being between 12 -25 nm [7, 19, 20] The aim of this study was to describe the physical properties of FCoV UPM C11/08 isolates and the ultrastructure of Fcwf-4 cell line following infection with this local isolate of feline coronavirus.
A prototype of local isolate FCoV designated as UPM 11C/08 was used in the study. The virus was isolated from a cat presented at the University Veterinary Hospital, Universiti Putra Malaysia (UVH-UPM) with effusive-form FIP. Ascitic fluid was obtained from the cat and screened for FCoV with RT-PCR assay using primers targeting the untranslated region of 3’UTR [22]. The sample was adapted and propagated in Fcwf-4 cell culture until characteristic CPE was observed and stored at -20oC (Sanyo, Malaysia) for further purification by sucrose gradient.

Cell culture
Fcwf-4 cell culture was obtained commercially (ATCC CRL-2787) and maintained in Eagle's minimal essential media, supplemented with 15% heat-inactivated fetal bovine serum (FBS) (Gibco,UK), 100 IU of penicillin/ml, 100µg of streptomycin/ml, and 2.5 µg of amphotericin/ml. Cultures were maintained in a humidified incubator at 37°C with 5% CO2 (Galaxy, UK ).

Virus inoculation
A total of 20 flasks (150cm2) (Nunc, Denmark) containing confluent monolayer of 3 days-old Fcwf-4 cell were infected with 100µl of purified virus stock for each flask, where 5 flasks were kept as control received only saline. All infected and control flasks were incubated at 37oC for 1 hour to allow virus adsorption before adding the maintenance media containing 2% FBS. The cells were further incubated and examined daily for cytopathic effect (CPE). For transmission electron microscopy (TEM) analysis, two infected and one control flask was removed consecutively at 6, 10, 15, 24 and 48 hours post infection (pi).
Virus purification
The virus stock was purified by sucrose gradient for the purpose of cell culture infection for...

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