Replication and plaque formation of mouse hepatitis virus (MHV-2) in mouse cell line DBT culture

Hirano, Norio; Fujiwara, Kôsaku; Hino, Shinjiro; Matumoto, Minoru

doi:10.1007/bf01240618

Cited by 229 publications

(161 citation statements)

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“…The DBT cell line was derived from a Schmidt-Ruppin Rous Sarcoma virus-induced murine astrocytoma (39) and was maintained as monolayer culture in Eagle's minimal essential medium (Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal bovine serum, 10% tryptose phosphate broth (Difco), and 0.06 mg/ml kanamycin. MHV-JHM and MHV-2 were kindly provided by Dr. Fujiwara (University of Tokyo) and were propagated on DBT cells.…”

Section: Cell Line and Virusesmentioning

confidence: 99%

A 12-Amino Acid Stretch in the Hypervariable Region of the Spike Protein S1 Subunit Is Critical for Cell Fusion Activity of Mouse Hepatitis Virus

Tsai

Chang

1999

Journal of Biological Chemistry

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The spike (S) glycoprotein of mouse hepatitis virus (MHV) plays a major role in the viral pathogenesis. It is often processed into the N-terminal S1 and the C-terminal S2 subunits that were evidently important for binding to cell receptor and inducing cell-cell fusion, respectively. As a consequence of cell-cell fusion, most of the naturally occurring infections of MHV are associated with syncytia formation. So far, only MHV-2 was identified to be fusion-negative. In this study, the S gene of MHV-2 was molecularly cloned, and the nucleotide sequence was determined. The MHV-2 S protein lacks a 12-amino acid stretch in the S1 hypervariable region Mouse hepatitis virus (MHV)1 is a member of the Coronaviridae family that causes inapparent enteric and respiratory infection, hepatitis, and acute and chronic demyelinating diseases of the central nervous system (1). It is an enveloped virus that contains a positive sense, single-stranded genomic RNA of approximately 31 kilobases in length (2, 3). The viral particle contains four major structural proteins: the nucleocapsid (N) protein that interacts with the viral genomic RNA and membrane (M) protein to form a spherical core (4, 5) and the spike (S) and small membrane (E) proteins that together with the M protein constitute the envelope (6, 7). In some strains of MHV, there exists an additional enveloped protein, the hemagglutinin esterase (8, 9). The E and M proteins were demonstrated to be required for virion assembly (5, 10). The S protein of MHV forms large characteristic projections of coronaviruses and plays a major role in viral pathogenesis.The S protein is cotranslationally glycosylated in the endoplasmic reticulum, giving a molecular mass of approximately 180 kDa, and trimerized (11). Following a transport through the Golgi apparatus, the high mannose oligosaccharides are trimmed, and the protein is further acylated (12). After the modifications, the S protein is often cleaved by a cellular protease into two noncovalently bound 90-kDa subunits, S1 and S2, derived from the N-terminal and C-terminal halves, respectively (13,14). S protein that is not assembled into virions is transported by the secretory system to the cell surface, where it may participate in inducing fusion between adjacent cells (15). In addition to the induction of pH independent cell-to-cell fusion, the S protein also mediates the viral attachment to the receptor of susceptible cells and is involved in the elicitation of neutralizing antibodies, the tissue tropism, and the variation of viral pathogenicity (7, 16 -21).The process of S protein-mediated membrane fusion has been intensively studied, but the mechanism is still poorly understood. Proteolytic cleavage of the S protein precursor into the S1 and S2 subunits was thought to be a prerequisite for the virus to induce cell fusion (14); cell fusion activity correlated with the cleavage level of S protein. The cleavage site was located at the C terminus of the amino acid sequence RRARR in the middle of the S protein (22). Nevertheless, muta...

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Section: Cell Line and Virusesmentioning

confidence: 99%

A 12-Amino Acid Stretch in the Hypervariable Region of the Spike Protein S1 Subunit Is Critical for Cell Fusion Activity of Mouse Hepatitis Virus

Tsai

Chang

1999

Journal of Biological Chemistry

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“…MHV-A59 [18] and MHV-JHM [15] were propagated in DBT cells as described previously [11]. MHV-KU was isolated from a sentinel nude mouse in 1996 by using CMT-93 cells, and then passaged in DBT cells twice.…”

Section: Methodsmentioning

confidence: 99%

“…The viral titer in the culture supernatants was determined by a conventional plaque assay on DBT cells [11] with minor modifications. Briefly, confluent cultures of DBT cells in 60-mm tissue culture dishes were inoculated with 0.2 ml of a 10-fold serial dilution of samples, and then adsorbed for 60 min.…”

Section: Plaque Assay On Dbt Cellsmentioning

confidence: 99%

“…Since the discovery of MHV-permissive cell lines, (e.g., delayed brain tumor (DBT) cells) [11], great advances have been made in the molecular biology and pathogenesis of MHV. On the other hand, little work has been carried out with enterotropic strains, considered to be the most prevalent in natural outbreaks in contemporary mouse colonies [13], since it has been difficult to prepare enterotropic coronaviruses due to the lack of an optimal in vitro replication system using cultured cell lines.…”

Section: Introductionmentioning

confidence: 99%

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Replication of Enterotropic and Polytropic Murine Coronaviruses in Cultured Cell Lines of Mouse Origin.

et al. 2000

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Abstract:To understand the virus-cell interactions that occur during murine coronavirus infection, six murine cell lines (A3-1M, B16, CMT-93, DBT, IC-21 and J774A.1) were inoculated with eight murine coronaviruses, including prototype strains of both polytropic and enterotropic biotypes, and new isolates. All virus strains produced a cytopathic effect (CPE) with cell-to-cell fusion in B16, DBT, IC-21 and J774A.1 cells. The CPE was induced most rapidly in IC-21 cells and was visible microscopically in all cell lines tested. In contrast, the coronaviruses produced little CPE in A3-1M and CMT-93 cells. Although most virus-infected cells, except KQ3E-infected A3-1M, CMT-93 and J774A.1 cells, produced progeny viruses in the supernatants when assayed by plaque formation on DBT cells, the kinetics of viral replication were dependent on both the cell line and virus strain; replication of prototype strains was higher than that of new isolates. There was no significant difference in replication of enterotropic and polytropic strains. B16 cells supported the highest level of viral replication. To determine the sensitivity of the cell lines to murine coronaviruses, the 50% tissue culture infectious dose of the coronaviruses was determined with B16, DBT, IC-21 and J774A.1 cells, and compared to that with DBT cells. The results indicate that IC-21 cells were the most sensitive to murine coronaviruses. These data suggest that B16 and IC-21 cells are suitable for large-scale preparation and isolation of murine coronaviruses, respectively.

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“…After passaging the tumor in the host animal, the tumors, blood and liver of the host in each passaging were subjected to virus isolation. We isolated MHV by plaque formation assay [10] with DBT cultured cells [4]. The specimens were homogenized with Dulbecco's phosphate buffered salts solution (magnesium and calcium free, pH 7.4, PBS) to make a 10% (wet tissue weight or blood volume/volume) solution, then centrifuged with 200G for 5 min.…”

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confidence: 99%

Decontamination of Human Xenotransplantable Tumor with Mouse Hepatitis Virus by Implantation in Nude Rat: A Case Report.

et al. 2000

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Abstract:A human tumor xenograft contaminated with mouse hepatitis virus (MHV) was implanted in a nude rat in order to decontaminate the tumor line. The decontamination failed in the first trial, but succeeded in the second trial. The difference between the two trials was the duration of implantation of the tumor in the nude rat, i.e., 12 days in the first and 24 days in the second trial. Duration of implantation might be a factor in the decontamination of transplantable tumors infected with MHV by passaging in the nude rat. Key words: human neoplasm, mouse hepatitis virus, xenograft Moreover, when HTX-bearing mice are infected with MHV, the HTX is easily contaminated with the virus. Therefore, the tumor line itself becomes one of the main sources for transmission of the virus into mouse colonies [6,9], and microbiological control of tumor lines including MHV is very important to assure the reliability and reproducibility of results of experiments using HTX/immune deficient animal models.We examined the microbiological status of established or introduced HTXs by using a quarantine system [14], and found that an HTX line introduced from a certain research institute was contaminated with MHV. In a previous report, Rülicke et al. [9] used nude rats as the host for xenotransplantation of tumors contaminated with MHV, and succeeded in decontamination of the tumors. To eliminate the virus contaminating the HTX, we also passaged the tumor in nude rats.

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Replication and plaque formation of mouse hepatitis virus (MHV-2) in mouse cell line DBT culture

Cited by 229 publications

References 13 publications

A 12-Amino Acid Stretch in the Hypervariable Region of the Spike Protein S1 Subunit Is Critical for Cell Fusion Activity of Mouse Hepatitis Virus

A 12-Amino Acid Stretch in the Hypervariable Region of the Spike Protein S1 Subunit Is Critical for Cell Fusion Activity of Mouse Hepatitis Virus

Replication of Enterotropic and Polytropic Murine Coronaviruses in Cultured Cell Lines of Mouse Origin.

Decontamination of Human Xenotransplantable Tumor with Mouse Hepatitis Virus by Implantation in Nude Rat: A Case Report.

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