Persistent infection promotes cross-species transmissibility of mouse hepatitis virus - PubMed (original) (raw)

Persistent infection promotes cross-species transmissibility of mouse hepatitis virus

R S Baric et al. J Virol. 1999 Jan.

Abstract

Persistent infection with mouse hepatitis virus (MHV) strain A59 in murine DBT (delayed brain tumor) cells resulted in the emergence of host range variants, designated V51A and V51B, at 210 days postinfection. These host range mutants replicated efficiently in normally nonpermissive Chinese hamster ovary (CHO), in human hepatocarcinoma (HepG2), and to a lesser extent in human breast carcinoma (MCF7) cell lines. Little if any replication was noted in baby hamster kidney (BHK), green African monkey kidney (COS-7), feline kidney (CRFK), and swine testicular (ST) cell lines. By fluorescent antibody (FA) staining, persistent viruses V10B and V30B, isolated at days 38 and 119 days postinfection, also demonstrated very low levels of replication in human HepG2 cells. These data suggest that persistence may rapidly select for host range expansion of animal viruses. Pretreatment of HepG2 cells with a polyclonal antibody directed against human carcinoembryonic antigens (CEA) or with some monoclonal antibodies (Col-1, Col-4, Col-12, and Col-14) that bind human CEA significantly inhibited V51B infection. Under identical conditions, little or no blockade was evident with other monoclonal antibodies (kat4c or Col-6) which also bind the human CEA glycoproteins. In addition, an antibody (EDDA) directed against irrelevant antigens did not block V51B replication. Pretreatment with the Col-4 and Col-14 antibodies did not block Sindbis virus replication in HepG2 cells or MHV infection in DBT cells, suggesting that one or more CEA glycoproteins likely functioned as receptors for V51B entry into human cell lines. To test this hypothesis, the human biliary glycoprotein (Bgp) and CEA genes were cloned and expressed in normally nonpermissive BHK cell lines by using noncytopathic Sindbis virus replicons (pSinRep19). By growth curves and FA staining, human CEA and to a much lesser extent human Bgp functioned as receptors for V51B entry. Furthermore, V51B replication was blocked with polyclonal antiserum directed against human CEA and Bgp. Under identical conditions, the parental MHV strain A59 failed to replicate in BHK cells expressing human Bgp or CEA. These data suggest that MHV persistence may promote virus cross-species transmissibility by selecting for virus variants that recognize phylogenetic homologues of the normal receptor.

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Figures

FIG. 1

FIG. 1

Persistent virus replication in HepG2 cells. Cultures of HepG2 cells (105) were infected with V8A, V10B, V30B, V51A, V51B, MHV-A59, or the MHV-H2 host range variant at an MOI of 5 for 1 h at room temperature. Virus samples were harvested at the designated times and assayed by plaque assay.

FIG. 2

FIG. 2

Persistent virus antigen expression in HepG2 cells. Cultures of HepG2 cell lines in eight-chamber LabTek chambers were infected with V51B (A), V10B (B), V30B (C), or MHV-A59 (D) at an MOI of 5 for 1 h. The cultures were stained with polyclonal MHV-A59 antiserum at 18 h postinfection.

FIG. 3

FIG. 3

Blockade experiments in HepG2 cells. Cultures of HepG2 cells in eight-chamber LabTek slides were untreated (w/o) or treated with various polyclonal or monoclonal antibodies against hCEA genes at a 1:4 dilution for 1 h at room temperature. The antibodies were removed, and the cultures were infected with V51B at an MOI of 5 for 1 h. The virus inoculum was removed, and complete medium containing a 1:20 dilution of the same antibody was added to the chamber. Virus samples were taken at the indicated times, and virus titers were determined by plaque assay in DBT-9 cells.

FIG. 4

FIG. 4

hCEA glycoprotein expression in HepG2 cells. HepG2 cells were pretreated with various antisera for 30 min at room temperature. After extensive washing, FITC-conjugated rabbit anti-mouse IgG antiserum was added for 30 min at room temperature. Following additional washing, the cells were analyzed by FACS techniques. (A) Col-1; (B) Col-12; (C) Col-14; (D) Col-6; (E) Kat4c.

FIG. 5

FIG. 5

pSinRep19 replicons do not inhibit MHV infection. To determine if the noncytopathic pSinRep19 replicons block MHV replication, cultures of BHK-MHVR cells were transfected with the pSinRep19/T7pol transcripts and selected with puromycin (5 μg/ml) for several days. Cultures of BHK-MHVR/SinT7pol, BHK-MHVR, and BHK cells were infected with MHV-A59 at an MOI of 5 for 1 h. Virus samples were harvested at the indicated times and assayed by plaque assay in DBT cells.

FIG. 6

FIG. 6

Expression of hCEA and hBgp in BHK cells. BHK cells were transfected with transcripts from the pSinRep19-hBgp and pSinRep19-hCEA replicons and selected with puromycin (5 μg/ml) for several days. By using monoclonal antibody Kat4c, the cultures were sorted into cell lines BHK-hBgp1 (A), BHK-hBgp1+ (B), and BHK-hCEA (C). These cell lines were grown into large stocks of cell lines expressing relatively uniform levels of hCEA or hBgp. FACS analysis was performed with monoclonal antibody Kat4c as previously described (12).

FIG. 6

FIG. 6

Expression of hCEA and hBgp in BHK cells. BHK cells were transfected with transcripts from the pSinRep19-hBgp and pSinRep19-hCEA replicons and selected with puromycin (5 μg/ml) for several days. By using monoclonal antibody Kat4c, the cultures were sorted into cell lines BHK-hBgp1 (A), BHK-hBgp1+ (B), and BHK-hCEA (C). These cell lines were grown into large stocks of cell lines expressing relatively uniform levels of hCEA or hBgp. FACS analysis was performed with monoclonal antibody Kat4c as previously described (12).

FIG. 7

FIG. 7

MHV replication in BHK cells expressing hCEA glycoproteins. Cultures of BHK, BHK-hCEA, BHK-hBgp1, and BHK-hBgp1+ cells were infected with V51B or MHV-A59, as indicated, at an MOI of 5 for 1 h at room temperature. In some experiments, the cells had been pretreated with a 1:4 dilution of pCEA or nonspecific antibody EDDA for 30 min prior to infection. Following infection, the cultures were washed extensively and virus were samples taken at the indicated times.

FIG. 7

FIG. 7

MHV replication in BHK cells expressing hCEA glycoproteins. Cultures of BHK, BHK-hCEA, BHK-hBgp1, and BHK-hBgp1+ cells were infected with V51B or MHV-A59, as indicated, at an MOI of 5 for 1 h at room temperature. In some experiments, the cells had been pretreated with a 1:4 dilution of pCEA or nonspecific antibody EDDA for 30 min prior to infection. Following infection, the cultures were washed extensively and virus were samples taken at the indicated times.

FIG. 7

FIG. 7

MHV replication in BHK cells expressing hCEA glycoproteins. Cultures of BHK, BHK-hCEA, BHK-hBgp1, and BHK-hBgp1+ cells were infected with V51B or MHV-A59, as indicated, at an MOI of 5 for 1 h at room temperature. In some experiments, the cells had been pretreated with a 1:4 dilution of pCEA or nonspecific antibody EDDA for 30 min prior to infection. Following infection, the cultures were washed extensively and virus were samples taken at the indicated times.

FIG. 8

FIG. 8

Virus antigen in BHK cells expressing hCEA glycoproteins. Cultures treated as described for Fig. 7 were fixed and FA stained for the presence of viral antigen. (A) BHK-hCEA cells infected with V51B; (B) BHK cells infected with V51B; (C) BHK-hCEA cells infected with MHV-A59; (D) BHK cells infected with MHV-A59.

FIG. 9

FIG. 9

Persistent virus replication in BHK-hCEA cells. Cultures of BHK-hCEA cells (106) were infected with MHV-A59, V10B, V30B, V51A, and V51B at an MOI of 5 for 1 h at room temperature. Virus titers were harvested at the designated times for analysis by plaque assay.

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