Full-length Genomic Clone Research Articles

Engineering Responses to Amino Acid Substitutions in the VP0- and VP3-Coding Regions of PanAsia-1 Strains of Foot-and-Mouth Disease Virus Serotype O.

The presence of sequence divergence through adaptive mutations in the major capsid protein VP1, and also in VP0 (VP4 and VP2) and VP3, of foot-and-mouth disease virus (FMDV) is relevant to a broad range of viral characteristics. To explore the potential role of isolate-specific residues in the VP0 and VP3 coding regions of PanAsia-1 strains in genetic and phenotypic properties of FMDV, a series of recombinant full-length genomic clones were constructed using Cathay topotype infectious cDNA as the original backbone. The deleterious and compensatory effects of individual amino acid substitutions at positions 4008 and 3060 and in several different domains of VP2 illustrated that the chain-based spatial interaction patterns of VP1, VP2, and VP3 (VP1-3), as well as between the internal VP4 and the three external capsid proteins of FMDV, might contribute to the assembly of eventually viable viruses. The Y2079H site-directed mutants dramatically induced a decrease in plaque size on BHK-21 cells and viral pathogenicity in suckling mice. Remarkably, the 2079H-encoding viruses displayed a moderate increase in acid sensitivity correlated with NH4Cl resistance compared to the Y2079-encoding viruses. Interestingly, none of all the 16 rescued viruses were able to infect heparan sulfate-expressing CHO-K1 cells. However, viral infection in BHK-21 cells was facilitated by utilizing non-integrin-dependent, heparin-sensitive receptor(s) and replacements of four uncharged amino acids at position 3174 in VP3 of FMDV had no apparent influence on heparin affinity. These results provide particular insights into the correlation of evolutionary biology with genetic diversity in adapting populations of FMDV.IMPORTANCE The sequence variation within the capsid proteins occurs frequently in the infection of susceptible tissue cultures, reflecting the high levels of genetic diversity of FMDV. A systematic study for the functional significance of isolate-specific residues in VP0 and VP3 of FMDV PanAsia-1 strains suggested that the interaction of amino acid side chains between the N terminus of VP4 and several potential domains of VP1-3 had cascading effects on the viability and developmental characteristics of progeny viruses. Y2079H in VP0 of the indicated FMDVs could affect plaque size and pathogenicity, as well as acid sensitivity correlated with NH4Cl resistance, whereas there was no inevitable correlation in viral plaque and acid-sensitive phenotypes. The high affinity of non-integrin-dependent FMDVs for heparin might be explained by the differences in structures of heparan sulfate proteoglycans on the surfaces of different cell lines. These results may contribute to our understanding of the distinct phenotypic properties of FMDV in vitro and in vivo.

Development of an infectious clone and replicon system of norovirus GII.4

Human norovirus (HuNoV) is one of the main causes of acute gastroenteritis worldwide and is responsible for at least 20% of all cases. The detailed molecular mechanism of this norovirus remains unknown due to the lack of a suitable in vitro culturing system. An infectious clone of HuNoV would be a useful tool for elucidating the processes of viral infection and the mechanisms of replication. We developed an infectious cDNA clone of HuNoV using the rapid technique of Gibson Assembly. The complete genome of the HuNoV GII.4 Sydney subtype was cloned into a previously modified pcDNA3.1-based plasmid vector downstream from a cytomegaloviral promoter. We monitored the viral infection in vitro by inserting the reporter gene of the green fluorescent protein (GFP) between the NTPase and p22 genes, also by Gibson Assembly, to construct a HuNoV-GFP replicon. Human Caco-2 cells were transfected with the full-length genomic clone and the replicon containing GFP. The gene encoding the VP1/VP2 capsid protein was expressed, which was indirect evidence of the synthesis of subgenomic RNAs and thus the negative strand of the genome. We successfully constructed the infectious clone and its replicon containing GFP for the HuNoV GII.4 Sydney subtype, a valuable tool that will help the study of noroviral infection and replication.

Rapid CRISPR/Cas9-Mediated Cloning of Full-Length Epstein-Barr Virus Genomes from Latently Infected Cells.

Herpesviruses have relatively large DNA genomes of more than 150 kb that are difficult to clone and sequence. Bacterial artificial chromosome (BAC) cloning of herpesvirus genomes is a powerful technique that greatly facilitates whole viral genome sequencing as well as functional characterization of reconstituted viruses. We describe recently invented technologies for rapid BAC cloning of herpesvirus genomes using CRISPR/Cas9-mediated homology-directed repair. We focus on recent BAC cloning techniques of Epstein-Barr virus (EBV) genomes and discuss the possible advantages of a CRISPR/Cas9-mediated strategy comparatively with precedent EBV-BAC cloning strategies. We also describe the design decisions of this technology as well as possible pitfalls and points to be improved in the future. The obtained EBV-BAC clones are subjected to long-read sequencing analysis to determine complete EBV genome sequence including repetitive regions. Rapid cloning and sequence determination of various EBV strains will greatly contribute to the understanding of their global geographical distribution. This technology can also be used to clone disease-associated EBV strains and test the hypothesis that they have special features that distinguish them from strains that infect asymptomatically.

Structural and functional characterisation of a class I endochitinase of the carnivorous sundew (Drosera rotundifolia L.).

Chitinase gene from the carnivorous plant, Drosera rotundifolia , was cloned and functionally characterised. Plant chitinases are believed to play an important role in the developmental and physiological processes and in responses to biotic and abiotic stress. In addition, there is growing evidence that carnivorous plants can use them to digest insect prey. In this study, a full-length genomic clone consisting of the 1665-bp chitinase gene (gDrChit) and adjacent promoter region of the 698bp in length were isolated from Drosera rotundifolia L. using degenerate PCR and a genome-walking approach. The corresponding coding sequence of chitinase gene (DrChit) was obtained following RNA isolation from the leaves of aseptically grown in vitro plants, cDNA synthesis with a gene-specific primer and PCR amplification. The open reading frame of cDNA clone consisted of 978 nucleotides and encoded 325 amino acid residues. Sequence analysis indicated that DrChit belongs to the class I group of plant chitinases. Phylogenetic analysis within the Caryophyllales class I chitinases demonstrated a significant evolutionary relatedness of DrChit with clade Ib, which contains the extracellular orthologues that play a role in carnivory. Comparative expression analysis revealed that the DrChit is expressed predominantly in tentacles and is up-regulated by treatment with inducers that mimick insect prey. Enzymatic activity of rDrChit protein expressed in Escherichia coli was confirmed and purified protein exhibited a long oligomer-specific endochitinase activity on glycol-chitin and FITC-chitin. The isolation and expression profile of a chitinase gene from D. rotundifolia has not been reported so far. The obtained results support the role of specific chitinases in digestive processes in carnivorous plant species.

Cloning and rescue of the genome of Bombyx mori bidensovirus, and characterization of a recombinant virus.

BackgroundBombyx mori bidensovirus (BmBDV), which belongs to the Bidnaviridae family established by the International Committee on Taxonomy of Viruses in 2011, was the first bidensovirus identified in insects. The structure of BmBDV is similar to that of parvoviruses, while its replication is similar to that of adenoviruses. Although BmBDV has the potential to be used as a tool in biological pest control and as an expression vector, virus rescue has been a bottleneck in the application of this virus.MethodsIn this study, we constructed a full-length genomic clone of BmBDV and showed that its terminal structure was restored. A recombinant BmBDV that expressed the green fluorescence protein (GFP) gene was constructed. Then, BmN cells, which are an ovarian cell line, were co-transfected with the linearized genome using continuous culture and expanded cell culture methods.ResultsThe results showed that the GFP gene was expressed successfully, and that cell lesions occurred in virus-infected cells. Furthermore, typical densonucleosis viruses were observed in reinfected silkworm larvae and larval midgut tissues infected by BmBDV, as evidenced by the emission of green fluorescence.ConclusionsOverall, these results suggest that the virus could be rescued from the infected BmN cells after co-transfection with the linear full length virus genome.

Use of Posttranscription Gene Silencing in Squash to Induce Resistance against the Egyptian Isolate of the Squash Leaf Curl Virus.

Squash leaf curl virus (SqLCV) is a bipartite begomovirus affecting squash plants. It is transmitted by whitefly Bemisia tabaci biotype B causing severe leaf curling, vein banding, and molting ending by stunting. In this study full-length genomic clone of SqLCV Egyptian isolated and posttranscriptional gene silencing (PTGS) has been induced to develop virus resistance. The Noubaria SqLCV has more than 95% homology with Jordon, Israel, Lebanon, Palestine, and Cairo isolates. Two genes fragment from SqLCV introduced in sense and antisense orientations using pFGC5049 vector to be expressed as hairpin RNA. The first fragment was 348 bp from replication associated protein gene (Rep). The second fragment was 879 bp representing the full sequence of the movement protein gene (BC1). Using real-time PCR, a silencing record of 97% has been recorded to Rep/TrAP construct; as a result it has prevented the appearance of viral symptoms in most tested plants up to two months after infection, while construct containing the BC1 gene scored a reduction in the accumulation of viral genome expression as appearing in real-time PCR results 4.6-fold giving a silencing of 79%, which had a positive effect on symptoms development in most tested plants.

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.

Retraction Note to: An infectious molecular clone in early infection with HIV-1 subtype CRF01_AE strains: construction and biological properties.

Our aim was to construct infectious molecular clones of the CRF01_AE subtype in the primary infection phase of an acute HIV-1 infections in people screened from MSM populations, as well as continue preliminary research on this virus and its biological properties pertaining to deriving viruses. Walking sequencing was performed on a half-molecular clone with target fragment inserted. Western Blot was used to detect protein expression in HIV-1 infected 293T cells. Sequence analysis of HIV-1 genomic clones showed full-length HIV-1 genomic clones without frame shift mutation or termination codon. HIV-1 p24 antigens generated from 08-IMC were slightly greater than those from infectious molecular clones pNL4-3 3 and 93JP-NH1, but without statistical difference (all P > 0.05). The relative light units of 08-ISO was higher than those of 08-IMC, but no significant difference was observed (all P > 0.05). 08-IMC-driven virus was linked to lower replication kinetics. The replication levels of pNL4-3 and 08-ISO were significantly higher than the 08-IMC replication level but close to NH1 replication level (all P < 0.05). 08-IMC could infect the cells expressing CCR5 and be replicated in the CCR5-expressing cells with a positive percentage of 24.3 %, 08-ISO may use CCR5-using macrophage-tropic isolates as coreceptor, while pNL4-3 viruses with T cell tropisms utilize the CXCR4 co-receptor. Our study showed that the infectious molecular clones of viruses in the primary infection phase have a close relationship with the major prevalent CRF01_AE strains and have high homology with the viral RNA in plasma.

Molecular characterization of the Tobacco rattle virus RNA2 genome isolated from Gladiolus.

Tobacco rattle virus (TRV-K) was first identified in a symptomatic Gladiolus plant cultivated in Korea. We analyzed the TRV-K genome and compared its phylogeny with other TRV isolates. After constructing of a full-length genomic RNA2 strand clone, a complete sequence was generated from several overlapping clones. The cloned genome was 3261 bases in length, identical to TRV-K, and had three open reading frames. TRV-K had the highest sequence identity with the American isolate TRV-ORY. Sequence analysis of the RNA2 genome showed that TRV-K contains an intact 2a, 2b, and 2c coding sequence and an RNA1-related 3' terminus, which is typical of TRV RNA2. Phylogenetic analysis revealed that TRV-K is in the same cluster as the American isolates and another Korean isolate, TRV-SK; however, it was in a different cluster than the European isolates.

An infectious molecular clone in early infection with HIV-1 subtype CRF01_AE strains: construction and biological properties.

Our aim was to construct infectious molecular clones of the CRF01_AE subtype in the primary infection phase of an acute HIV-1 infections in people screened from MSM populations, as well as continue preliminary research on this virus and its biological properties pertaining to deriving viruses. Walking sequencing was performed on a half-molecular clone with target fragment inserted. Western Blot was used to detect protein expression in HIV-1 infected 293T cells. Sequence analysis of HIV-1 genomic clones showed full-length HIV-1 genomic clones without frame shift mutation or termination codon. HIV-1 p24 antigens generated from 08-IMC were slightly greater than those from infectious molecular clones pNL4-3 3 and 93JP-NH1, but without statistical difference (all P > 0.05). The relative light units of 08-ISO was higher than those of 08-IMC, but no significant difference was observed (all P > 0.05). 08-IMC-driven virus was linked to lower replication kinetics. The replication levels of pNL4-3 and 08-ISO were significantly higher than the 08-IMC replication level but close to NH1 replication level (all P < 0.05). 08-IMC could infect the cells expressing CCR5 and be replicated in the CCR5-expressing cells with a positive percentage of 24.3 %, 08-ISO may use CCR5-using macrophage-tropic isolates as coreceptor, while pNL4-3 viruses with T cell tropisms utilize the CXCR4 co-receptor. Our study showed that the infectious molecular clones of viruses in the primary infection phase have a close relationship with the major prevalent CRF01_AE strains and have high homology with the viral RNA in plasma.

Molecular characterization and genetic diversity of the starch branching enzyme (SBE) gene from Amaranthus: the evolutionary origin of grain amaranths

Full-length genomic DNA clones encoding starch branching enzymes (SBEs) from three cultivated grain amaranths were isolated and characterized. A sequence analysis showed that the SBE genes from Amaranthus caudatus, A. cruentus, and A. hypochondriacus, viz. SBE-ca, SBE-cr, and SBE-hy, respectively, contained the same exon and intron structure and consisted of 961 amino acids with molecular weights of approximately 108.4 kDa. The lengths of the genes were 7,446 (SBE-ca), 7,453 (SBE-cr), and 7,443 bp (SBE-hy), and they are highly homologous. We also tested, for the first time, the potential use of SBE from diverse genetic sources to answer long-standing questions on the origin of grain amaranths. The maximum parsimony tree based on the SBE sequence data suggests that A. hybridus is the progenitor wild species of grain amaranths. The results could be summarized as follows: (1) the A. hybridus species comprise all grain amaranths, which can then be divided into two subgroups; (2) A. caudatus and A. hypochondriacus and/or A. caudatus and A. quitensis appear to be closely related to each other; (3) A. powellii was the most distantly related to the other five species in the A. hybridus complex. These results indicate that A. hybridus is the ancestor species in at least two separate domestication events, lending support to the theory of three separate, rather than one, domestication events involving A. hybridus as the common ancestor. A new approach to investigating the genetic diversity in Amaranthus using the nuclear gene SBE provided useful information on the evolutionary origin of three cultivated species.

Insertion and stable expression of Gaussia luciferase gene by the genome of bovine viral diarrhea virus

As a tool to address selected issues of virus biology, we constructed a recombinant cDNA clone of bovine viral diarrhea virus (BVDV) expressing Gaussia luciferase (Gluc) reporter gene. A full-length genomic cDNA clone of a non-cytopathic BVDV isolate was assembled by recombination in yeast Saccharomyces cerevisiae. The Gluc gene was inserted between the Npro and Core protein coding regions by recombination. The cDNA transcribed in vitro was infectious upon transfection of MDBK cells, resulting in reporter gene expression and productive virus replication. The rescued viruses were stable for 15 passages in cell culture, maintaining the replication kinetics, focus size and morphology similar to those of the parental virus. Expression and correct processing of the reporter protein were also maintained, as demonstrated by Gluc activity. These results demonstrate that genes up to 555 bp are simply assembled by a single step in yeast recombination and are stably expressed by this cDNA clone.

Characterization of a full-length infectious clone of bovine foamy virus 3026

The biological features of most foamy viruses (FVs) are poorly understood, including bovine foamy virus (BFV). BFV strain 3026 (BFV3026) was isolated from the peripheral blood mononuclear cells of an infected cow in Zhangjiakou, China. A full-length genomic clone of BFV3026 was obtained from BFV3026-infected cells, and it exhibited more than 99% amino acid (AA) homology to another BFV strain isolated in the USA. Upon transfection into fetal canine thymus cells, the full-length BFV3026 clone produced viral structural and auxiliary proteins, typical cytopathic effects, and virus particles. These results demonstrate that the full-length BFV3026 clone is fully infectious and can be used in further BFV3026 research.

Recovery of a chemically synthesized Japanese encephalitis virus reveals two critical adaptive mutations in NS2B and NS4A

A full-length genome infectious clone is a powerful tool for functional assays in virology. In this study, using a chemical synthesized complete genome of Japanese encephalitis virus (JEV) strain SA14 (GenBank accession no. U14163), we constructed a full-length genomic cDNA clone of JEV. The recovered virus from the cDNA clone replicated poorly in baby hamster kidney (BHK-21) cells and in suckling mice brain. Following serial passage in BHK-21 cells, adaptive mutations within the NS2B and NS4A proteins were recovered in the passaged viruses leading to viruses with a large-plaque phenotype. Mutagenesis analysis, using a genome-length RNA and a replicon of JEV, demonstrated that the adaptive mutations restored replication to different degrees, and the restoration efficiencies were in the order: NS2B-T102M<NS4A-R79K<NS2B-T102M+NS4A-R79K. An in vivo virulence assay in mice showed that the recombinant virus containing double mutations showed similar virulence to the WT SA14 (GenBank accession no. M55506). This study reports the first chemically synthesized JEV. A reverse genetics assay demonstrated that substitutions of NS2B-T102M and NS4A-R79K altered JEV replication.

Loss of AtCRK1 gene function in Arabidopsis thaliana decreases tolerance to salt

Calcium serves as an important second messenger in many adaptational and developmental processes of plants. The CDPK-related kinases (CRKs) are a type of serine-threonine kinases belonging to the CDPK-SnRK superfamily. In this study, a T-DNA insertion mutant of AtCRK1 (crk1-1) was employed to explore the possible function of AtCRK1 in salt tolerance. Our results indicated that crk1-1 transformants had a decreased tolerance to salt stress during seed germination compared with both wild-type plants and the complemented transgenic lines, in which the full-length genomic AtCRK1 clone with a 2.3 kb promoter was inserted into crk1-1 to drive CRK1 expression under the control of the CRK1 promoter. Furthermore, a higher malondialdehyde (MDA) level and lower free proline content were measured in crk1-1 plants. The qRT-PCR analyses revealed that the transcription of several stress-related genes was decreased in crk1-1 plants. These results indicate that AtCRK1 is involved in the tolerance to salt stress by changing both MDA and proline levels in Arabidopsis, and may act as a positive regulator in the induction of stress-response gene transcription.

Development of a reverse genetics system to generate recombinant Marburg virus derived from a bat isolate

Recent investigations have shown the Egyptian fruit bat (Rousettus aegyptiacus) to be a natural reservoir for marburgviruses. To better understand the life cycle of these viruses in the natural host, a new reverse genetics system was developed for the reliable rescue of a Marburg virus (MARV) originally isolated directly from a R. aegyptiacus bat (371Bat). To develop this system, the exact terminal sequences were first determined by 5′ and 3′ RACE, followed by the cloning of viral proteins NP, VP35, VP30 and L into expression plasmids. Novel conditions were then developed to efficiently replicate virus mini-genomes followed by the construction of full-length genomic clones from which recombinant wild type and GFP-containing MARVs were rescued. Surprisingly, when these recombinant MARVs were propagated in primary human macrophages, a dramatic difference was found in their ability to grow and to elicit anti-viral cytokine responses.

HPV-16 E2 contributes to induction of HPV-16 late gene expression by inhibiting early polyadenylation

We provide evidence that the human papillomavirus (HPV) E2 protein regulates HPV late gene expression. High levels of E2 caused a read-through at the early polyadenylation signal pAE into the late region of the HPV genome, thereby inducing expression of L1 and L2 mRNAs. This is a conserved property of E2 of both mucosal and cutaneous HPV types. Induction could be reversed by high levels of HPV-16 E1 protein, or by the polyadenylation factor CPSF30. HPV-16 E2 inhibited polyadenylation in vitro by preventing the assembly of the CPSF complex. Both the N-terminal and hinge domains of E2 were required for induction of HPV late gene expression in transfected cells as well as for inhibition of polyadenylation in vitro. Finally, overexpression of HPV-16 E2 induced late gene expression from a full-length genomic clone of HPV-16. We speculate that the accumulation of high levels of E2 during the viral life cycle, not only turns off the expression of the pro-mitotic viral E6 and E7 genes, but also induces the expression of the late HPV genes L1 and L2.

Effects of hepatitis B virus precore and basal core promoter mutations on the expression of viral antigens: genotype B vs C

Hepatitis B virus (HBV) genotypes/mutants are known to affect natural outcomes. The virologic differences among HBV genotype, precore and basal core promoter (BCP) mutations were investigated. HBV strains were isolated from 18 hepatitis B e antigen (HBeAg)-positive patients (nine genotype B and nine genotype C). All had precore and BCP wild-type sequences. After cloning of full-length HBV genome, the effects of viral genotype, precore and BCP mutations singly or additively on the expression of viral DNA and antigens were investigated by mutagenesis and transfection assays in Huh7 cells. Significant findings included the following: (i) expression of intracellular core protein increased when precore or BCP mutation was introduced in genotype C strains; (ii) expression of intracellular surface protein was lower in genotype C precore wild-type strain compared with genotype B; (iii) precore mutation was associated with a lower extracellular expression level of HBV DNA; (iv) secretion of hepatitis B surface antigen in genotype C was lower than that in genotype B; and (v) secretion of HBeAg in genotype B was lower than that in genotype C. No additive effect was observed by combining precore and BCP mutations. Hence, HBV genotype and precore/BCP mutations correlate with intrahepatic expression of viral antigens in vitro.

Distribution of the phenotypic effects of random homologous recombination between two virus species.

Recombination has an evident impact on virus evolution and emergence of new pathotypes, and has generated an immense literature. However, the distribution of phenotypic effects caused by genome-wide random homologous recombination has never been formally investigated. Previous data on the subject have promoted the implicit view that most viral recombinant genomes are likely to be deleterious or lethal if the nucleotide identity of parental sequences is below 90%. We decided to challenge this view by creating a bank of near-random recombinants between two viral species of the genus Begomovirus (Family Geminiviridae) exhibiting 82% nucleotide identity, and by testing infectivity and in planta accumulation of recombinant clones randomly extracted from this bank. The bank was created by DNA-shuffling—a technology initially applied to the random shuffling of individual genes, and here implemented for the first time to shuffle full-length viral genomes. Together with our previously described system allowing the direct cloning of full-length infectious geminivirus genomes, it provided a unique opportunity to generate hundreds of “mosaic” virus genomes, directly testable for infectivity. A subset of 47 randomly chosen recombinants was sequenced, individually inoculated into tomato plants, and compared with the parental viruses. Surprisingly, our results showed that all recombinants were infectious and accumulated at levels comparable or intermediate to that of the parental clones. This indicates that, in our experimental system, despite the fact that the parental genomes differ by nearly 20%, lethal and/or large deleterious effects of recombination are very rare, in striking contrast to the common view that has emerged from previous studies published on other viruses.

Infectious Full-Length Clones of Calibrachoa Mottle Virus (CbMV)

Full-length cDNA clones derived from genomic RNA (gRNA) and subgenomic RNAs (sgRNAs) of Calibrachoa mottle virus (CbMV) were constructed under the control of the T7 RNA promoter and ligated into plasmid pUC-18. The capped and uncapped in vitro transcripts, synthesized from full length genomic cDNA clone pUC-CbMV-FL generated chlorotic local lesions on mechanical inoculated Chenopodium quinoa within 10 to 15 days post-inoculation. The progeny virions recovered from inoculated symptomatic leaves had the same morphological properties as the parental virions. These virions are also reacted positively with CbMV antiserum in double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and the viral coat protein was detected by Western blotting. The presence of five open reading frames (ORFs) on the CbMV genome and synthesis of corresponding proteins by gRNA and sgRNAs were confirmed by Northern blotting and in vitro translation. The transcription start site of three sgRNAs of CbMV was also determined. Successful construction of full-length infectious cDNA clone of CbMV makes it possible to develop molecular tools that can be used to understand the gene functions of this virus.