key: cord-257974-kllqjn68
authors: Woods, Roger D.; Wesley, Ronald D.
title: Cultivation techniques for animal coronaviruses: Emphasis on feline infectious peritonitis virus, canine coronavirus, transmissible gastroenteritis virus, and porcine hemagglutinating encephalomyelitis virus
date: 1988
journal: J Tissue Cult Methods
DOI: 10.1007/bf01404139
sha: 
doc_id: 257974
cord_uid: kllqjn68

Techniques are described for the growth and characterization of some mammalian coronaviruses. Because of the fastidious nature of their growth requirements, most will replicate only in cells derived from the natural host or a closely related species. Fetal cat cells are used to grow FIPV, and porcine cells are used to grow TGEV and HEV. However, CCV will replicate in both feline and canine cells. Although all four of these viruses prefer to replicate in a cell in the stationary phase of growth, FIPV is able to replicate in an actively growing cell. Each virus causes a cytopathic effect in monolayer cell cultures under agar or media 18 to 72 h postinfection. Primary isolation of each virus from field specimens is difficult, although most can usually be isolated after 1 to 3 blind passages in the cell culture.

The Coronaviridae family of viruses has a worldwide distribution. These viruses cause economically important diseases in man and in domestic and laboratory animals {33). At the present time the group consists of 14 recognized viruses and 4 or more unclassified isolates. The viruses and their abbreviations are listed in Table 1 . These viruses comprise four distinct antigenic groups plus a miscellaneous group (35) . Generally these viruses infect epithelial cells of the respiratory tract [human coronavirus (HCV), infectious bronchitis virus (IBV), rat coronavirus (RCV), porcine respiratory coronavirus (PCV)] and epithelial cells of the gastrointestinal tract [bovine coronavirus (BCV), canine eoronavirus (CCV), transmissible gastroenteritis virus {TGEV), turkey coronavirus (TCV), feline enteric coronavirus (FECV), human enteric coronavirus (HECV)]. In addition, feline infectious peritonitis virus tFIPV), mouse hepatitis virus (MHV), rabbit coronavirus (RbCV), and hemagglutinating encephalomyelitis virus (HEV) exhibit other tissue tropisms and alternate pathogenic mechanisms (40) . The feline, murine, and avian coronaviruses will usually cause a mild or inapparent infection in adults, hut usually a severe diarrheal disease and often death in neonatal animals (40) . Basic studies on these viruses have been limited because of their fastidious growth in cell culture (14) . Most coronaviruses will grow only in cells derived from the natural host animal or in cells from a closely related species (33, 35) . Growth of HCV, HECV, and RbCV in cell culture is difficult, whereas (Fig. 1 c, d) . After CPE is observed, freeze each flask at --75 ° C. When grown under these conditions, TGEV will have a plaque titer of approximately 7 X 107 pfu/ml 18 to 20 h after inoculation, and HEV will have a plaque titer of approximately 3 X 107 pfu/ml 24 to 48 h after inoculation.

To study the pathogenesis and molecular biology of coronaviruses, it is beneficial to identify cell lines in which the viruses will grow to titers greater than 5 X 10 s pfu/ml, thus providing sufficient viral mass for molecular studies. At the present time, most of the animal coronaviruses have been adapted to cell culture (14,33,35}. The methods presented in this report are easy to reproduce and could be adapted by laboratories worldwide for in vitro growth of FIPV, CCV, HEV, and TGEV. Such procedures have been used to grow these viruses to titers ranging from about 1 X 106 pfu/ml for FIPV (43) to 1 X 109 pfu/ml for TGEV ~16L

Although most coronaviruses can now be grown in cell culture, their primary isolation from field specimens is still difficult I14}. No one isolation technique or method can be used for the entire group. Probably the most successful isolation method is to blindly passage 0.2-pm-filtered fluids 1 to 3 times in a host organ cell line known to support growth of suspected virus, until a CPE is observed. The use of monospecific fluorescent antibodies is recommended to follow in vitro growth of the virus and to confirm virus identity. Several techniques have been used to enhance or improve the chances of isolating these viruses. Some of these techniques include addition of pancreatin 10.5%}, trypsin (10 pg/ml}, or DEAE-dextran to cell culture media at the time of initial inoculation, maintaining a slightly acidic pH in the culture media, the use of primary cell cultures instead of secondary cultures, and incorporation of high titer hyperimmune antisera in the culture media to suppress the growth of contaminating viruses.

Coronavirus-induced CPE is dependent on the virus, cell line, and isolate. The CPE ranges from an inap-parent infection in persistently infected cell lines to nearly complete cell disruption with several cell cultureadapted viruses 114,42). In a typical coronavirusinfected cell culture the first sign of CPE will be the appearance of granular and refractile cells, followed by formation of enlarged rounded cells, ballooned cells, and finally the detachment of infected cells from the culture flask 119). When grown under the conditions described, optimal viral titers are usually obtained when approximately 25% of the cell sheet is observed with CPE.

Both FIPV and CCV induce a similar CPE in CRFK ceils. Thirty-six to 48 h postinfection (PI), FIPVinfected CRFK cells are granular and refractile. Over the next 24 h small multinucleated ~8 to 10 nuclei~ cells are formed by fusion and then they detach from the flask 19~. The CPE is focal and under 0.5% agar-MEM-2% FBS, distinct plaques up to 1 mm in diameter form in the cell sheet in 48 to 72 h. Initial CPE in CCV-infected CRFK appears within 12 h and consist of granular, refractile, and amorphorus multinucleated tgreater than 10 nuclei) cells. During the next 12 to 24 h the CPE will spread over the entire sheet producing numerous ballooned cells and finally infected cells detaching from the flask. Under 0.5% agar-MEM, CCV will form distinct plaques 1.5 mm diameter within 48 h ~5).

Initial CPE produced by attenuated and virulent TGEV in ST cells is similar to that observed with FIPV and CCV in CRFK cells, whereas CPE produced by HEV in SPTh cells is slightly different. Sixteen to 18 h PI, TGEV infected ST cells are granular, refractile, and greatly enlarged. Over the next 12 to 16 h the infection will spread over the cell sheet, and with advancing infection the round cells become ballooned and detach from the flask. Under 0.5% agar-MEM, attenuated TGEV produces clear uniform plaques up to 3 mm in diameter within 48 h, whereas the virulent virus produces diffuse, irregular plaques approximately 0.5 to 1 mm in diameter within 48 h I19,42). In 24 to 48 h PI. HEV infected SPTh ceils the CPE will show small areas of syncytia, which are easily visible with an inverted microscope. Over the next 24 h the syncytia degenerates, producing syncytial debris in the culture medium and clear holes with an opaque irregular shape in the cell sheet (2) . After disruption of the syncytia, ballooned structures appear and the formation of new syncytia is seldom observed. A hemadsorption plaque assay has been developed for titration of infectivity of HEV t24L

The CRFK cell line has been used for the primary isolation and growth of FIPV. Isolation of FIPV from clinical specimens is very difficult using cell lines. However, virus can frequently be recovered if CRFK cells are inoculated and blindly passaged 1 to 3 times in the presence of trypsin (43i. The incorporation of trypsin into the FIPV growth medium enhances both the isolation and growth of FIPV. The reason for this enhancement is unknown, but a similar observation has been recorded for BCV, IBV, and MHV t39,40~. In addition to CRFK cells, a fetal cat whole fibroblast (FCWF) cell line will support the growth of FIPV, as well as FECV, CCV, and TGEV (28,43t, and corn-Journal of Tissue Culture Methods Vol. 11, No. 2, 1988 parison of antigenic and serologic relatedness of the enteric coronaviruses was done in this cell line ~43). One to three cell culture passages of virulent FIPV in CRFK or FCWF cells do not destroy its virulence for susceptible cats. A vaccine produced with a CRFK cell culture attenuated strain was unable to protect cats against the original virulent strain, although vaccinated cats do develop a neutralizing antibody response t27). Using cDNA clones of cell-culture-adapted FIPV, the complete nucleotide sequence of the FIPV peplomer protein has been determined ( 15}.

The CRFK and A-72 (5) cell lines can be used for the isolation and propagation of CCV. This virus will grow to a titer of 1 X 107 pfu/ml or higher in either cell line. In addition, CCV can be adapted to grow in FCWF and ST cells. However, neither CCV nor FIPV will grow on primary isolation in ST cells, and this property may allow one to biologically differentiate these antigenically related coronaviruses. A single passage of CCV in CRFK cells does not decrease the infectivity for the natural host; however, prolonged serial passage in any cell line may result in an attenuated virus that grows well in cell culture but is avirutent for dogs. A cellculture-adapted CCV vaccine has been prepared and provides dogs vaccinated either i.m. or s.c. with neutralizing antibodies that are protective against a virulent CCV challenge (11 ) .

The SPTh cell line can be used for the primaryisolation of virus from clinical specimens and for the in vitro growth of HEV (2~. Although virus grown in cell culture is still infective for pigs, it is less virulent than field strains (W. L. Mengeling, Ames, IA, personal communication). No vaccine is currently available for this virus.

The ST cell line has been used for more than 20 yr to grow TGEV 122). This cell line can be used for primary isolation of virus from clinical specimens and in vitro growth of TGEV It0}. Virus from clinically positive animals will usually have a titer near 1 )< 10 s pfu/ml, and after 1 or 2 passages in ST cells it may have a titer of 1 to 2 X l07 pfu/ml. Passage of the virus in serum-free media in 5-d-old ST cells at an MOI of 0.5 produces a maximum virus titer 18 to 24 h postinoculation. Subpassage of the virus under the same conditions in ST cells that are less than 4 d old or more than 7 d old will produce a lower virus titer t38}. Cell-culture-adapted TGEV is still infective for pigs after 150 passages in ST cells, but pig virulence is usually reduced after only l0 to 15 passages in ST cells. Three tissue-cnlture-adapted, modified-live TGEV vaccine strains are sold for use against virulent TGEV in pregnant swine. Two of the vaccine viruses are grown in porcine cell lines, and the source of the third vaccine virus is unknown. Although the safety and ability of these vaccines to elicit virusneutralizing antibodies in pregnant swine has been proven, their efficacy has been questioned 16, 26) . The complete nucleotide sequence of the three major structural proteins of TGEV has been determined on cDNA clones of attenuated virus grown in ST cell culture (15,17,18,32L 9O

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