INFO:The In vitro-synthesized RNA from a cDNA clone of hepatitis E virus is infectious

[Guest guest]

J Virol 2000 May;74(5):2430-7

The In vitro-synthesized RNA from a cDNA clone of hepatitis E virus is

infectious.

Panda SK, Ansari IH, Durgapal H, Agrawal S, Jameel S

Department of Pathology, All India Institute of Medical Sciences, Ansari

Nagar, New Delhi 110029, India.

[Medline record in process]

Hepatitis E virus (HEV) is an important etiological agent of epidemic and

sporadic hepatitis, which is endemic to the Indian subcontinent and

prevalent in most of the developing parts of the world. The infection is

often associated with acute liver failure and high mortality, particularly

in pregnant women. In order to develop methods of intervention, it is

essential to understand the biology of the virus. This is particularly

important as no reliable in vitro culture system is available. We have

constructed a cDNA clone encompassing the complete HEV genome from

independently characterized subgenomic fragments of an Indian epidemic

isolate. Transfection studies were carried out with HepG2 cells using in

vitro-transcribed RNA from this full-length HEV cDNA clone. The presence of

negative-sense RNA, indicative of viral replication, was demonstrated in the

transfected cells by strand-specific reverse transcription-PCR and slot blot

hybridization. The viral proteins pORF2 and pORF3 and processed components

of the pORF1 polyprotein (putative methyltransferase, helicase, and

RNA-dependent RNA polymerase) were identified in the transfected cells by

metabolic pulse-labeling with [(35)S]methionine-cysteine, followed by

immunoprecipitation with respective antibodies. The expression of viral

proteins in the transfected cells was also demonstrated by

immunofluorescence microscopy. Viral replication was detected in the

transfected cells up to 33 days posttransfection (six passages). The culture

supernatant from the transfected cells was able to produce HEV infection in

a rhesus monkey (Macaca mulatta) following intravenous injection, indicating

the generation of viable HEV particles following transfection of cells with

in vitro-synthesized genomic RNA. This transient cell culture model using in

vitro-transcribed RNA should facilitate our understanding of HEV biology.

PMID: 10666275, UI: 20130435

---------------

[Guest guest]

J Virol 2000 May;74(5):2430-7

The In vitro-synthesized RNA from a cDNA clone of hepatitis E virus is

infectious.

Panda SK, Ansari IH, Durgapal H, Agrawal S, Jameel S

Department of Pathology, All India Institute of Medical Sciences, Ansari

Nagar, New Delhi 110029, India.

[Medline record in process]

Hepatitis E virus (HEV) is an important etiological agent of epidemic and

sporadic hepatitis, which is endemic to the Indian subcontinent and

prevalent in most of the developing parts of the world. The infection is

often associated with acute liver failure and high mortality, particularly

in pregnant women. In order to develop methods of intervention, it is

essential to understand the biology of the virus. This is particularly

important as no reliable in vitro culture system is available. We have

constructed a cDNA clone encompassing the complete HEV genome from

independently characterized subgenomic fragments of an Indian epidemic

isolate. Transfection studies were carried out with HepG2 cells using in

vitro-transcribed RNA from this full-length HEV cDNA clone. The presence of

negative-sense RNA, indicative of viral replication, was demonstrated in the

transfected cells by strand-specific reverse transcription-PCR and slot blot

hybridization. The viral proteins pORF2 and pORF3 and processed components

of the pORF1 polyprotein (putative methyltransferase, helicase, and

RNA-dependent RNA polymerase) were identified in the transfected cells by

metabolic pulse-labeling with [(35)S]methionine-cysteine, followed by

immunoprecipitation with respective antibodies. The expression of viral

proteins in the transfected cells was also demonstrated by

immunofluorescence microscopy. Viral replication was detected in the

transfected cells up to 33 days posttransfection (six passages). The culture

supernatant from the transfected cells was able to produce HEV infection in

a rhesus monkey (Macaca mulatta) following intravenous injection, indicating

the generation of viable HEV particles following transfection of cells with

in vitro-synthesized genomic RNA. This transient cell culture model using in

vitro-transcribed RNA should facilitate our understanding of HEV biology.

PMID: 10666275, UI: 20130435

---------------

[Guest guest]

J Virol 2000 May;74(5):2430-7

The In vitro-synthesized RNA from a cDNA clone of hepatitis E virus is

infectious.

Panda SK, Ansari IH, Durgapal H, Agrawal S, Jameel S

Department of Pathology, All India Institute of Medical Sciences, Ansari

Nagar, New Delhi 110029, India.

[Medline record in process]

Hepatitis E virus (HEV) is an important etiological agent of epidemic and

sporadic hepatitis, which is endemic to the Indian subcontinent and

prevalent in most of the developing parts of the world. The infection is

often associated with acute liver failure and high mortality, particularly

in pregnant women. In order to develop methods of intervention, it is

essential to understand the biology of the virus. This is particularly

important as no reliable in vitro culture system is available. We have

constructed a cDNA clone encompassing the complete HEV genome from

independently characterized subgenomic fragments of an Indian epidemic

isolate. Transfection studies were carried out with HepG2 cells using in

vitro-transcribed RNA from this full-length HEV cDNA clone. The presence of

negative-sense RNA, indicative of viral replication, was demonstrated in the

transfected cells by strand-specific reverse transcription-PCR and slot blot

hybridization. The viral proteins pORF2 and pORF3 and processed components

of the pORF1 polyprotein (putative methyltransferase, helicase, and

RNA-dependent RNA polymerase) were identified in the transfected cells by

metabolic pulse-labeling with [(35)S]methionine-cysteine, followed by

immunoprecipitation with respective antibodies. The expression of viral

proteins in the transfected cells was also demonstrated by

immunofluorescence microscopy. Viral replication was detected in the

transfected cells up to 33 days posttransfection (six passages). The culture

supernatant from the transfected cells was able to produce HEV infection in

a rhesus monkey (Macaca mulatta) following intravenous injection, indicating

the generation of viable HEV particles following transfection of cells with

in vitro-synthesized genomic RNA. This transient cell culture model using in

vitro-transcribed RNA should facilitate our understanding of HEV biology.

PMID: 10666275, UI: 20130435

---------------

[Guest guest]

J Virol 2000 May;74(5):2430-7

The In vitro-synthesized RNA from a cDNA clone of hepatitis E virus is

infectious.

Panda SK, Ansari IH, Durgapal H, Agrawal S, Jameel S

Department of Pathology, All India Institute of Medical Sciences, Ansari

Nagar, New Delhi 110029, India.

[Medline record in process]

Hepatitis E virus (HEV) is an important etiological agent of epidemic and

sporadic hepatitis, which is endemic to the Indian subcontinent and

prevalent in most of the developing parts of the world. The infection is

often associated with acute liver failure and high mortality, particularly

in pregnant women. In order to develop methods of intervention, it is

essential to understand the biology of the virus. This is particularly

important as no reliable in vitro culture system is available. We have

constructed a cDNA clone encompassing the complete HEV genome from

independently characterized subgenomic fragments of an Indian epidemic

isolate. Transfection studies were carried out with HepG2 cells using in

vitro-transcribed RNA from this full-length HEV cDNA clone. The presence of

negative-sense RNA, indicative of viral replication, was demonstrated in the

transfected cells by strand-specific reverse transcription-PCR and slot blot

hybridization. The viral proteins pORF2 and pORF3 and processed components

of the pORF1 polyprotein (putative methyltransferase, helicase, and

RNA-dependent RNA polymerase) were identified in the transfected cells by

metabolic pulse-labeling with [(35)S]methionine-cysteine, followed by

immunoprecipitation with respective antibodies. The expression of viral

proteins in the transfected cells was also demonstrated by

immunofluorescence microscopy. Viral replication was detected in the

transfected cells up to 33 days posttransfection (six passages). The culture

supernatant from the transfected cells was able to produce HEV infection in

a rhesus monkey (Macaca mulatta) following intravenous injection, indicating

the generation of viable HEV particles following transfection of cells with

in vitro-synthesized genomic RNA. This transient cell culture model using in

vitro-transcribed RNA should facilitate our understanding of HEV biology.

PMID: 10666275, UI: 20130435

---------------