Construction of Infectious cDNA Clones for Dengue 2 Virus: Strain 16681 and Its Attenuated Vaccine Derivative, Strain PDK-53

Kinney, Richard M.; Butrapet, Siritorn; Chang, Gwong‐Jen J.; Tsuchiya, Kiyotaka R.; Roehrig, John T.; Bhamarapravati, Natth; Gubler, Duane J.

doi:10.1006/viro.1997.8500

Cited by 291 publications

(274 citation statements)

References 34 publications

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“…The plasmid, D2/IC-30P-A (Fig. 1A) (Kinney et al, 1997), containing fulllength DENV-2 cDNA derived from wild type (wt) strain 16681, and its subclones containing different parts of DENV-2 cDNA were used for construction of the clones in this study. The pD2/IC-30P-A was also used to derive wt virus for this study.…”

Section: Cells and Denv-2 Cdna Plasmidmentioning

confidence: 99%

“…Plasmid containing wt DENV-2 cDNA nt 1-1380 (Kinney et al, 1997) was used for insertion of the GFP fragment at the BclI site (DEN-2 cDNA nt-407 position). The DENV-2/GFP fusion fragment of the subclone was then cloned into the fulllength DEN-2 cDNA clone, pD2/IC-30P-A by SstI and SphI restriction sites.…”

Section: Construction Of a Full-length Dengue Virus Plasmid Expressinmentioning

confidence: 99%

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Development of Dengue type-2 virus replicons expressing GFP reporter gene in study of viral RNA replication

et al. 2012

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Insertion of green fluorescent protein (GFP) encoding-gene into virus genes has provided a valuable tool for flavivirus research. This study aimed to develop dengue virus (DENV) replicons expressing GFP reporter that would provide a fast in vitro system to analyze functional roles of specific DENV sequences in viral replication. Two classes of recombinant replicon constructs were generated; one was a RNA-launched replicon with a GFP gene directly inserted into a fulllength DENV genome (FL-DENV/GFP), and the other consisted of 4 types of DNA-launched DENV subgenomic replicons with GFP replacement at various structural genes (Δ-DENV/GFP). The FL-DENV/GFP resulted in GFP expression in transfected cells with no viable DENV being recovered from the transfection. The Δ-DENV/GFP constructs with partial structural gene deletion (ΔC-, ΔCprM/M-, ΔprM/M-, or ΔE-) expressed bright and long lasting GFP. The GFP expression intensity in living cells correlated well with the level of RNA replication. Various mutations in the 5′noncoding region of DENV-2 previously shown to be important genetic determinants for virus replication and mouse virulence were incorporated into the 5 different replicon constructs. Characterizations of 29 mutants demonstrated that these replicons can provide a useful platform for a quick and powerful in vitro system to analyze genetic determinants of DENV replication. These constructs can also be useful for development of vectors expressing foreign genes for various researches.

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Section: Cells and Denv-2 Cdna Plasmidmentioning

confidence: 99%

Section: Construction Of a Full-length Dengue Virus Plasmid Expressinmentioning

confidence: 99%

Development of Dengue type-2 virus replicons expressing GFP reporter gene in study of viral RNA replication

et al. 2012

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“…The influence of cell typespecific electroporation efficiencies on IV production is an obvious consideration; however, efficiencies are known to be higher for mammalian cells (30-40 %) than for C6/36 cells (2-10 %), which is counter-intuitive to our DENV-2 results. Viral titres were collected over time for P-0 viruses and the growth-curve patterns (data not shown) resembled that of the standardized growth curve described below, as well as that of other DENV-2 viruses (Kinney et al, 1997).…”

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confidence: 99%

“…For genome sequencing and infectious clone (ic) construction, five cDNA fragments (f1-f5) representing the entire 1409hp genome were generated by RT-PCR (Titan RT-PCR kit; Roche Biochemicals), each of the fragments was inserted into a modified pBR322 plasmid (Kinney et al, 1997) and the five plasmids (p1-p5; Fig. 1a) were transfected by electroporation into competent Escherichia coli XL-1 Blue cells (Stratagene).…”

mentioning

confidence: 99%

Infectious clone construction of dengue virus type 2, strain Jamaican 1409, and characterization of a conditional E6 mutation

Pierro

Salazar

Beaty

et al. 2006

Journal of General Virology

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A full-length infectious cDNA clone (ic) was constructed from the genome of the dengue virus type 2 (DENV-2) Jamaica83 1409 strain, pBAC1409ic, by using a bacterial artifical chromosome plasmid system. Infectious virus was generated and characterized for growth in cell culture and for infection in Aedes aegypti mosquitoes. During construction, an isoleucine to methionine (IleRMet) change was found at position 6 in the envelope glycoprotein sequence between low-and high-passage DENV-2 1409 strains. In vitro-transcribed genomic RNA of 1409ic with E6-Ile produced infectious virions following electroporation in mosquito cells, but not mammalian cells, while 1409ic RNA with an E6-Met mutation produced virus in both cell types. Moreover, DENV-2 1409 with the E6-Ile residue produced syncytia in C6/36 cell culture, whereas viruses with E6-Met did not. However, in vitro cell culture-derived growth-curve data and in vivo mosquito-infection rates revealed that none of the analysed DENV-2 strains differed from each other.The four dengue virus (DENV) serotypes are mosquitoborne members of the genus Flavivirus (family Flaviviridae) and are endemic in the tropical and subtropical regions of the world. Transmission to humans is by peridomestic Aedes spp. mosquitoes, primarily Aedes aegypti. Manifestations of a DENV infection range from subclinical to a self-limited fever and rash [dengue fever (DF)] to a severe and sometimes deadly illness characterized by capillary leakage, thrombocytopenia and hypovolaemic shock [dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS)]. Taken together, these viruses are estimated each year to cause 100 million cases of DF, 500 000 cases of DHF/DSS and 25 000 deaths, and 40 % of the world population (i.e. 2?5 billion people) is at risk of infection (Monath, 1994).Epidemiological studies have identified dengue virus type 2 (DENV-2) as the most important dengue virus serotype worldwide. The DENV-2 genome is a single-stranded, positive-sense RNA molecule of approximately 10 700 nt that contains a type I 59-cap structure, but lacks a 39 poly(A) tail. The genome is translated directly as a single polyprotein containing the three structural genes (NH 2 -CapsidprM-Envelope) fused to the seven non-structural genes (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-COOH) that is processed co-and post-translationally (Chambers et al., 1990).Molecular analysis of the envelope gene from DENV-2 field isolates originally identified five major DENV-2 genotypes: Caribbean (American), South Pacific, Middle East and Indonesia, Vietnam and Thailand (Asian), and West African (Lewis et al., 1993;Rico-Hesse, 1990;Rico-Hesse et al., 1997;Wang et al., 2000). Additional analysis of more DENV-2 samples has revealed a total of six DENV-2 genotypes: American, American/Asian, Asian I, Asian II, Sylvatic and Cosmopolitan (Twiddy et al., 2002). The new DENV-2 American/Asian genotype, which includes the DENV-2 Jamaican 1409 isolate (1409), is an Asian genotype DENV-2 introduced into the Americas, with subsequent proliferation and circ...

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“…There is a common difficulty in the construction of full-length clones of flaviviruses, because the plasmids containing a full-length cDNA of these viruses are often unstable during propagation in E. coli. Therefore, by using low-copy-number plasmids and specific bacterial hosts, stable full-length infectious clones have been developed for several flaviviruses (Gritsun and Gould, 1998;Hayasaka et al, 2004;Kinney et al, 1997;Shi et al, 2002;Yamshchikov et al, 2001;Yun et al, 2003). In this study, the low-copy-number plasmid pACNR was used for the construction of the full-length OHFV cDNA.…”

Section: Discussionmentioning

confidence: 99%

Construction of an infectious cDNA clone for Omsk hemorrhagic fever virus, and characterization of mutations in NS2A and NS5

et al. 2011

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Omsk hemorrhagic fever virus (OHFV) is a member of the tick-borne encephalitis serocomplex of flaviviruses, and causes hemorrhagic disease in humans. In this study, an infectious cDNA of OHFV was constructed to investigate the molecular mechanisms involved in OHFV pathogenesis for the first time. Our cDNA clone was capable of producing infectious virus which is genetically identical to the parental Guriev strain, and the recombinant virus showed similar biological properties to the parental virus including growth kinetics and virulence characteristics. While characterizing the cDNAs, fortuitous mutations at NS2A position 46 and NS5 position 836 were found to affect viral production. By using a viral replicon expressing luciferase, it was shown that both of the mutations produced a defect in RNA replication and that the NS5 mutation induced a temperature-sensitive phenotype, indicating the importance of these residues in RNA replication.This infectious cDNA will be a useful tool to study the replication and pathogenesis of OHFV.

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Construction of Infectious cDNA Clones for Dengue 2 Virus: Strain 16681 and Its Attenuated Vaccine Derivative, Strain PDK-53

Cited by 291 publications

References 34 publications

Development of Dengue type-2 virus replicons expressing GFP reporter gene in study of viral RNA replication

Development of Dengue type-2 virus replicons expressing GFP reporter gene in study of viral RNA replication

Infectious clone construction of dengue virus type 2, strain Jamaican 1409, and characterization of a conditional E6 mutation

Construction of an infectious cDNA clone for Omsk hemorrhagic fever virus, and characterization of mutations in NS2A and NS5

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