Infectious Japanese encephalitis virus RNA can be synthesized from in vitro-ligated cDNA templates

Abstract

Japanese encephalitis virus (JEV) is a positive-stranded enveloped RNA virus that belongs to the family Flaviviridae. Genomic JEV RNA is approximately 11 kb long and encodes 10 proteins, 3 structural and 7 nonstructural. A fIl-length cDNA copy of the JEV genome was constructed by in vitro ligation of two cDNA fragments which encode the 5' (nucleotide positions 1 to 5576) and 3' (nucleotide positions 5577 to 10976) halves of the genome. T7 RNA polymerase transcripts of the ligated full-length cDNA template were infectious when transfected into BHK-21 cells. To identify the recombinant virus, a silent mutation was introduced into the clone encoding the 3' half of the genome, which abolished an XbaI site at nucleotide position 9131. Virus recovered by transfection with the transcripts contained this silent mutation, confirming its identity. Recombinant and parent viruses were identical with respect to growth and plaque production in BHK-21 cells, envelope protein expression in C6/36 cells, and neurovirulence and immunogenicity in mice. Repeated attempts to obtain infectious RNA by transcription from full-length JEV genome cDNA templates cloned into plasmid vectors were unsuccessful. Synthesis of infectious JEV RNA from in vitro-ligated JEV cDNA templates will be useful for molecular and genetic studies of flavivirus replication and virulence.

A ‘minimal’ approach in design of flavivirus infectious DNA

“…Modern (+)RNA virus studies increasingly rely on the infectious clone methodology (reviewed, Boyer and Haenni, 1994), which allows targeted manipulation of viral genomes. In the classical approach, (+ )RNA viruses are recov-ered from cells transfected with synthetic RNA made by in vitro transcription of infectious clone cDNA templates (Campbell and Pletnev, 2000;Gritsun and Gould, 1995;Kapoor et al, 1995;Polo et al, 1997;Rice et al, 1989;Sumiyoshi et al, 1992). In a layered DNA/RNA approach, also known as 'infectious DNA' (Herweijer et al, 1995), (+ )RNA viruses are recovered directly after transfection of plasmids carrying viral genome cDNA into susceptible cells.…”

“…As a model, we have used a highly unstable infectious clone of Japanese encephalitis flavivirus (JE) (Sumiyoshi et al, 1992;Yamshchikov et al, 2001). JE is a member of the Fla6i6irus genus of the family Fla6i6iridae, which also includes yellow fever (YF), West Nile, tick borne encephalitis, dengue, and hepatitis C viruses (Kuno et al, 1998).…”

An inactivated West Nile Virus vaccine derived from a chemically synthesized cDNA system

“…Genomic RNA is infectious when introduced into susceptible cells by transfection [3]. For replication and pathogenesis studies, reverse genetic systems have been established for several members of the genus [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. These systems comprise one or two plasmids encoding cDNA of viral genomic sequence under control of bacteriophage promoters allowing transcription of full-length infectious RNA in vitro.…”

“…These systems comprise one or two plasmids encoding cDNA of viral genomic sequence under control of bacteriophage promoters allowing transcription of full-length infectious RNA in vitro. For YFV [4], DEN-1 [17], DEN-2 [6,8,10], DEN-4 [11], TBEV [13,15], KUN [9], MVE [7] and WNV lineage I [19] and II [21], cDNA comprising the full genome was stably cloned into bacterial expression plasmids, whereas in other reports [5,8,13,18,20] cDNA was split in two fragments, each integrated in individual plasmids, from which cDNA can be fused together before RNA transcription. An alternative approach was applied to construct a JEV infectious clone, in which the viral coding sequence was put under the control of a eukaryotic promoter and split by introns to circumvent instability during propagation in bacteria [22].…”

Generation of a recombinant avian coronavirus infectious bronchitis virus using transient dominant selection

“…Molecular analysis of the structure and the role of individual genes in pathogenesis of RNA viruses has been advanced by the availability of full-length cDNAs, for the generation of infectious RNA transcripts that can replicate and result in infectious viruses from permissive cell lines (Boyer and Haenni, 1994). Such reverse genetics systems have resulted in the recovery of a number of infectious positive-stranded RNA viruses including, picornaviruses, caliciviruses, alphaviruses, flaviviruses, arteriviruses and Closterovirus, whose RNA genomes range in size from 7 to 20 kb in length (Agapov Davis et al, 1989;Racaniello and Baltimore, 1981;Rice et al, 1987Rice et al, , 1989Satyanarayana et al, 2001;Sosnovtsev and Green, 1995;Sumiyoshi et al, 1992;van Dinten et al, 1997). Recently full-length cDNAs capable of producing infectious RNA transcripts for several coronaviruses, viruses with the largest RNA genomes, Transmissible gastroenteritis virus (TGEV; (Almazán et al, 2000;Yount et al, 2000)), Human coronavirus 229E (HCoV; ), IBV (Casais et al, 2001), Murine hepatitis virus (MHV; (Yount et al, 2002)), and severe acute respiratory syndrome coronavirus (SARS-CoV; Yount et al, 2003) have been produced.…”