Construction Of Infectious cDNA Clone Research Articles

Construction of Infectious cDNA Clone of Brassica Yellows Virus Isolated from Strawberry and Establishment of TaqMan RT-qPCR

The natural host range for brassica yellows virus (BrYV) is generally limited to Cruciferae. However, we found that BrYV can naturally infect strawberry. The full-length genome sequences of BrYV-MB (accession No. MZ666129) and BrYV-HY (accession No. ON060762) identified in strawberry from Yantai and Beijing, China, were obtained by high-throughput sequencing (HTS) combined with the RT-PCR and RACE techniques. The complete genome sequences of BrYV-MB and BrYV-HY are 5666 nt and contain six open reading frames (ORFs). The two isolates have the highest nucleotide (nt) sequence identity of 99.0%. The infectious cDNA clone of BrYV-HY was constructed through homologous recombination and used to agroinfiltrate Nicotiana benthamiana and Arabidopsis thaliana. The inoculated leaves of N. benthamiana showed necrotic symptoms after 4 days of inoculation (dpi), and the systematic leaves of A. thaliana exhibited purple symptoms at 14 dpi. To develop a rapid and high-sensitive method for the detection of BrYV, a TaqMan real-time fluorescence quantitative RT-PCR method (TaqMan RT-qPCR) was established. Under optimum reaction conditions, the sensitivity of the detection was as low as 100 fg and approximately 100-fold more sensitive than the conventional RT-PCR, so it can be used in large-scale testing.

Construction and characterization of an infectious cDNA clone of potato virus S developed from selected populations that survived genetic bottlenecks

BackgroundInfectious cDNA clones are a powerful tool for studies on RNA viruses using reverse genetics. Potato virus S (PVS) is a carlavirus with a worldwide distribution. Although the complete genome sequences of many PVS isolates have been reported, the construction of an infectious cDNA clone of PVS is yet to be reported. The aim of this study is the development and molecular characterization of an infectious cDNA clone of PVS.MethodsA full-length cDNA clone pPVS-H-FL-AB was constructed by connecting eight cDNA clones of PVS isolate H95. Capped RNA transcripts from pPVS-H-FL-AB and a modified clone pPVS-H-FL-H, containing the consensus genome sequence of PVS-H95, proved to be non-infectious. Therefore, a full-length cDNA clone pPVS-H-FL-β was reconstructed from PVS-H00, isolated from PVS-H95 populations by repeating a single local lesion isolation in Chenopodium quinoa three times; PVS-H00 appeared to be a selected variant that survived genetic bottlenecks. The sequence of cDNA clone pPVS-H-FL-β was determined as the genome sequence of PVS-H00 and compared with the consensus sequence of PVS-H95 genome.ResultsAll Nicotiana occidentalis plants inoculated with ≥0.2 μg capped RNA transcripts from pPVS-H-FL-β developed symptoms on upper leaves, as observed with PVS-H00 inoculation. Similar levels of viral genomic and subgenomic RNAs and coat protein were detected in systemically infected leaves. Sequence comparison of PVS-H95 and PVS-H00 revealed 370 nucleotide polymorphisms (4.4% of the entire genome sequence), causing 91 amino acid substitutions in six open reading frames (ORFs). The infectivity of chimeric RNAs derived from recombinants between the two cDNA clones revealed that the lack of infectivity of pPVS-H-FL-H transcripts was due to ORF1, which encodes replicase and harbors 80 amino acid substitutions compared with pPVS-H-FL-β. Approximately 71.3% amino acid substitutions in replicase were located within the variable region of unknown function between the putative methyltransferase and ovarian tumor-like protease domains.ConclusionsThis is the first report of the development of an infectious cDNA clone of PVS. Our analyses suggest that PVS population within a plant exists as quasispecies and the replicase sequence diversity of PVS obstruct the construction of a full-length infectious cDNA clone.

Construction of infectious cDNA clone derived from a classical swine fever virus field isolate in BAC vector using in vitro overlap extension PCR and recombination

To develop reverse genetics system of RNA viruses, cloning of full-length viral genome is required which is often challenging due to many steps involved. In this study, we report cloning of full-length cDNA from an Indian field isolate (CSFV/IVRI/VB-131) of classical swine fever virus (CSFV) using in vitro overlap extension PCR and recombination which drastically reduced the number of cloning steps. The genome of CSFV was amplified in six overlapping cDNA fragments, linked by overlap extension PCR and cloned in a bacterial artificial chromosome (BAC) vector using in vitro recombination method to generate full-length cDNA clone. The full-length CSFV cDNA clone was found stable in E. coli Stellar and DH10B cells. The full-length RNA was transcribed in vitro using T7 RNA polymerase and transfected in PK15 cells using Neon-tip electroporator to rescue infectious CSFV. The progeny CSFV was propagated in PK15 cells and found indistinguishable from the parent virus. The expression of CSFV proteins were detected in cytoplasm of PK15 cells infected with progeny CSFV at 72h post-infection. We concluded that the in vitro overlap extension PCR and recombination method is useful to construct stable full-length cDNA clone of RNA virus in BAC vector.

Construction of Infectious cDNA Clone of a Chrysanthemum stunt viroid Korean Isolate.

Chrysanthemum stunt viroid (CSVd), a noncoding infectious RNA molecule, causes seriously economic losses of chrysanthemum for 3 or 4 years after its first infection. Monomeric cDNA clones of CSVd isolate SK1 (CSVd-SK1) were constructed in the plasmids pGEM-T easy vector and pUC19 vector. Linear positive-sense transcripts synthesized in vitro from the full-length monomeric cDNA clones of CSVd-SK1 could infect systemically tomato seedlings and chrysanthemum plants, suggesting that the linear CSVd RNA transcribed from the cDNA clones could be replicated as efficiently as circular CSVd in host species. However, direct inoculation of plasmid cDNA clones containing full-length monomeric cDNA of CSVd-SK1 failed to infect tomato and chrysanthemum and linear negative-sense transcripts from the plasmid DNAs were not infectious in the two plant species. The cDNA sequences of progeny viroid in systemically infected tomato and chrysanthemum showed a few substitutions at a specific nucleotide position, but there were no deletions and insertions in the sequences of the CSVd progeny from tomato and chrysanthemum plants.

Engineering a Replication-Competent, Propagation-Defective Middle East Respiratory Syndrome Coronavirus as a Vaccine Candidate

ABSTRACTMiddle East respiratory syndrome coronavirus (MERS-CoV) is an emerging coronavirus infecting humans that is associated with acute pneumonia, occasional renal failure, and a high mortality rate and is considered a threat to public health. The construction of a full-length infectious cDNA clone of the MERS-CoV genome in a bacterial artificial chromosome is reported here, providing a reverse genetics system to study the molecular biology of the virus and to develop attenuated viruses as vaccine candidates. Following transfection with the cDNA clone, infectious virus was rescued in both Vero A66 and Huh-7 cells. Recombinant MERS-CoVs (rMERS-CoVs) lacking the accessory genes 3, 4a, 4b, and 5 were successfully rescued from cDNA clones with these genes deleted. The mutant viruses presented growth kinetics similar to those of the wild-type virus, indicating that accessory genes were not essential for MERS-CoV replication in cell cultures. In contrast, an engineered mutant virus lacking the structural E protein (rMERS-CoV-ΔE) was not successfully rescued, since viral infectivity was lost at early passages. Interestingly, the rMERS-CoV-ΔE genome replicated after cDNA clone was transfected into cells. The infectious virus was rescued and propagated in cells expressing the E protein in trans, indicating that this virus was replication competent and propagation defective. Therefore, the rMERS-CoV-ΔE mutant virus is potentially a safe and promising vaccine candidate to prevent MERS-CoV infection.IMPORTANCE Since the emergence of MERS-CoV in the Arabian Peninsula during the summer of 2012, it has already spread to 10 different countries, infecting around 94 persons and showing a mortality rate higher than 50%. This article describes the development of the first reverse genetics system for MERS-CoV, based on the construction of an infectious cDNA clone inserted into a bacterial artificial chromosome. Using this system, a collection of rMERS-CoV deletion mutants has been generated. Interestingly, one of the mutants with the E gene deleted was a replication-competent, propagation-defective virus that could only be grown in the laboratory by providing E protein in trans, whereas it would only survive a single virus infection cycle in vivo. This virus constitutes a vaccine candidate that may represent a balance between safety and efficacy for the induction of mucosal immunity, which is needed to prevent MERS-CoV infection.

Application of the ligation-independent cloning (LIC) method for rapid construction of a minigenome rescue system for Newcastle disease virus VG/GA strain

Newcastle disease virus (NDV) can cause serious diseases and substantial economic losses to the poultry industry. To gain a better understanding of NDV pathogenesis, several reverse genetics systems for different NDV strains have been established. However, the construction of infectious cDNA clone by conventional restriction digestion/ligation cloning methods is a time-consuming process and has many drawbacks by its nature. To address the problems, we employed a novel and robust ligation-independent cloning (LIC) method for efficient assembly of multiple DNA fragments. Using this method, we successfully generated a NDV minigenome construct within three weeks by assembling RT-PCR products of the VG/GA strain genomic termini and a cDNA coding for the green fluorescence protein (GFP), as a reporter, into a modified pBluescript vector. Co-transfection of the NDV minigenome with three supporting plasmids expressing the N, P, and L proteins into MVA-T7 infected HEp-2 cells and followed by infection with NDV VG/GA resulted in the minigenome replication, transcription, and packaging as evidenced by the reporter gene GFP expression. These results suggest that this LIC approach is a powerful tool for all sequence-independent DNA cloning and multi-DNA fragment assembly, which has a potential application for rapid development of gene therapy and recombinant vaccines.

Construction of an infectious cDNA clone of radish mosaic virus, a crucifer-infecting comovirus

Radish mosaic virus (RaMV) is a crucifer-infecting comovirus that has been detected worldwide. Here, we report the successful construction of a full-length infectious cDNA clone of RaMV. The full-length cDNA clones corresponding to RNA1 and RNA2 of a Japanese isolate of RaMV were cloned into the pBlueScript plasmid or the binary vector pCAMBIA1301 downstream of the cauliflower mosaic virus 35S promoter. Mechanical inoculation or agroinoculation of Nicotiana benthamiana with these vectors resulted in systemic RaMV infections causing symptoms similar to those caused by the wild-type parental virus. The presence of progeny virus was verified by western blot analysis and electron microscopy.

Construction of an infectious cDNA clone of Culex flavivirus, an insect-specific flavivirus from Culex mosquitoes

Culex flavivirus (CxFV) is an insect-specific flavivirus that has recently been detected in various Culex spp. mosquitoes worldwide. Here, we report the successful construction of a full-length infectious cDNA clone of a Tokyo strain, CxFV-NIID21. The full-length CxFV-NIID21 cDNA was cloned into the low-copy-number plasmid pMW119, which was stably amplified in Escherichia coli. Transfection of a mosquito cell line with in vitro-transcribed RNA from the cDNA clone resulted in the production of recombinant progeny virus with growth properties, cytopathogenicity, and virion morphology similar to the parental virus.

Construction of full-length complementary DNA of hepatitis C virus genome from an HCV infected patient

To construct the full-length complementary DNA of HCV genome from an HCV infected patient. Four HCV gene fragments (1.6, 3.5, 2.4 kb and 2.6 kb) were amplified by RT-PCR from serum of a Chinese patient and fused and connected together to produce a 9.2 kb subgenomic fragment, which was further cloned into a cassette vector with fixed 5-prime and 3-prime termini of HCV to make the full-length cDNA. The cDNA heterogeneity was analyzed by comparing the sequences of 4 isolated HVR1 regions. The prokaryotic expressed Core, NS3 protease, NS3 helicase were detected for their specific reactivities with patient serum by Western blot analysis. And the protease activity of NS3 was evaluated in a cell-based NS3/4A-SEAP expression system. The cDNA covered the near full-length of HCV genome from the patient's serum. The difference among HVR1 regions indicated no selection of HCV variants during RT-PCR and the quasi-species characteristic of the amplified cDNA. The prokaryotic expressed viral proteins could be identified by patient serum. In the NS3/4A-SEAP system, NS3 could cleave the 4A-4B site between NS4A and SEAP proteins and resulted in the secretion of SEAP in culture media. These results suggest that the cloned HCV cDNA encodes a complete and functional open reading frame and will be useful for further construction of infectious cDNA clone.