cDNA clone of hepatitis A virus encoding a virulent virus: induction of viral hepatitis by direct nucleic acid transfection of marmosets

Emerson, Suzanne U.; Lewis, M.; Govindarajan, Sugantha; Shapiro, Max; Moskal, Thomas J.; Purcell, Robert H.

doi:10.1128/jvi.66.11.6649-6654.1992

Cited by 43 publications

(35 citation statements)

References 21 publications

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“…cDNA clones and in vitro transcription. Three infectious cDNA clones in pGEM1 (Promega Corporation, Madison, Wis.) were used as the basis for new chimeric viruses: an infectious cDNA clone which encodes an attenuated virus adapted to efficient growth in AGMK cells (pHAV/7) (7), one which encodes a virulent virus which can be consistently recovered in cell culture (p8Y) (15), and one which represents most of the genome of the MRC-5-adapted virus (pMR8), constructed by sequential replacement of pHAV/7 restriction fragments with those generated by reverse transcription-PCR (RT-PCR) amplification of the MRC-5/9 virus genome ( Fig. 1).…”

Section: Cellsmentioning

confidence: 99%

“…Transfection of at least two sister clones was performed to confirm the phenotype. In vivo transfection of tamarins with a mixture of cDNA and transcribed RNA was performed by laparotomy with direct injection of the liver as previously described (15). One clone of each mutant cDNA was tested by inoculation of two animals with equal aliquots of the same transfection mixture.…”

Section: Cellsmentioning

confidence: 99%

“…Therefore, it is also necessary to determine whether mutations which are required for propagation in cell culture affect virulence. The tamarin Saguinus mystax has been used in many animal studies (15,25) because of previous work demonstrating the virulence of wild-type HAV in these animals (26) and because of their availability. Because wild-type HAV grows extremely slowly in cell culture (16), may mutate in the process, and sometimes cannot be recovered, it has been difficult to test in animals the virulence phenotype of virus amplified in tissue culture.…”

mentioning

confidence: 99%

“…Because wild-type HAV grows extremely slowly in cell culture (16), may mutate in the process, and sometimes cannot be recovered, it has been difficult to test in animals the virulence phenotype of virus amplified in tissue culture. Therefore, an important advancement for studying the virulence of mutant viruses was achieved with the successful infection of tamarins by direct intrahepatic inoculation of cDNA and transcribed RNA genomes of p8Y, which encoded a virus that differed from the wild type by a single mutation that permitted consistent recovery of the virus in cell culture but had no discernible effect on the virulence phenotype (15).…”

mentioning

confidence: 99%

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Progress toward the development of a genetically engineered attenuated hepatitis A virus vaccine

Funkhouser

Raychaudhuri

Purcell

et al. 1996

J Virol

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Mutations which positively affect growth of hepatitis A virus in cell culture may negatively affect growth in vivo. Therefore, development of an attenuated vaccine for hepatitis A may require a careful balancing of mutations to produce a virus that will grow efficiently in cells suitable for vaccine production and still maintain a satisfactory level of attenuation in vivo. Since such a balance could be achieved most directly by genetic engineering, we are analyzing mutations that accumulated during serial passage of the HM-175 strain of hepatitis A virus in MRC-5 cell cultures in order to determine the relative importance of the mutations for growth in MRC-5 cells and for attenuation in susceptible primates. Chimeric viral genomes of the HM-175 strain were constructed from cDNA clones derived from a virulent virus and from two attenuated viruses adapted to growth in African green monkey kidney (AGMK) and MRC-5 cells, respectively. Viruses encoded by these chimeric genomes were recovered by in vitro or in vivo transfection and assessed for their ability to grow in cultured MRC-5 cells or to cause hepatitis in primates (tamarins). The only MRC-5-specific mutations that substantially increased the efficiency of growth in MRC-5 cells were a group of four mutations in the 5 noncoding (NC) region. These 5 NC mutations and a separate group of 5 NC mutations that accumulated during earlier passages of the HM-175 virus in primary AGMK cells appeared, independently and additively, to result in decreased biochemical evidence of hepatitis in tamarins. However, neither group of 5 NC mutations had a demonstrable effect on the extent of virus excretion or liver pathology in these animals.

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Section: Cellsmentioning

confidence: 99%

Section: Cellsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Progress toward the development of a genetically engineered attenuated hepatitis A virus vaccine

Funkhouser

Raychaudhuri

Purcell

et al. 1996

J Virol

Self Cite

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show abstract

“…In the past, common marmosets have been used as an animal model for the disease. They can be infected by the oral route [78], via intragastric infection [79], or by direct inoculation of the virus in the liver [80]. Infected animals develop an acute hepatitis 2 weeks after infection and shed the virus in feces from day 7 onward.…”

Section: Hepatitis a Virusmentioning

confidence: 99%