Construction Of Full-length cDNA Clone Research Articles

Strategy for efficient generation of numerous full-length cDNA clones of classical swine fever virus for haplotyping

BackgroundDirect molecular cloning of full-length cDNAs derived from viral RNA is an approach to identify the individual viral genomes within a virus population. This enables characterization of distinct viral haplotypes present during infection.ResultsIn this study, we recover individual genomes of classical swine fever virus (CSFV), present in a pig infected with vKos that was rescued from a cDNA clone corresponding to the highly virulent CSFV Koslov strain. Full-length cDNA amplicons (ca. 12.3 kb) were made by long RT-PCR, using RNA extracted from serum, and inserted directly into a cloning vector prior to detailed characterization of the individual viral genome sequences. The amplicons used for cloning were deep sequenced, which revealed low level sequence variation (< 5%) scattered across the genome consistent with the clone-derived origin of vKos. Numerous full-length cDNA clones were generated using these amplicons and full-genome sequencing of individual cDNA clones revealed insights into the virus diversity and the haplotypes present during infection. Most cDNA clones were unique, containing several single-nucleotide polymorphisms, and phylogenetic reconstruction revealed a low degree of order.ConclusionsThis optimized methodology enables highly efficient construction of full-length cDNA clones corresponding to individual viral genomes present within RNA virus populations.

A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy.

The Zika virus (ZIKV) has recently attracted major research interest as infection was unexpectedly associated with neurological manifestations in developing foetuses and with Guillain-Barré syndrome in infected adults. Understanding the underlying molecular mechanisms requires reverse genetic systems, which allow manipulation of infectious cDNA clones at will. In the case of flaviviruses, to which ZIKV belongs, several reports have indicated that the construction of full-length cDNA clones is difficult due to toxicity during plasmid amplification in Escherichia coli. Toxicity of flaviviral cDNAs has been linked to the activity of cryptic prokaryotic promoters within the region encoding the structural proteins leading to spurious transcription and expression of toxic viral proteins. Here, we employ an approach based on in silico prediction and mutational silencing of putative promoters to generate full-length cDNA clones of the historical MR766 strain and the contemporary French Polynesian strain H/PF/2013 of ZIKV. While for both strains construction of full-length cDNA clones has failed in the past, we show that our approach generates cDNA clones that are stable on single bacterial plasmids and give rise to infectious viruses with properties similar to those generated by other more complex assembly strategies. Further, we generate luciferase and fluorescent reporter viruses as well as sub-genomic replicons that are fully functional and suitable for various research and drug screening applications. Taken together, this study confirms that in silico prediction and silencing of cryptic prokaryotic promoters is an efficient strategy to generate full-length cDNA clones of flaviviruses and reports novel tools that will facilitate research on ZIKV biology and development of antiviral strategies.

Infectious cDNA clones of the crinivirus Tomato chlorosis virus are competent for systemic plant infection and whitefly-transmission

Tomato chlorosis virus (ToCV) (genus Crinivirus, family Closteroviridae) causes important emergent diseases in tomato and other solanaceous crops. ToCV is not transmitted mechanically and is naturally transmitted by whiteflies. The ToCV genome consists of two molecules of linear, positive-sense RNA encapsidated into long flexuous virions. We present the construction of full-length cDNA clones of the ToCV genome (RNA1 and RNA2) fused to the SP6 RNA polymerase promoter and under the control of the CaMV 35S promoter. RNA1 replicated in the absence of RNA2 in Nicotiana benthamiana and tomato protoplasts after inoculation with cDNA-derived in vitro transcripts. Agroinfiltration of RNA1 and RNA2 under the 35S promoter resulted in systemic infection in N. benthamiana plants. In addition, tomato plants were infected by grafting with agroinfected N. benthamiana scions, showing the typical ToCV symptoms. The viral progeny generated in tomato was transmissible by the whitefly Bemisia tabaci.

An infectious full-length cDNA clone of duck Tembusu virus, a newly emerging flavivirus causing duck egg drop syndrome in China

Duck Tembusu virus (TMUV) is a recently identified pathogenic flavivirus that causes severe egg drop and encephalitis in Chinese ducks and geese. It has been found to be most closely related to the mosquito-origin Tembusu virus and chicken Sitiawan virus reported in Malaysia. However, the ecological characteristics and the pathogenesis of duck TMUV are largely unknown. We report the construction of full-length cDNA clone of duck TMUV strain JXSP. The virus genome was reverse transcribed, amplified as seven overlapping fragments and successively ligated into the low copy number vector pWSK29 under the control of a T7 promoter. Transfection of BHK-21 cells with the transcribed RNA from the full-length cDNA clone resulted in production of highly infectious progeny virus. In vitro growth characteristics in BHK-21 cells and virulence in ducklings and BALB/c mice were similar for the rescued and parental viruses. This stable infectious cDNA clone will be a valuable tool for studying the genetic determinants of duck TMUV.

Construction of Full-Length cDNA Clones to Soil-Borne Wheat Mosaic Virus RNA1 and RNA2, from Which Infectious RNAs Are Transcribed In Vitro: Virion Formation and Systemic Infection without Expression of the N-Terminal and C-Terminal Extensions to the Capsid Protein

The 19-kDa capsid protein (CP) of Soil-borne wheat mosaic furovirus (SBWMV) is encoded in the 5′-terminal region of RNA2. In addition to CP, two CP-related proteins are translated from SBWMV RNA2: (1) a 24-kDa protein (N-CP) with an N-terminal 40-amino-acid extension initiated at an upstream in-frame CUG codon; and (2) an 83-kDa protein (CP-RT) with an about 580-amino-acid, C-terminal extension by partial translational readthrough at the UGA termination codon at the end of the CP gene. We examined requirements for N-CP and CP-RT on virion formation and systemic infection in wheat plants using full-length cDNA clones, from which infectious RNA can be transcribed in vitro. RNA2 mutants, which could not synthesize N-CP, CP-RT, or either infected wheat plants systemically in combination with the wild-type RNA1 transcripts, produced rod-shaped virus particles in uninoculated upper leaves. Original mutations which abolished translation of N-CP and CP-RT were confirmed on RNA2 extracted from purified virus from the upper leaves by nucleotide sequence analysis. These results indicate that neither N-terminal nor C-terminal extensions to the CP are required for virion formation and systemic infection of SBWMV in wheat plants.

Construction of full-length cDNA clones of lettuce mosaic virus (LMV) and the effects of intron-insertion on their viability in Escherichia coli and on their infectivity to plants.

A full length cDNA copy of the genomic RNA of lettuce mosaic virus (LMV) was constructed under the control of an enhanced CaMV 35S promoter and of the NOS terminator. This construct was found infectious when inoculated to lettuce plants. The intron II of the bean nitrite reductase gene was engineered into the LMV FL cDNA in order to relieve possible deleterious effects of viral sequences to Escherichia coli cells and to evaluate the effects of the presence of the intron on the FL cDNA infectivity. The intron-less FL cDNA was found to be as stable as its intron-containing counterpart in E. coli. Sequence analysis of progeny RNA derived from plants inoculated with the intron-containing FL cDNA demonstrated that the inserted intron was perfectly spliced out. The symptoms induced in lettuce by either the intron-less or the intro-containing constructs were identical to those caused by the wild-type virus. However a slight delay in the establishment of infection in lettuce and a more obvious lag in Nicotiana benthamiana were observed with the intron-containing FL cDNA.

Construction of full-length cDNA clones of cucumber mosaic virus RNAs 1, 2 and 3: Generation of infectious RNA transcripts

Full-length cDNA copies of cucumber mosaic virus (CMV) RNAs 1 and 2 of the Fny strain were constructed from partial cDNA clones and were cloned downstream of bacteriophage T7 promoters. In one pair of clones, transcription proceeded from an unaltered T7 promoter such that in vitro transcripts representing RNAs 1 and 2 contained an additional 17 nucleotides at their 5' termini. In a second pair of clones, the T7 promoter/cDNA junction was altered by oligonucleotide-directed mutagenesis such that the in vitro transcripts contained only an additional G residue at their 5' ends. In addition, a full-length cDNA copy of Fny-CMV RNA 3 was constructed from two overlapping cDNA clones and was cloned downstream of an altered T7 promoter such that the resultant in vitro transcripts also contained only an additional G residue at their 5' ends. In vitro transcripts derived from all clones contained an additional C residue at their 3' ends. In vitro transcripts representing RNAs 1, 2 and 3 which contained an additional residue at each terminus were shown to be infectious together in several hosts of CMV.

Construction of full-length cDNA copies of viral double-stranded RNA.

A method is described for the construction of full-length cDNA clones of dsRNAs. All dsRNA viruses have a capsid-associated transcriptase that is responsible for synthesis of the plus strand that is then extruded from viral particles. We have used in vitro transcripts synthesized by the segmented Saccharomyces cerevisiae virus (ScV) as templates for first-strand cDNA synthesis. Synthesis was primed by a 33-base synthetic oligonucleotide. This contained 27 nucleotides complementary to the 3' end of the plus strand from one ScV viral dsRNA segment (S14), and 6 additional nucleotides encoding an XbaI restriction site at the 5' end. The second cDNA strand was synthesized using a similar XbaI linker-synthetic oligonucleotide and the ds cDNA was cloned by standard ligation techniques. All four cDNA plasmid isolates characterized by sequence analysis contained the complete 5' end sequence of S14. Two of these were complete at the 3' end, and one lacked a single base here. Of these four clones, one also retained the XbaI sites at either end. Preparing full-length cDNA clones with unique restriction-site linkers by the use of synthetic oligonucleotides allows for easier screening for complete cDNA clones if neither the vector nor the cDNA has the chosen restriction site. It also provides for easier sequence analysis and manipulation of the genome for later studies, such as cloning into expression vectors. This method is more efficient than any previously described for production of full-sized cDNA clones.