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At least 19 recordsLinked to original sources
PubMed2003

[Rapid construction of infectious clones of infectious bursal disease virus].

A rapid procedure was established for rescuing infectious bursal disease virus (IBDV), an important pathogen in poultry. A full-length cDNA clone of the segment B of a CEF-adapted IBDV strain HZ2 was constructed by long RT-PCR, and the 2827 bp nucleotide sequence, including the 5 - and 3 -noncoding regions (NCR), was established. Then the cDNA clone of segment B was engineered to make it contain three silent nucleotide changes, creating a new EcoRV site that was different from the parent virus sequences, by site-directed silent mutagenesis. Cotransfection of eukaryotic expression recombinants containing modified segment A and segment B with Lipofectamine into Vero cells resulted in the expression of IBDV RNA and proteins, as confirmed by Northern RNA dot hybridization and indirect immunofluorescence assay analysis. The change of cell morphology after cotransfection and passages of cell cultures was similar to that of cells infected by authentic IBDV, causing cellular pathogenic effects (CPE). The virus-like particles at 55-60 nm were observed under electron microscopy, affirming the rescue of IBDV. The genetic markers were retained in the recovered progeny virus.

Animals↗
PubMed2000

A strategy for cloning infectious molecular clones of retroviruses from serum or plasma.

To enable biological characterisation of lentiviral variants which emerge during infection and development of AIDS, a method was developed to construct molecular clones from circulating simian immunodeficiency virus (SIV) particles present in as little as 20 microl of serum from infected rhesus monkeys. This technique uses a long distance RT-PCR method optimised for the amplification of partly overlapping 5-kb SIV (half genome) amplimers. Ligation of the genome halves resulted in the construction of full-length clones which, after transfection, were able to replicate well in rhesus peripheral blood mononuclear cells (PBMCs) and in various human T-cell lines inducing syncytia. In addition to the study of molecular cloned virus quasispecies emerging in circulation as a result of immune escape, this method may also be applied to obtain entire genes or full-length molecular clones. These clones may be present in other extracellular body fluids such as urine, saliva, tears, lymph, and bronchial or cerebral spinal fluid. Genes amplified in this way can be inserted quickly in new recombinant expression vectors and may then be applied for DNA vaccination approaches.

AIDS Vaccines↗
PubMed1993

Pseudorecombination between infectious cloned DNA components of tomato mottle and bean dwarf mosaic geminiviruses.

A newly described whitefly-transmitted geminivirus infecting tomato plants in Florida induces yellow mottling symptoms on leaves, and stunted and distorted growth. The DNA-A and DNA-B components were cloned from extracts of field-infected tomato tissue; excised monomers or uncut tandem dimers of these clones were infectious when co-inoculated on to Nicotiana benthamiana by rub-inoculation. Tomato plants inoculated directly with the DNA-A and DNA-B dimers, or indirectly by sap or graft transmission from N. benthamiana plants previously infected with the dimers, developed symptoms similar to those observed in field-infected plants. This tomato geminivirus is different from previously characterized geminiviruses, and has been named tomato mottle geminivirus (ToMoV). DNA sequence comparisons revealed that ToMoV is closely related to bean dwarf mosaic geminivirus (BDMV) and abutilon mosaic geminivirus. Infectious pseudorecombinants were made by exchanging the cloned infectious DNA components of ToMoV and BDMV and inoculating N. benthamiana plants. The presence of the inoculated DNA components in systemically infected plants was confirmed by characterization of DNA-A and DNA-B fragments amplified by the polymerase chain reaction. This is the first report of pseudorecombination between two distinct geminiviruses. The implications of this finding in geminivirus evolution are discussed.

Base Sequence↗
PubMed2005

Construction of a gene inactivation library for Bovine herpesvirus 1 using infectious clone technology.

The application of infectious clone technology to herpesvirus biology has revolutionized the study of these viruses. Previously the ability to manipulate these large DNA viruses was limited to methods dependent on homologous recombination in mammalian cells. However, the construction of herpesvirus infectious clones using bacterial artificial chromosome vectors has permitted the application of powerful bacterial genetics for the manipulation of these viruses. A method is described for the construction and characterization of a gene inactivation library of Bovine herpesvirus 1 using an infectious clone. The method utilizes transposon-mediated gene inactivation, which permits gene inactivation without any prior knowledge of the viral genomic sequence. Furthermore, as the genetic manipulation is performed in bacteria the inactivation of those viral genes that are essential for viral replication is also possible. The method described here can be readily applied to any herpesvirus clone and provides the tools for precise characterization of all the genes contained within a herpesvirus genome.

Cloning, Molecular↗
PubMed2001

A new strategy in design of +RNA virus infectious clones enabling their stable propagation in E. coli.

Infectious clone methodology is a valuable tool of modern experimental virology. However, its use is often constrained by the instability of infectious clone constructs during propagation in E. coli. To circumvent this problem, we have devised a strategy that could be suitable for design of +RNA virus molecular clones in general. An infectious clone is assembled as "infectious DNA," and expression of problem regions present in the viral cDNA is prevented during propagation in E. coli by insertion of short introns. To demonstrate the feasibility of this approach, a highly unstable Japanese encephalitis flavivirus infectious clone has been successfully converted into a remarkably stable infectious DNA construct with the specific infectivity of 10(6) pfu/microg in cell culture. The proposed strategy may be useful in the design of self-amplifying gene therapy vectors and development of new immunization methodologies, and could facilitate creation of molecular repositories of existing viral vaccines.

Base Sequence↗
PubMed2005

Characterization of an infectious clone of the wild-type yellow fever virus Asibi strain that is able to infect and disseminate in mosquitoes.

Infectious clone technology provides an opportunity to study the molecular basis of arthropod-virus interactions in detail. This study describes the development of an infectious clone of the prototype yellow fever virus Asibi strain (YFV-As) with the purpose of identifying sequences or domains that influence infection dynamics in the mosquito vector. The full-length cDNA of YFV-As virus was produced from RT-PCR products of parental viral RNA. These were cloned into a low-copy-number plasmid previously used to develop the YFV-17D infectious clone (pACNR/FLYF-17D). Virus recovered from the infectious clone exhibited biological characteristics similar to those of the parental YFV-As, including replication kinetics, reactivity to flavivirus cross-reactive and YFV-specific antibodies and infection and dissemination rates in Aedes aegypti, the principal mosquito vector of YFV. These data provide the basis for future studies with chimeric Asibi/17D viruses to identify the determinants of vaccine attenuation in the vector.

Aedes↗
PubMed2005

Amino acid mutations of the infectious clone from Chinese EIAV attenuated vaccine resulted in reversion of virulence.

The Chinese equine infectious anemia virus (EIAV) donkey-leukocyte attenuated vaccine (DLV) provides a unique natural model system by which attenuated mechanism and immunological control of lentivirus replication may be studied. We analyzed the critical consensus mutations that occurred during the viral passages in vitro and in vivo for vaccine's preparation. Based on the full-length infectious clone pLGFD3 (EIAV vaccine background) and according to mutations displayed during viral attenuation, we successfully constructed an infectious clones pLG5-3-l in which gag and env genes were point-mutated by overlap PCR mutagenesis strategy. pLG5-3-l was proved to have the ability of effective replication in vitro cells culture systems by Reverse Transcriptase Assay and virion observation under electron microscopy. Results of the in vivo experiments indicated that marked differences occurred between the mutated virus and their parental virus in clinical manifestation and plasma viral replication during 6-month observation period. In contrast to asymptom of animals infected with pLGFD3-V, the mutated virus (pLG5-3-l-V) developed typical clinical progression in the corresponding experimentally infected animals. The results of the distinct differences in clinical profiles and viral dynamics before and after mutation of EIAV infectious clone will help to understand the protective mechanism of Chinese EIAV vaccine and shed light on novel HIV vaccine design.

Amino Acid Substitution↗
PubMed2001

An infectious clone of the West Nile flavivirus.

West Nile (WN) virus is the most widespread among flaviviruses, but until recently it was not known on the American continent. We describe here design of a subgenomic replicon, as well as a full-length infectious clone of the lineage II WN strain, which appeared surprisingly stable compared to other flavivirus infectious clones. This infectious clone was used to investigate effects of 5'- and 3'-nonrelated sequences on virus replication and infectivity of synthetic RNA. While a long nonrelated sequence at the 3'-end delayed but did not prevent establishment of the productive infectious cycle, a much shorter extra sequence at the 5'-end completely abrogated virus replication. Replacement of the conserved 5'-adenosine residue substantially delayed, but did not prevent, establishment of virus infection. In all cases, the recovered virus had restored its authentic 5'- and 3'-end genome sequences. However, the presence of extensive nonrelated sequences at both 5'- and 3'-ends could not be repaired.

3' Untranslated Regions↗
PubMed2025

Construction and Segmental Reconstitution of Full-Length Infectious Clones of Milk Vetch Dwarf Virus.

The construction of infectious clones (ICs) is essential for studying viral replication, pathogenesis, and host interactions. Milk vetch dwarf virus (MDV), a nanovirus with a multipartite, single-stranded DNA genome, presents unique challenges for IC development due to its segmented genome organization. To enable functional analysis of its genome, we constructed full-length tandem-dimer-based ICs for all eight MDV genomic segments. Each segment was cloned into a binary vector and co-delivered into Nicotiana benthamiana, Nicotiana tabacum, Vicia faba, and Vigna unguiculata plants via Agrobacterium-mediated inoculation. Systemic infection was successfully reconstituted in all host plants, with PCR-based detection confirming the presence of all viral segments in the infected leaves of nearly all tested plants. Segmental accumulation in infected plants was quantified using qPCR, revealing non-equimolar distribution across hosts. This study establishes the first complete IC system for MDV, enabling reproducible infection, replication analysis, and quantitative segment profiling. It provides a foundational tool for future molecular investigations into MDV replication, host interactions, and viral movement, advancing our understanding of nanovirus biology and transmission dynamics.

Nicotiana↗
PubMed2000

Construction of a full length infectious clone for dengue-1 virus Western Pacific,74 strain.

The flavivirus dengue 1 Western Pacific,74 (DEN1 WP) virus has a positive-stranded RNA genome of 10,735 nucleotides. DEN1 WP genomic RNA was amplified into three overlapping fragments by RT-PCR. These fragments were assembled into a full-length cDNA clone in the yeast-E. coli shuttle vector pRS424, using homologous recombination in yeast. RNA produced by in vitro transcription of this clone was infectious upon electroporation into LLCMK2 cells, as shown by cytopathic effects and detection of viral antigens by indirect immunofluorescence, and by propagation of the virus released into the culture media. Biological properties of the transcript-derived virus, such as the pattern of dengue-specific protein synthesis and growth rate in LLCMK2 or C6/36 cells, resembled those of the parent DEN1 WP virus.

Animals↗
PubMed2026

Construction of an infectious clone of Spodoptera frugiperda densovirus and its biological characteristics.

Densoviruses are highly pathogenic to their insect hosts and have great potential for biocontrol. Spodoptera frugiperda densovirus (SfDV) was isolated from diseased larvae of Spodoptera frugiperda, while its biological functions remain unclear. Herein, we successfully constructed an infectious clone of SfDV. The S. frugiperda larvae transfected with the infectious clone exhibited anorexia, stunted growth, and reduced activity. Histopathological analysis further showed that the epidermis, fat body and trachea were infected instead of muscle and midgut tissues. Transmission electron microscopy (TEM) revealed that numerous virions of about 22 nm were distributed within both the nucleoplasm and cytoplasm of epidermal cells. Moreover, many virions were also found contained within vesicles in the cytoplasm. The replication kinetics of the rescued SfDV (rSfDV) was similar to that of the parental SfDV. The median lethal dose (LD50) and median lethal time (LT50) values of rSfDV were 6.63 × 107 viral genome copies (vgc), 5.23 d, respectively, which were also comparable to those of the parental SfDV. Taken together, the infectious clone of SfDV provides an important tool for further exploring the genome function, pathogenesis, and interactions with its hosts.

Animals↗
PubMed1986

The nucleotide sequence of an infectious clone of the geminivirus beet curly top virus.

A number of infectious clones of a Californian isolate of the leafhopper-transmitted geminivirus beet curly top virus (BCTV) have been constructed from virus-specific double-stranded DNA isolated from infected Beta vulgaris and used to demonstrate a single component genome. The nucleotide sequence of one infectious clone has been determined (2993 nucleotides). Comparison with other geminiviruses has shown that the organisation of the genome closely resembles DNA 1 of the whitefly-transmitted members. The four conserved coding regions of DNA 1 have highly homologous counterparts in BCTV with the exception of the putative coat protein which is more closely related to those of the leafhopper-transmitted geminiviruses suggesting a strong interrelationship between coat protein and insect vector. A BCTV component equivalent to DNA 2 is not required for virus infection or transmission and has not been isolated from infected plants.

Journal Article↗
PubMed1997

Plasmid vector for cloning infectious cDNAs from plant RNA viruses: high infectivity of cDNA clones of tomato aspermy cucumovirus.

An improved version of the previously obtained cloning vector pCass was constructed by partially duplicating the 35S promoter used to drive the transient transcription of cloned viral cDNAs. Full-length cDNAs of the three genomic RNAs of tomato aspermy cucumovirus (TAV) cloned in this improved pCass (designated pCass2) gave a 3-fold higher infectivity in two plant species tested than the same cDNAs cloned in pCass1 with only a single 35S promoter. Host range, symptoms, morphology of viral particles and viral progeny RNAs induced by these sets of infectious cDNA clones analysed were identical to those induced by the wild-type virus. A mutant of genomic TAV RNA 3 containing a 163 nt deletion in the 3' untranslated region was stably maintained in the progeny RNAs, indicating that these cDNA clones may facilitate a study of virus function. This is the first report of infectious cDNA clones of TAV as well as of infectious cDNA clones with a duplicated 35S promoter of CaMV.

Cloning, Molecular↗
PubMed1998

Development and analysis of a tick-borne encephalitis virus infectious clone using a novel and rapid strategy.

In less than 1 month we have constructed an infectious clone of attenuated tick-borne encephalitis virus (strain Vasilchenko) from 100 microl of unpurified virus suspension using long high fidelity PCR and a modified bacterial cloning system. Optimization of the 3' antisense primer concentration was essential to achieve PCR synthesis of an 11 kb cDNA copy of RNA from infectious virus. A novel system utilising two antisense primers, a 14-mer for reverse transcription and a 35-mer for long PCR, produced high yields of genomic length cDNA. Use of low copy number Able K cells and an incubation temperature of 28 degrees C increased the genetic stability of cloned cDNA. Clones containing 11 kb cDNA inserts produced colonies of reduced size, thus providing a positive selection system for full length clones. Sequencing of the infectious clone emphasised the improved fidelity of the method compared with conventional PCR and cloning methods. A simple and rapid strategy for genetic manipulation of the infectious clone is also described. These developments represent a significant advance in recombinant technology and should be applicable to positive stranded RNA viruses which cannot easily be purified or genetically manipulated.

Animals↗
PubMed2026

Infectious Clone Development of Zucchini Green Mottle Mosaic Virus Infecting Medicinal Plant Trichosanthes kirilowii and Establishment of a Serological Assay System.

Trichosanthes kirilowii has long been cultivated for application in traditional Chinese medicine. In this study, we identified two isolates of zucchini green mottle mosaic virus (ZGMMV; species Tobamovirus cucurbitae) from T. kirilowii plants. We determined the complete genome sequences of the ZGMMV isolates named ZGMMV-GL-1 and ZGMMV-GL-2. Each ZGMMV genome was 6,517 nucleotides in length, with only a single nucleotide variation detected between two sequences. Sequence analysis revealed that the ZGMMV isolates from this study shared 88.07 to 91.62% nucleotide identity with five other ZGMMV isolates deposited in GenBank. Phylogenetic analysis indicated that ZGMMV isolates can be clustered into two distinct groups; our two isolates shared the highest sequence similarity with the ZGMMV isolate from Nanning (GenBank accession number MF066176) and clustered within Group II. The coat protein (CP) gene was cloned from ZGMMV-infected T. kirilowii samples, and the CPZGMMV was expressed using the pET28(a) vector. Specific polyclonal antiserum CPZGMMV was generated by immunizing rabbits with the purified protein, and its sensitivity was determined to be satisfactory. Leveraging the high accuracy and sensitivity of the CPZGMMV antiserum, we developed a rapid, precise, and scalable diagnostic method for ZGMMV. We then constructed the full-length cDNA clones (ZGMMV-GL-1 and ZGMMV-GL-2). Additionally, the ZGMMV cDNA infectious clones from T. kirilowii were also able to infect Nicotiana benthamiana and Cucumis sativus systemically, inducing rough-textured and curled leaves in N. benthamiana and mosaic symptoms in C. sativus and T. kirilowii. In this study, we produced an antiserum against the ZGMMV CP and developed a sensitive, rapid, and reliable diagnostic assay, which lays a technical foundation for the detection and monitoring of ZGMMV. Therefore, the establishment of the ZGMMV infectious clone facilitates further research on viral protein functions, plant-pathogen interactions, and the formulation of effective ZGMMV management strategies.

Nicotiana benthamiana↗
PubMed2006

Infectious clones of Chikungunya virus (La Réunion isolate) for vector competence studies.

The recent outbreak of Chikungunya virus (CHIKV) on several islands in the Indian Ocean and in India has focused attention on this reemerging virus and highlighted the need for development of new tools to study vector-virus-host interactions. We have constructed and characterized, in cell culture, Aedes aegypti and Ae. albopictus mosquitoes, infectious cDNA clones of CHIKV using a recent isolate from La Réunion Island. Comparison of the growth kinetics and infection rates of the viral isolate CHIKV strain LR2006 OPY1 (CHIKV-LR) and a full-length infectious clone (CHIKV-LR ic) indicate that the infectious clone has retained the viral phenotypes of the original isolate. Infectious clones that express green fluorescent protein (GFP) were also produced and characterized in cell culture and in Aedes mosquitoes. The CHIKV-LR 5'GFP infected Ae. aegypti and Ae. albopictus mosquitoes at a similar rate to the original virus and to the full length infectious clone. The CHIKV-LR 3'GFP only infected Ae. albopictus mosquitoes at similar rates. The development of these authentic infectious clones will enable targeted studies of the molecular determinants of infection, pathogenesis and transmission competence by Ae. aegypti and Ae. albopictus mosquitoes.

Aedes↗
PubMed1996

Nucleotide sequencing and generation of an infectious clone of adeno-associated virus 3.

We have determined the complete nucleotide sequences of adeno-associated virus 3 (AAV-3) and generated an infectious clone. The single-stranded DNA genome of AAV-3 is 4726 nucleotides in length. The positive strand contains two large open reading frames; the left open reading frame encodes the nonstructural proteins and the right open reading frame encodes the structural proteins. The coding regions are flanked by identical inverted terminal repeat sequences containing palindromes. AAV-3 has little homology with the autonomous parvoviruses or erythroviruses but has 82% overall sequence homology with AAV-2. At the amino acid level there was 88% homology with AAV-2 nonstructural (Rep) proteins and 87% homology with AAV-2 capsid proteins. In addition, AAV-3 differed importantly from AAV-2 in the lack of a typical promoter sequence (TATA box) at p40 and the presence of the consensus sequence for adenovirus-related transcription factor E4F binding within the upstream region of the p5 promoter. These results suggest that AAV-3 not only consists of serologically distinct structural proteins but that viral propagation also may be controlled by different gene regulatory elements at the transcription level. The infectious clone confirmed the sequence and may be useful for developing new vectors for gene therapy.

Amino Acid Sequence↗
PubMed2001

Cloning and sequence analysis of an infectious clone of Citrus yellow mosaic virus that can infect sweet orange via Agrobacterium-mediated inoculation.

Citrus yellow mosaic virus (CYMV), a member of the family Caulimoviridae, genus Badnavirus, causes citrus mosaic disease, a disease that occurs commonly in India. The CYMV genome has been cloned and its complete nucleotide sequence determined. Its DNA genome is 7559 bp in length and contains six putative open reading frames (ORFs), all on the plus-strand of the genome and each capable of encoding proteins with a molecular mass of greater than 10 kDa. ORF 3, the largest ORF, encodes a putative polyprotein for functions involved in virus movement, assembly and replication. The other ORFs encode proteins whose exact functions are not completely understood. The genome also contains a plant tRNA(met)-binding site, which may serve as a primer for minus-strand DNA synthesis, in its intergenic region. Phylogenetic analysis of the badnaviruses revealed that CYMV is most closely related to Cacao swollen shoot virus. It was demonstrated that a construct containing 1.4 copies of the cloned CYMV genome could infect sweet orange via Agrobacterium-mediated inoculation.

Amino Acid Sequence↗