Full-length cDNA Clone Research Articles

Use of an Infectious cDNA Clone of Pepper Veinal Mottle Virus to Confirm the Etiology of a Disease in Capsicum chinense.

The pepper cultivar Yellow Lantern, one of the spiciest pepper varieties, is a local germplasm of Capsicum chinense, cultivated exclusively on Hainan Island, China. However, this variety is susceptible to viral diseases that severely affect its production. In this study, we report that pepper veinal mottle virus (PVMV) is associated with foliar chlorosis and rugosity symptoms in Yellow Lantern. To verify this correlation, we constructed a full-length cDNA clone of a PVMV isolate named HNu. The virus progeny derived from the cDNA clone replicated and moved systemically in the pepper, inducing the same symptoms as those induced by PVMV-HNu in Yellow Lantern peppers in the field. The results support that PVMV-HNu is the causal agent of foliar chlorosis and rugosity disease in Yellow Lantern. This knowledge will help in the diagnosis and prevention of disease caused by PVMV. Furthermore, the cDNA clone serves as a reverse genetic tool to study the molecular pathogenesis of PVMV.

Rapid manipulation of the porcine epidemic diarrhea virus genome by CRISPR/Cas9 technology

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus causing lethal watery diarrhea in suckling piglets. Reverse genetics is a valuable tool to study the functions of viral genes and to generate vaccine candidates. In this study, a full-length infectious cDNA clone of the highly virulent PEDV strain AJ1102 was assembled in a bacterial artificial chromosome (BAC). The rescued virus (rAJ1102) exhibited similar proliferation characteristics in vitro to the wildtype AJ1102. Using CRISPR/Cas9 technology, a recombinant virus rAJ1102-ΔORF3-EGFP in which the ORF3 gene was replaced with an EGFP gene, was successfully generated, and its proliferation characteristics were compared with the parental rAJ1102. Importantly, it just took one week to construct the recombinant PEDV rAJ1102-ΔORF3-EGFP using this method, providing a more efficient platform for PEDV genome manipulation, which could also be applied to other RNA viruses.

Molecular characterization and In Vitro synthesis of infectious RNA of a Turnip vein-clearing virus isolated from Alliaria petiolata in Hungary.

A tobamovirus was isolated from leaves of Alliaria petiolata plants, showing vein-clearing, interveinal chlorosis, and moderate deformation. Host range experiments revealed a high similarity of isolate ApH both to ribgrass mosaic viruses and turnip vein-clearing viruses. The complete nucleotide sequence of the viral genome was determined. The genomic RNA is composed of 6312 nucleotides and contains four open reading frames (ORF). ORF1 is 3324 nt-long and encodes a polypeptide of about 125.3 kDa. The ORF1 encoded putative replication protein contains an Alphavirus-like methyltransferase domain. ORF2 is 4806 nt-long and encodes a polypeptide of about 182 kDa. The ORF2 encoded putative replication protein contains an RNA-dependent RNA polymerase, catalytic domain. ORF3 encodes the putative cell-to-cell movement protein with a molecular weight of 30.1 kDa. ORF4 overlaps with ORF3 and encodes the coat protein with a size of 17.5 kDa. Sequence comparisons revealed that the ApH isolate has the highest similarity to turnip vein-clearing viruses and should be considered an isolate of Turnip vein-clearing virus (TVCV). This is the first report on the occurrence of TVCV in Hungary. In vitro transcripts prepared from the full-length cDNA clone of TVCV-ApH were highly infectious and induced typical symptoms characteristic to the original isolate of the virus. Since infectious clones of TVCV-ApH and crTMV (another isolate of TVCV) markedly differed in respect to recovery phenotype in Arabidopsis thaliana, it is feasible to carry out gene exchange or mutational studies to determine viral factors responsible for the symptom recovery phenotype.

Construction of full-length infectious cDNA clones of Apple stem grooving virus using Gibson Assembly method

Apple stem grooving virus (ASGV) belongs to the genus Capillovirus within the family Betaflexiviridae. In this work, we described the construction of full-length infectious cDNA clones of ASGV isolate jilin-shaguo (JL-SG) using the Gibson Assembly approach (New England BioLabs). The isolate was previously detected in a Chinese pear-leaf crab apple (Malus asiatica Nakai.) in Baicheng, Jilin province, China. Two full-length cDNA clones of ASGV JL-SG were obtained, and they are identical to each other in sequence. The full-length cDNA clone was infectious on Chenopodium quinoa, Nicotiana glutinosa, and N. occidentalis 37B via agroinfiltration. Through sap inoculation, the infection was additionally spread to C. amaranticolor. N. benthamiana could not be infected, neither through agroinfiltration nor sap inoculation. In infected herbaceous plants, typical ASGV particles with morphology of flexuous filaments were observed by transmission electron microscope (TEM). Moreover, seeds of infected N. glutinosa and N. occidentalis 37B were collected and germinated, the seedlings were ASGV-free in RT-PCR test, suggesting ASGV JL-SG is not seed-transmissible in the tested Nicotiana species. In addition, the cDNA clone was agroinfiltrated into seedlings of Malus pumila cv. Fuji. The infection was symptomless, and can be spread to C. quinoa via sap inoculation, causing typical symptoms. ASGV JL-SG was also detected by RT-PCR in the infected Fuji plants, however, no virion was observed by TEM.

Establishment of a Virulent Full-Length cDNA Clone for Type I Feline Coronavirus Strain C3663.

Feline infectious peritonitis (FIP) is one of the most important infectious diseases in cats and is caused by feline coronavirus (FCoV). Tissue culture-adapted type I FCoV shows reduced FIP induction in experimental infections, which complicates the understanding of FIP pathogenesis caused by type I FCoV. We previously found that the type I FCoV strain C3663 efficiently induces FIP in specific-pathogen-free cats through the naturally infectious route. In this study, we employed a bacterial artificial chromosome-based reverse genetics system to gain more insights into FIP caused by the C3633 strain. We successfully generated recombinant virus (rC3663) from Fcwf-4 cells transfected with infectious cDNA that showed growth kinetics similar to those shown by the parental virus. Next, we constructed a reporter C3663 virus carrying the nanoluciferase (Nluc) gene to measure viral replication with high sensitivity. The inhibitory effects of different compounds against rC3663-Nluc could be measured within 24 h postinfection. Furthermore, we found that A72 cells derived from canine fibroblasts permitted FCoV replication without apparent cytopathic effects. Thus, our reporter virus is useful for uncovering the infectivity of type I FCoV in different cell lines, including canine-derived cells. Surprisingly, we uncovered aberrant viral RNA transcription of rC3663 in A72 cells. Overall, we succeeded in obtaining infectious cDNA clones derived from type I FCoV that retained its virulence. Our recombinant FCoVs are powerful tools for increasing our understanding of the viral life cycle and pathogenesis of FIP-inducing type I FCoV.IMPORTANCE Feline coronavirus (FCoV) is one of the most significant coronaviruses, because this virus induces feline infectious peritonitis (FIP), which is a lethal disease in cats. Tissue culture-adapted type I FCoV often loses pathogenicity, which complicates research on type I FCoV-induced feline infectious peritonitis (FIP). Since we previously found that type I FCoV strain C3663 efficiently induces FIP in specific-pathogen-free cats, we established a reverse genetics system for the C3663 strain to obtain recombinant viruses in the present study. By using a reporter C3663 virus, we were able to examine the inhibitory effect of 68 compounds on C3663 replication in Fcwf-4 cells and infectivity in a canine-derived cell line. Interestingly, one canine cell line, A72, permitted FCoV replication but with low efficiency and aberrant viral gene expression.

Transcriptome Analysis and Metabolic Profiling of Lycoris Radiata.

Lycoris radiata belongs to the Amaryllidaceae family and is a bulbous plant native to South Korea, China, and Japan. Galantamine, a representative alkaloid of Amaryllidaceae plants, including L. radiata, exhibits selective and dominant acetylcholinesterase inhibition. In spite of the economic and officinal importance of L. radiata, the molecular biological and biochemical information on L. radiata is relatively deficient. Therefore, this study provides functional information of L. radiata, describe galantamine biosynthesis in the various organs, and provide transcriptomic and metabolic datasets to support elucidation of galantamine biosynthesis pathway in future studies. The results of studies conducted in duplicate revealed the presence of a total of 325,609 and 404,019 unigenes, acquired from 9,913,869,968 and 10,162,653,038 raw reads, respectively, after trimming the raw reads using CutAdapt, assembly using Trinity package, and clustering using CD-Hit-EST. All of the assembled unigenes were aligned to the public databases, including National Center for Biotechnology Information (NCBI) non-redundant protein (NR) and nucleotide (Nt) database, SWISS-PROT (UniProt) protein sequence data bank, The Arabidopsis Information Resource (TAIR), the Swiss-Prot protein database, Gene Ontology (GO), and Clusters of Orthologous Groups (COG) database to predict potential genes and provide their functional information. Based on our transcriptome data and published literatures, eight full-length cDNA clones encoding LrPAL2, LrPAL3, LrC4H2, LrC3H, LrTYDC2, LrNNR, LrN4OMT, and LrCYP96T genes, involved in galantamine biosynthesis, were identified in L. radiata. In order to investigate galantamine biosynthesis in different plant parts of L. radiata grown in a growth chamber, gene expression levels were measured through quantitative real-time polymerase chain reaction (qRT-PCR) analysis using these identified genes and galantamine levels were quantified by high-performance liquid chromatography (HPLC) analysis. The qRT-PCR data revealed high expression levels of LrNNR, LrN4OMT, and LrCYP96T in the bulbs, and, as expected, we observed higher amounts of galantamine in the bulbs than in the root and leaves. Additionally, a total of 40 hydrophilic metabolites were detected in the different organs using gas-chromatography coupled with time-of-flight mass spectrometry. In particular, a strong positive correlation between galantamine and sucrose, which provides energy for the secondary metabolite biosynthesis, was observed.

Establishment of an efficient reverse genetic system of Mumps virus S79 from cloned DNA

BackgroundMumps is a common type of respiratory infectious disease caused by mumps virus (MuV), and can be effectively prevented by vaccination. In this study, a reverse genetic system of MuV that can facilitate the rational design of safer, more efficient mumps vaccine candidates is established.MethodsMuV-S79 cDNA clone was assembled into a full-length plasmid by means of the GeneArt™ High-Order Genetic Assembly System, and was rescued via reverse genetic technology. RT-PCR, sequencing, and immunofluorescence assays were used for rMuV-S79 authentication. Viral replication kinetics and in vivo experimental models were used to evaluate the replication, safety, and immunogenicity of rMuV-S79.ResultsA full-length cDNA clone of MuV-S79 in the assembly process was generated by a novel plasmid assemble strategy, and a robust reverse genetic system of MuV-S79 was successfully established. The established rMuV-S79 strain could reach a high virus titer in vitro. The average viral titer of rMuV-S79 in the lung tissues was 2.68 ± 0.14 log10PFU/g lung tissue, and rMuV-S79 group did not induce inflammation in the lung tissues in cotton rats. Neutralizing antibody titers induced by rMuV-S79 were high, long-lasting and could provide complete protection against MuV wild strain challenge.ConclusionWe have established a robust reverse genetic system of MuV-S79 which can facilitate the optimization of mumps vaccines. rMuV-S79 rescued could reach a high virus titer and the safety was proven in vivo. It could also provide complete protection against MuV wild strain challenge.

Rescue of Recombinant Zika Virus from a Bacterial Artificial Chromosome cDNA Clone.

The association of Zika virus (ZIKV) infection with neurological complications during the recent worldwide outbreak and the lack of approved vaccines and/or antivirals have underscored the urgent need to develop ZIKV reverse genetic systems to facilitate the study of ZIKV biology and the development of therapeutic and/or prophylactic approaches. However, like with other flaviviruses, the generation of ZIKV full-length infectious cDNA clones has been hampered due to the toxicity of viral sequences during its amplification in bacteria. To overcome this problem, we have developed a nontraditional approach based on the use of bacterial artificial chromosomes (BACs). Using this approach, the full-length cDNA copy of the ZIKV strain Rio Grande do Norte Natal (ZIKV-RGN) is generated from four synthetic DNA fragments and assembled into the single-copy pBeloBAC11 plasmid under the control of the human cytomegalovirus (CMV) immediate-early promoter. The assembled BAC cDNA clone is stable during propagation in bacteria, and infectious recombinant (r)ZIKV is recovered in Vero cells after transfection of the BAC cDNA clone. The protocol described here provides a powerful technique for the generation of infectious clones of flaviviruses, including ZIKV, and other positive-strand RNA viruses, particularly those with large genomes that have stability problems during bacterialpropagation.

Generation of Full-Length Infectious cDNA Clones of Middle East Respiratory Syndrome Coronavirus

Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in Saudi Arabia in 2012 and related infection cases have been reported in over 20 countries. Roughly 10,000 human cases have so far been reported in total with fatality rates at up to 40%. The majority of cases have occurred in Saudi Arabia with mostly sporadic outbreaks outside the country except for the one in South Korea in 2015. The Korean MERS-CoV strain was isolated from the second Korean patient and its genome was fully sequenced and deposited. To develop virusspecific protective and therapeutic agents against the Korean isolate and to investigate molecular determinants of virus-host interactions, it is of paramount importance to generate its full-length cDNA. Here we report that two full-length cDNAs from a Korean patientisolated MERS-CoV strain were generated by a combination of conventional cloning techniques and efficient Gibson assembly reactions. The full-length cDNAs were validated by restriction analysis and their sequence was verified by Sanger method. The resulting cDNA was efficiently transcribed in vitro and the T7 promoter-driven expression was robust. The resulting reverse genetic system will add to the published list of MERS-CoV cDNAs and facilitate the development of Korean isolate-specific antiviral measures.

MTORC1/rpS6 signaling complex modifies BTB transport function: an in vivo study using the adjudin model.

Studies have shown that the mTORC1/rpS6 signaling cascade regulates Sertoli cell blood-testis barrier (BTB) dynamics. For instance, specific inhibition of mTORC1 by treating Sertoli cells with rapamycin promotes the Sertoli cell barrier, making it "tighter." However, activation of mTORC1 by overexpressing a full-length rpS6 cDNA clone (i.e., rpS6-WT, wild type) in Sertoli cells promotes BTB remodeling, making the barrier "leaky." Also, there is an increase in rpS6 and p-rpS6 (phosphorylated and activated rpS6) expression at the BTB in testes at stages VIII-IX of the epithelial cycle, and it coincides with BTB remodeling to support the transport of preleptotene spermatocytes across the barrier, illustrating that rpS6 is a BTB-modifying signaling protein. Herein, we used a constitutively active, quadruple phosphomimetic mutant of rpS6, namely p-rpS6-MT of p-rpS6-S235E/S236E/S240E/S244E, wherein Ser (S) was converted to Glu (E) at amino acid residues 235, 236, 240, and 244 from the NH2 terminus by site-directed mutagenesis, for its overexpression in rat testes in vivo using the Polyplus in vivo jet-PEI transfection reagent with high transfection efficiency. Overexpression of this p-rpS6-MT was capable of inducing BTB remodeling, making the barrier "leaky." This thus promoted the entry of the nonhormonal male contraceptive adjudin into the adluminal compartment in the seminiferous epithelium to induce germ cell exfoliation. Combined overexpression of p-rpS6-MT with a male contraceptive (e.g., adjudin) potentiated the drug bioavailability by modifying the BTB. This approach thus lowers intrinsic drug toxicity due to a reduced drug dose, further characterizing the biology of BTB transport function.

Enhanced patient care, reduced costs and measurable better healthcare performance through implementation of intelligent liver function testing (ILFT)

Triterpene saponins include bioactive compounds with structures consisting of triterpene aglycones (sapogenins) and one or more sugar moieties linked through acetal or ester glycosidic linkages at one or more sites. Centella asiatica (L.) Urban is a medicinal plant that contains bioactive ursane-type saponins, such as madecassoside and asiaticoside. In this work, glucosylation of triterpenoids in C. asiatica was investigated starting with plant extracts. An enzyme capable of glucosylating asiatic and madecassic acids was partially purified. Proteomics methods and cDNA sequence data were employed as tools to obtain a full-length cDNA clone encoding a glucosyltransferase. The recombinant gene product, UGT73AD1, was functionally expressed in Escherichia coli and purified by immobilized metal-affinity chromatography. Purified recombinant UGT73AD1 was found to have a narrow specificity, glucosylating asiatic and madecassic acids at the C28 carboxyl. mRNA accumulated in all tissues tested (leaves, stems, roots and flowers), with highest expression in leaves. Thus, UGT73AD1 was identified as a triterpenoid carboxylic acid: UDP-glucose 28-O-glucosyltransferase that appears to be involved in saponin biosynthesis in C. asiatica.

The N-Terminal Domain of Spike Protein Is Not the Enteric Tropism Determinant for Transmissible Gastroenteritis Virus in Piglets.

Transmissible gastroenteritis virus (TGEV) is the etiologic agent of transmissible gastroenteritis in pigs, and the N-terminal domain of TGEV spike protein is generally recognized as both the virulence determinant and enteric tropism determinant. Here, we assembled a full-length infectious cDNA clone of TGEV in a bacterial artificial chromosome. Using a novel approach, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems efficiently and rapidly rescued another recombinant virus with a 224-amino-acid deletion in the N-terminal domain of the TGEV Spike gene (S_NTD224), which is analogous to the N-terminal domain of porcine respiratory coronavirus. S_NTD224 notably affected the TGEV growth kinetics in PK-15 cells but was not essential for recombinant virus survival. In animal experiments with 13 two-day-old piglets, the TGEV recombinant viruses with/without S_NTD224 deletion induced obvious clinical signs and mortality. Together, our results directly demonstrated that S_NTD224 of TGEV mildly influenced TGEV virulence but was not the enteric tropism determinant and provide new insights for the development of a new attenuated vaccine against TGEV. Importantly, the optimized reverse genetics platform used in this study will simplify the construction of mutant infectious clones and help accelerate progress in coronavirus research.

A single nucleotide change in the overlapping MP and CP reading frames results in differences in symptoms caused by two isolates of Youcai mosaic virus.

Two isolates of Youcai mosaic virus (YoMV) were obtained, and their full-length genomic sequences were determined. Full-length infectious cDNA clones of each isolate were generated in which the viral sequence was under the control of dual T7 and 35S promoters for both in vitro transcript production and agro-infiltration. Comparison of the predicted amino acid sequences of the encoded proteins revealed only four differences between the isolates: three in the RNA-dependent RNA polymerase (RdRp) (V383I and M492I in the 125-kDa protein and T1245M in the 182-kDa protein); and one in the overlapping region of the movement protein (MP) and coat protein (CP) genes, affecting only the N-terminal domain of CP (CP M17T). One of the isolates caused severe symptoms in Nicotiana benthamiana plants, while the other caused only mild symptoms. In order to identify the amino acid residues associated with symptom severity, chimeric constructs were generated by combining parts of the two infectious YoMV clones, and the symptoms in infected plants were compared to those induced by the parental isolates. This allowed us to conclude that the M17T substitution in the N-terminal domain of CP was responsible for the difference in symptom severity. The M17T variation was found to be unique among characterized YoMV isolates. A difference in potential post-translational modification resulting from the presence of a predicted casein kinase II phosphorylation site only in the CP of isolate HK2 may be responsible for the symptom differences.

Rescue and characterization of a recombinant HY12 bovine enterovirus carrying a foreign HA epitope in the 3A nonstructural protein.

Full-length infectious cDNA clones for recombinant HY12 bovine enteroviruses designated as rHY12-3A-2-HA, rHY12-3A-3-HA, and rHY12-3A-9-HA were constructed by the insertion of an epitope from influenza virus hemagglutinin (HA) at the N-terminus of the HY12-encoded 3A protein at amino acid positions 2, 3, and 9. The recombinant HY12 viruses expressing the HA epitope were rescued and characterized using immunoperoxidase monolayer assay, western blotting, and electron microscopy. The three rescued recombinant marker viruses showed similar characteristics, such as TCID50 titer, plaque size, and growth properties, to those of parental rHY12 virus. Comparative analysis of the nucleotide sequences demonstrated the three recombinant marker viruses remained stable for 15 passages with no genetic changes. The recombinant viruses remained viable in various permissive cell lines, including BHK-21, Vero, and PK15 cells, suggesting that the insertion of the HA epitope tag had no effect on virus infectivity. Mice infected with the recombinant marker viruses and the parental virus produced anti-HY12-virus antibodies, while the recombinant marker viruses also produced anti-HA-epitope-tag antibodies. Taken together, these results demonstrate that HY12 viruses containing genetic markers may be useful tools for future investigations of the mechanisms of viral pathogenesis and virus replication, as well as for vaccine development.

Functional expression of a peritrophin A-like SfPER protein is required for larval development in Spodoptera frugiperda (Lepidoptera: Noctuidae)

Peritrophins are associated with structural and functional integrity of peritrophic membranes (PM), structures composed of chitin and proteins. PM lines the insect midgut and has roles in digestion and protection from toxins. We report the full-length cDNA cloning, molecular characterization and functional analysis of SfPER, a novel PM peritrophin A protein, in Spodoptera frugiperda. The predicted amino acid sequence indicated SfPER’s domain structure as a CMCMC-type, consisting of a signal peptide and three chitin-binding (C) domains with two intervening mucin-like (M) domains. Phylogenetic analysis determined a close relationship between SfPER and another S. frugiperda PM peritrophin partial sequence. SfPER transcripts were found in larvae and adults but were absent from eggs and pupae. Chitin affinity studies with a recombinant SfPER-C1 peritrophin A-type domain fused to SUMO/His-tag confirmed that SfPER binds to chitin. Western blots of S. frugiperda larval proteins detected different sized variants of SfPER along the PM, with larger variants found towards the posterior PM. In vivo suppression of SfPER expression did not affect susceptibility of larvae to Bacillus thuringiensis toxin, but significantly decreased pupal weight and adult emergence, possibly due to PM structural alterations impairing digestion. Our results suggest SfPER could be a novel target for insect control.

The Three Essential Motifs in P0 for Suppression of RNA Silencing Activity of Potato leafroll virus Are Required for Virus Systemic Infection.

Higher plants exploit posttranscriptional gene silencing as a defense mechanism against virus infection by the RNA degradation system. Plant RNA viruses suppress posttranscriptional gene silencing using their encoded proteins. Three important motifs (F-box-like motif, G139/W140/G141-like motif, and C-terminal conserved region) in P0 of Potato leafroll virus (PLRV) were reported to be essential for suppression of RNA silencing activity. In this study, Agrobacterium-mediated transient experiments were carried out to screen the available amino acid substitutions in the F-box-like motif and G139/W140/G141-like motif that abolished the RNA silencing suppression activity of P0, without disturbing the P1 amino acid sequence. Subsequently, four P0 defective mutants derived from a full-length cDNA clone of PLRV (L76F and W87R substitutions in the F-box-like motif, G139RRR substitution in the G139/W140/G141-like motif, and F220R substitution in the C-terminal conserved region) were successfully generated by reverse PCR and used to investigate the impact of these substitutions on PLRV infectivity. The RT-PCR and western blot analysis revealed that these defective mutants affected virus accumulation in inoculated leaves and systemic movement in Nicotiana benthamiana as well as in its natural hosts, potato and black nightshade. These results further demonstrate that the RNA silencing suppressor of PLRV is required for PLRV accumulation and systemic infection.

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.

Complete genome sequence and construction of an infectious full-length cDNA clone of celery latent virus – an unusual member of a putative new genus within the Potyviridae

Celery latent virus (CeLV) is an incompletely described plant virus known to be sap and seed transmissible and to possess flexuous filamentous particles measuring about 900 nm in length, suggesting it as a possible member of the family Potyviridae. Here, an Italian isolate of CeLV was transmitted by sap to a number of host plants and shown to have a single-stranded and monopartite RNA genome being 11 519 nucleotides (nts) in size and possessing some unusual features. The RNA contains a large open reading frame (ORF) that is flanked by a short 5' untranslated region (UTR) of 13 nt and a 3' UTR consisting of 586 nt that is not polyadenylated. CeLV RNA shares nt sequence identity of only about 40 % with other members of the Potyviridae (potyvirids). The CeLV polyprotein is notable in that it starts with a signal peptide, has a putative P3N-PIPO ORF and shares low aa sequence identity (about 18 %) with other potyvirids. Although potential cleavage sites were not identified for the N-terminal two-thirds of the polyprotein, the latter possesses a number of sequence motifs, the identity and position of which are characteristic of other potyvirids. Attempts at constructing an infectious full-length cDNA clone of CeLV were successful following Rhizobium radiobacter infiltration of Nicotiana benthamiana and Apium graveolens. CeLV appears to have the largest genome of all known potyvirids and some unique genome features that may warrant the creation of a new genus, for which we propose the name 'celavirus'.

Validation of a site-specific recombination cloning technique for the rapid development of a full-length cDNA clone of a virulent field strain of vesicular stomatitis New Jersey virus

This study reports the use of a site-specific recombination cloning technique for rapid development of a full-length cDNA clone that can produce infectious vesicular stomatitis New Jersey virus (VSNJV). The full-length genome of the epidemic VSNJV NJ0612NME6 strain was amplified in four overlapping cDNA fragments which were linked together and cloned into a vector plasmid by site-specific recombination. Furthermore, to derive infectious virus, three supporting plasmid vectors containing either the nucleoprotein (N), phosphoprotein (P) or polymerase (L) genes were constructed using the same cloning methodology. Recovery of recombinant VSNJV was achieved after transfecting all four vectors on into BSR-T7/5 cells, a BHK-derived cell line stably expressing T7 RNA polymerase (PMID: 9847328). In vitro characterization of recombinant and parental viruses revealed similar growth kinetics and plaque morphologies. Furthermore, experimental infection of pigs with the recombinant virus resulted in severe vesicular stomatitis with clinical signs similar to those previously reported for the parental field strain. These results validate the use of site-directed specific recombination cloning as a useful alternative method for rapid construction of stable full-length cDNA clones from vesicular stomatitis field strains. The approach reported herein contributes to the improvement of previously published methodologies for the development of full-length cDNA clones of this relevant virus.

Amplification and Cloning of Large cDNA Fragments of the Citrus tristeza virus Genome.

Citrus tristeza virus (CTV) is probably the most destructive viral pathogen of citrus. It causes chronic losses to commercial citrus production in all citrus-growing areas. The complete sequences of at least 42 genomes of different CTV strains have been obtained using different technologies including sequencing of multiple overlapped RT-PCR-amplified fragments with sizes of less than 4kb, or from small viral RNA (svRNA), through next-generation high-throughput sequencing (NGS) technologies. The large size of CTV genome (>19.2kb) makes it impractical to obtain and amplify full-length cDNA in a single step. The strategy of ligation of multiple cDNA fragments to assemble a full-length cDNA clone involves several serial cloning steps and sometimes subcloning phases using enzymatic digestion with restriction nucleases and ligation reactions. In this protocol, we describe a strategy to clone the entire genome of CTV obtained from two RT-PCR amplified products. These 5'- and 3'-genomic halves, which were designed to be overlapped in 15nt in their 3'- and 5'-ends, respectively, were used as templates for further overlapped PCR to amplify the entire ~20kb CTV genome. The resultant full cDNA PCR product was then inserted into pCAMBIA-binary vector.