key: cord-0004990-g574zjyn
authors: Ishii, H.; Watanabe, I.; Mukamoto, M.; Kobayashi, Y.; Kodama, Y.
title: Adaptation of transmissible gastroenteritis virus to growth in non-permissive Vero cells
date: 1992
journal: Arch Virol
DOI: 10.1007/bf01321128
sha: 1c593ff9a3c832c2ed6cd2f98ee5d65eaae9be39
doc_id: 4990
cord_uid: g574zjyn

The CPK cells derived from swine kidney were infected with the attenuated TO-163 strain of transmissible gastroenteritis (TGE) virus, and fused with uninfected Vero cells in the presence of polyethylene glycol. Repeated cocultivation of the fused cells with uninfected Vero cells rendered the virus to grow in Vero cells. The Vero cell-adapted virus acquired the ability to infect and produce cytopathic effects in several other non-permissive cell lines of non-porcine origin. No major differences in viral polypeptides were shown between the Vero cell-adapted TO-163 strain and its parent strain by indirect immunofluorescence and Western blotting using monoclonal and polyclonal antibodies to TGE virus.

Summary. The CPK cells derived from swine kidney were infected with the attenuated TO-163 strain of transmissible gastroenteritis (TGE) virus, and fused with uninfected Vero cells in the presence of polyethylene glycol. Repeated cocultivafion of the fused cells with uninfected Vero cells rendered the virus to grow in Vero cells. The Vero cell-adapted virus acquired the ability to infect and produce cytopathic effects in several other non-permissive cell lines of nonporcine origin. No major differences in viral polypeptides were shown between the Vero cell-adapted TO-163 strain and its parent strain by indirect immunofluorescence and Western blotting using monoclonal and polyclonal antibodies to TGE virus.

Transmissible gastroenteritis (TGE) virus is a coronavirus with an envelope and positive strand RNA genome. The virion contains 3 structural proteins, i.e., peplomer (S or E 2), transmembrane (M or E 1), and nucleocapsid (N) [4] .

The virus replicates predominantly in villous epithelial cells of the small intestines, and results in diarrhea in infected pigs [11, 17] . Although clinical symptoms are not clearly demonstrated, the respiratory tract and the tonsils are also susceptible to infection [3, 8, 15] . The host range of TGE virus is restricted like the other coronaviruses [16] , and for the propagation of the virus, swine cell cultures are commonly used, although canine [12] and feline [6] cell cultures are less often used.

An attempt was made to adapt TGE virus to Vero cells non-permissive to TGE virus infection. The CPK cells derived from swine kidney [10] were inoculated with the avirulent TO-163 strain of TGE virus [5] , at a multiplicity of infection of 10, and incubated at 37 °C for 18 h. The infected cells were fused with uninfected Vero cells in the presence of 45% (w/v) polyethylene glycol (PEG 4000 Wako Pure Chemical Co., Japan) as described previously [7] . The PEG treated cells were incubated at 37 °C in Eagle's minimum essential medium (Nissui Seiyaku, Tokyo) containing 10% bovine serum. The culture was further passaged by the cocultivation with uninfected Vero cells without the presence of PEG at intervals of 4 or 5 days until the appearance of cytopathogenic effects (CPE). To detect CPE and viral replication, cell cultures were examined by the light microscopy and indirect immunofluorescence using rabbit antiserum against TGE virus and fluorescein isothiocyanate-conjugated antibody against rabbit IgG (Miles). At the 5th passage, some small clusters of round cells appeared in limited areas of the monolayer in which the virus-specific immunofluorescence was detected (Fig. 1) . Thereafter specific fluorescence and CPE with degenerative changes spread over the monolayer, The culture fluid from each viral passage was titrated by the TCIDs0 method using CPK and Vero cell monolayers in microplates. Infectious virus was first detected on the 6th and 8th passage at a titer of 104STCIDs0/mt with CPK cells and at a titer of 10°7TCIDs0/ml with Vero cells, respectively. The titer increased rapidly in the following 3 passages, and reached more than 105°TCIDs0/ml in the 24th passage (Fig. 2) .

In order to compare the Veto cell-adapted TO-163 strain with its parent strain, the 39th and 70th passages of TO-163 strain and the parental TO-163 (Fig. 3) . Western blot analysis revealed no major difference in antigenicity and electrophoretic mobility of the viral proteins between the Vero cell-adapted and the parental viruses (Fig.4) . No antigenic difference between the two viruses was also detected by immunofluorescent staining of infected Vero, CV-1, Hela and MA 104 cells with a series of monoclonal antibodies against E 2, E 1 and N (data not shown). Thus, it appeared that no gross change of antigenicity and structure of viral proteins might have occurred during virus adaptation to Vero cell.

In the present study the TO-163 strain of TGE virus was adapted to grow in Vero cells by fusing the CPK cells infected with the virus with Vero cells. Polyethylene glycol-mediated infection of non-permissive cells has been reported for retroviruses [13] and paramyxovirus [7] . Further studies are needed to clarify their mechanism.

The Vero cell-adapted TO-163 strain acquired infectivity for the other nonpermissive cells. This result may suggest that functional or structural changes have occurred in the viral proteins responsible for virus replication, although such changes were not detected in the present study. The glycoproteins of enveloped viruses play an important role in the virus adsorption and penetration, and are important determinants of the host range and their cleavage with (2 and 4) TO-163 strain. These viruses were propagated in Vero and CPK cells, respectively, partially purified, subjected to SDS-PAGE and examined using a mixture of monoclonal antibodies to E t and N (1 and 2) and a polyclonal antiserum to E 2 (3 and 4) by Western blotting proteolytic enzymes of the host cell is the important determinant for the host range and virulence [1, 9] . For the mouse hepatitis coronavirus the proteolytic cleavage of E 2 glycoprotein by host enzymes has been shown to activate the cell-fusion activity [-2,14] . Like the other coronaviruses, TGE virus exhibits a high degree of host dependence in the replication [16] . However, little is known of the function of glycoproteins of the virus. The Vero cell-adapted TO-163 strain would be useful in studies of TGE virus.

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