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Growth of wild-type hepatitis a virus in cell culture

A hepatitis A virus and cell technology, applied in the direction of viruses, microorganisms, tissue culture, etc.

Inactive Publication Date: 2009-01-28
美利坚合众国政府，由卫生与人类服务部部长代表
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

but it does not cause cytopathic effects in culture

Method used

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  • Growth of wild-type hepatitis a virus in cell culture
  • Growth of wild-type hepatitis a virus in cell culture
  • Growth of wild-type hepatitis a virus in cell culture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1: Recovery of wt HAV from cells transfected with in vitro transcripts.

[0084] To rescue HAV8Y-Bsd, the SP6 transcript was transfected into Huh7, FRhK4, GL37, HeLa, Vero, CHO, MMH-D3, and Jurkat cells. One day after transfection, cells were passaged 1:6 and grown in medium containing 1, 2, 4, or 5 μg / ml blasticidin. After 14 days of selection with 1 μg / ml blasticidin, few blasticidin-resistant colonies grew in HAV8Y-Bsd RNA transfected Huh7 cells, but not mock-transfected cells. Transfected Huh7 cells did not survive selection with higher concentrations (2, 4, or 5 μg / ml) of blasticidin. All other transfected cell lines were not viable in the presence of blasticidin treatment, showing that HAV8Y-Bsd could only be rescued from Huh7 transfected cells. IF analysis (Figure 2) showed that blasticidin-resistant Huh7 cells had cytoplasmic granular fluorescence characteristic of HAV-infected cells (B), which was not observed in mock-transfected cells (A). To assess...

Embodiment 2

[0086] Example 2: Stable growth of wt HAV in Huh7 cells.

[0087] It was of interest to determine whether the strong selection pressure for cell culture-adapted and attenuating mutations observed in most cell lines is also present in Huh7 cells. To investigate whether wt HAV containing the Bsd selectable marker could grow stably in Huh7 cells without accumulating cell culture-adapted mutations and retain the selectable marker, we performed HAV8Y-Bsd in the presence of 1 μg / ml blasticidin and nine serial passages of HAV.WT-Bsd in Huh7 cells. RT-PCR amplification and nucleotide sequence analysis of HAV RNA extracted from nine serial passages showed that the inserted bsd gene was stable in both viruses. The nucleotide sequences of the 2B-2C and 5'-NTR regions of the ninth passage HAV8Y-Bsd and HAV.WT-Bsd were identical to the parental cDNAs, suggesting that these viruses do not accumulate cell culture-adapted mutations at these two hotspots. To further assess the stability of w...

Embodiment 3

[0088] Example 3: HAV8Y-Bsd-infected Huh7 cells cured / eliminated by IFN-αA / D are sensitive to wt HAV infection.

[0089] Blasticidin-resistant cells infected with HAV.WT-Bsd in Example 1 were cured using interferon (9). To achieve the above, blasticidin-resistant HAV8Y-Bsd-infected cultures were subcultured several times in the presence of 100, 250, or 500 IU / ml IFN-αA / D in medium lacking blasticidin Huh7 cells. After 7 passages, IF analysis showed that cells treated with 250 (Fig. 4A) or 500 U / ml (data not shown) IFN-α A / D lost HAV antigen, while untreated control cells (Fig. 4A) and some cells cultured in the presence of 100 IU / ml IFN-αA / D had the characteristic cytoplasmic fluorescence of HAV-infected cells (data not shown). To determine whether interferon-treated cells that had lost HAV antigens also became sensitive to blasticidin, the interferon-treated cells were cultured in the presence of 0.5-10 μg / ml blasticidin for 10 days, and the cultures were observed by micro...

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Abstract

The invention provides recombinant Hepatitis A Virus (HAV) nucleic acids and host cells that are permissive for their growth and replication. The recombinant Hepatitis A Virus nucleic acids not particularly limited, except that they incorporate at least one heterologous nucleic acid fragment. The heterologous nucleic acid can encode a selectable marker gene and such recombinant HAV nucleic acids are useful for selecting cells that are permissive for growth and replication of wild type HAV. Alternatively, the heterologous nucleic acid may encode a vaccine antigen or other expression product that is desirable to express in a cell harboring the recombinant HAV nucleic acid. The invention further provides cell lines permissive for growth and replication of wild type HAV or HAV having minimal mutations for growth in cell culture. The invention further provides methods for producing HAV vaccines and for monitoring environmental and patient samples for the presence of HAV.

Description

[0001] U.S. GOVERNMENT RIGHTS IN THIS INVENTION [0002] Funding used to develop this invention was provided, at least in part, by the United States Government (represented by the Department of Health and Human Services). Accordingly, the United States Government may have certain rights in this invention. technical field [0003] The present invention relates to recombinant hepatitis A virus (HAV). The present invention comprises recombinant HAV genomes and assembled virus particles which are useful as vaccines and as vectors for introducing the recombinant HAV genomes they contain into cells for various purposes. The invention also relates to cells and cell lines that can be used to grow wild-type and engineered HAV viruses in culture for a variety of purposes, including diagnostic and environmental monitoring purposes. Background technique [0004] Hepatitis A virus (HAV) is a picornavirus that causes acute hepatitis in humans, a preventable but still prevalent infectiou...

Claims

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Application Information

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IPC IPC(8): C12N15/86C12N5/00
CPCC12N2770/32443C12N15/86C12N7/00C12N2770/32461
Inventor 格拉尔多·卡普兰克里希纳默西·孔杜鲁
Owner 美利坚合众国政府，由卫生与人类服务部部长代表
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