Construction Of Infectious Clone Research Articles

Rapid one-step construction of a Middle East Respiratory Syndrome (MERS-CoV) infectious clone system by homologous recombination

BackgroundViral Infectious clone systems serve as robust platforms to study viral gene or replicative function by reverse genetics, formulate vaccines and adapt a wild type-virus to an animal host. Since the development of the first viral infectious clone system for the poliovirus, novel strategies of viral genome construction have allowed for the assembly of viral genomes across the identified viral families. However, the molecular profiles of some viruses make their genome more difficult to construct than others. Two factors that affect the difficulty of infectious clone construction are genome length and genome complexity. ResultsThis work examines the available strategies for overcoming the obstacles of assembling the long and complex RNA genomes of coronaviruses and reports one-step construction of an infectious clone system for the Middle East Respiratory Syndrome coronavirus (MERS-CoV) by homologous recombination in S. cerevisiae. ConclusionsFuture use of this methodology will shorten the time between emergence of a novel viral pathogen and construction of an infectious clone system. Completion of a viral infectious clone system facilitates further study of a virus’s biology, improvement of diagnostic tests, vaccine production and the screening of antiviral compounds.

Genomic deletions and mutations resulting in the loss of eight genes reduce the in vivo replication capacity of Meleagrid herpesvirus 1.

Meleagrid herpesvirus 1 (MeHV-1 or turkey herpesvirus) has been widely used as a vaccine in commercial poultry. Initially, these vaccine applications were for the prevention of Marek's disease resulting from Gallid herpesvirus 2 infections, while more recently MeHV-1 has been used as recombinant vector for other poultry infections. The construction of herpesvirus infectious clones that permit propagation and manipulation of the viral genome in bacterial hosts has advanced the studies of herpesviral genetics. The current study reports the construction of five MeHV-1 infectious clones. The in vitro properties of viruses recovered from these clones were indistinguishable from the parental MeHV-1. In contrast, the rescued MeHV-1 viruses were significantly attenuated when used in vivo. Complete sequencing of the infectious clones identified the absence of two regions of the MeHV-1 genome compared to the MeHV-1 reference sequence. These analyses determined the rescued viruses have seven genes, UL43, UL44, UL45, UL56, HVT071, sorf3 and US2 either partially or completely deleted. In addition, single nucleotide polymorphisms were identified in all clones compared with the MeHV-1 reference sequence. As a consequence of one of the polymorphisms identified in the UL13 gene, four of the rescued viruses were predicted to encode a serine/threonine protein kinase lacking two of three domains required for activity. Thus four of the recovered viruses have a total of eight missing or defective genes. The implications of these findings in the context of herpesvirus biology and infectious clone construction are discussed.

Construction of an infectious clone of a plant RNA virus in a binary vector using one-step Gibson Assembly

The construction of full-length infectious clones of RNA viruses is often laborious due to the many cloning steps required and the DNA exclusion within the plasmid during Escherichia coli transformation. We demonstrate single-step cloning procedure of an infectious cDNA of the tomato blistering mosaic virus (ToBMV) using Gibson Assembly (GA), which drastically reduces the number of cloning steps. By agro-inoculation with the construct obtained by this procedure, ToBMV was recovered six days post-inoculation in Nicotiana benthamiana plants. The symptoms induced by the recovered virus were indistinguishable from those caused by the wild-type virus. We conclude that the GA is very useful method particularly to construct a full-length cDNA clone of a plant RNA virus in a binary vector.

Construction of infectious clones of tomato torrado virus and their delivery by agroinfiltration.

The first biologically active infectious clones of tomato torrado virus (ToTV) were generated and delivered into Nicotiana benthamiana and Solanum lycopersicum plants via Agrobacterium tumefaciens. The engineered constructs consisted of PCR-amplified complementary DNAs derived from the ToTV RNA1 and RNA2 components, individually inserted into an engineered pGreen binary vector between the CaMV 35S promoter and nopaline synthase terminator. These constructs were introduced into the plant hosts by means of A. tumefaciens-mediated infiltration. In the presence of the progeny virus, typical symptoms of ToTV infection developed in N. benthamiana and S. lycopersicum. Moreover, the virus was sap-transmissible when isolated from agroinfiltrated plants and induced symptoms similar to those caused by the wild-type virus. The presence of viral particles and viral genetic material was confirmed by electron microscopy and re-inoculation to S. lycopersicum and N. benthamiana, as well as by reverse transcription polymerase chain reaction and high-resolution melt analysis.Electronic supplementary materialThe online version of this article (doi:10.1007/s00705-014-2266-1) contains supplementary material, which is available to authorized users.

Simplified methods for the construction of RNA and DNA virus infectious clones.

Infectious virus clones are one of the most powerful tools in plant pathology, molecular biology, and biotechnology. The construction of infectious clones of RNA and DNA viruses, however, usually requires laborious cloning and subcloning steps. In addition, instability of the RNA virus genome is frequently reported after its introduction into the vector and transference to Escherichia coli. These difficulties hamper the cloning procedures, making it tedious and cumbersome. This chapter describes two protocols for a simple construction of infectious viruses, an RNA virus, the tobamovirus Pepper mild mottle virus, and a DNA virus, a bipartite begomovirus. For this purpose, the strategy of overlap-extension PCR was used for the construction of infectious tobamovirus clone and of rolling circle amplification (RCA) for the construction of a dimeric form of the begomovirus clone.

A Rapid and Efficient Method for Construction of an Infectious Clone of Tomato yellow leaf curl virus

Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus, is responsible for one of the most devastating viral diseases in tomato-growing countries and is becoming a serious problem in many subtropical and tropical countries. The climate in Korea is getting warmer and developing subtropical features in response to global warming. These changes are being accompanied by TYLCV, which is now becoming a large problem in the Korean tomato industry. The most effective way to reduce damage caused by TYLCV is to breed resistant varieties of tomatoes. To accomplish this, it is necessary to establish a simple inoculation technique for the efficient evaluation of resistance to TYLCV. Here, we present the rolling circle amplification (RCA) method, which employs a bacteriophage using phi-29 DNA polymerase for construction of infectious TYLCV clones. The RCA method is simple, does not require sequence information for cloning, and is less expensive and time consuming than conventional PCR based-methods. Furthermore, RCA-based construction of an infectious clone can be very useful to other emerging and unknown geminiviruses in Korea.

Establishment of reverse genetics system for infectious bronchitis virus attenuated vaccine strain H120

Infectious bronchitis virus (IBV) strain H120 was successfully rescued as infectious clone by reverse genetics. Thirteen 1.5–2.8kb fragments contiguously spanning the virus genome were amplified and cloned into pMD19-T. Transcription grade complete length cDNA was acquired by a modified “No See’m” ligation strategy, which employed restriction enzyme Bsa I and BsmB I and ligated more than two fragments in one T4 ligase reaction. The full-length genomic cDNA was transcribed and its transcript was transfected by electroporation into BHK-21 together with the transcript of nucleocapsid gene. At 48h post transfection, the medium to culture the transfected BHK-21 cells was harvested and inoculated into 10-days old SPF embryonated chicken eggs (ECE) to replicate the rescued virus. After passage of the virus in ECE five times, the rescued H120 virus (R-H120) was successfully recovered. R-H120 was subsequently identified to possess the introduced silent mutation site in its genome. Some biological characteristics of R-H120 such as growth curve, EID50 and HA titers, were tested and all of them were very similar to its parent strain H120. In addition, both R-H120 and H120 induced a comparable titer of HA inhibition (HI) antibody in immunized chickens and also provided up to 85% of immune protection to the chickens that were challenged with Mass41 IBV strain. The present study demonstrated that construction of infectious clone from IBV vaccine strain H120 is possible and IBV-H120 can be use as a vaccine vector for the development of novel vaccines through molecular recombination and the modified reverse genetics approach.

The Infectious Clone Construction of Tomato Yellow Leaf Curl Virus Isolate from Tianjin

The Infectious Clone Construction of Tomato Yellow Leaf Curl Virus Isolate from Tianjin

Parental LTRs Are Important in a Construct of a Stable and Efficient Replication-Competent Infectious Molecular Clone of HIV-1 CRF08_BC

Circulating recombinant forms (CRFs) of HIV-1 have been identified in southern China in recent years. CRF08_BC is one of the most predominant subtypes circulating in China. In order to study HIV subtype biology and to provide a tool for biotechnological applications, the first full-length replication-competent infectious molecular clone harboring CRF08_BC is reported. The construction of this clone pBRGX indicates that a moderate-copy number vector is required for its amplification in E. coli. In addition, it is shown that the parental CRF08_BC LTRs are important for generating this efficient replication-competent infectious clone. These observations may aid in the construction of infectious clones from other subtypes. Both the pBRGX-derived virus and its parental isolate contain CCR5 tropism. Their full-length genomes were also sequenced, analyzed, compared and deposited in GenBank (JF719819 and JF719818, respectively). The availability of pBRGX as the first replication-competent molecular clone of CRF08_BC provides a useful tool for a wide range of studies of this newly emergent HIV subtype, including the development of HIV vaccine candidates, antiviral drug screening and drug resistance analysis.

Molecular characterization and an infectious clone construction of sweet potato leaf curl virus (SPLCV) isolated from Korea

Sweet potato leaf curl disease (SPLCD) was primarily identified in sweet potato fields in Korea in 2003, and the complete genomic sequence of sweet potato leaf curl virus (SPLCV) has been cloned. The genome of the Korean SPLCV isolate (SPLCV-KR) comprises 2,828 nucleotides with six open reading frames in DNA-A, similar to a monopartite begomovirus. Additionally, neither the genome B genomic component nor the DNA beta sequence was detected. The results of phylogenetic analysis using the maximum parsimony method showed that SPLCV-KR is more closely related to SPLCV-US (US) than SPLCV-CN (China) and SPLCV-JP (Japan). A tandem repeat dimer of SPLCV-KR was cloned and found to be infectious in sweet potatoes (Ipomoea batatas) via biolistic inoculation. The SPLCV-infected sweet potatoes exhibited mild leaf curl symptoms of SPLCD, and the newly-replicated viral DNA was detected via Southern blot analysis. Results of biotic, molecular, and phylogenetic characterization suggest that SPLCV-KR is a new strain of SPLCV and is importantly placed in the evolutionary progression from curtoviruses to begomoviruses. sweet potato leaf curl virus; sweet potato leaf curl disease; phylogenetic analysis; infectious clone; biolistic infection.

First construction of infectious clone for newly emerging mutation porcine circovirus type 2 (PCV2) followed by comparison with PCV2a and PCV2b genotypes in biological characteristics in vitro

BackgroundPorcine circovirus type 2 (PCV2), the causative agent of postweaning multisystemic wasting syndrome (PMWS), is a serious economic problem in the swine industry. Different genotypes (PCV2a, PCV2b and PCV2d) of the virus are present in the clinical cases in China, and it is necessary to elucidate the pathogenic difference among different genotypes of PCV2. In this study, four strains of different genotypes were isolated, two were ordinary strains and another two were mutation strains, which there are one and two amino acids elongation in the capsid protein (Cap) of PCV2, respectively. Representative strains of different genotypes of the virus were constructed by infectious molecular clone and biological characterization of the rescued viruses were identified in vitro.ResultsFour PCV2 isolates (PCV2a/CL, PCV2b/YJ, PCV2b/JF and PCV2d/BDH) of different genotypes were isolated from the clinical cases of PMWS in China. Four infectious clones of PCV2 were constructed and the rescued viruses were harvested after transfection into PK15 cells. The rescued viruses were verified by nucleotide sequence analysis, morphology of the viruses and immunoperoxidase monolayer assay (IPMA). The rescued viruses propagated stably after consecutive incubation for more than ten passages, and virus propagation reached its peak 72h post infection (PI), and the virus titers were up to 105.7 TCID50/ml. By using neutralizing 1D2 monoclonal antibody (mAb) of PCV2, the antigen capture ELISA showed that only the PCV2a/rCL and PCV2b/rJF strains has immunoreactivity with the 1D2 mAb, however, another two rescued strains (PCV2b/rYJ and PCV2d/rBDH) do not, which indicated the antigenic difference among the rescued viruses of different genotypes. In addition, here is the first report of obtaining the newly emerging PCV2 with mutation in vitro by infectious molecular clone technology.ConclusionsConclusions drawn from this study show that PCV2 has prevailing differences in genomic and ORF2 gene length and antigen in swine herds in China. Four representative clones for different genotypes were constructed and rescued, which will facilitate further studies on the pathogenic differences resulting from different subtypes of PCV2.

Dicistroviruses

Dicistroviruses are members of a recently defined and rapidly growing family of picornavirus-like RNA viruses called the Dicistroviridae. Dicistroviruses are pathogenic to beneficial arthropods such as honey bees and shrimp and to insect pests of medical and agricultural importance. Our understanding of these viruses is uneven. We present highly advanced studies of the virus particle structure, remarkable mechanisms of internal ribosome entry in translation of viral RNA, and the use of dicistroviruses to study the insect immune system. However, little is known about dicistrovirus RNA replication mechanisms or gene function, except by comparison with picornaviruses. The recent construction of infectious clones of dicistrovirus genomes may fill these gaps in knowledge. We discuss economically important diseases caused by dicistroviruses. Future research may lead to protection of beneficial arthropods from dicistroviruses and to application of dicistroviruses as biopesticides targeting pestiferous insects.

Detection of Truncated Circular DNA Species in Escherichia coli with a PCV2-Containing Plasmid with a Single PCV2 Origin of Replication

Objective: Porcine circovirus type 2 (PCV2) is a small circular single-stranded DNA virus that causes postweaning multisystemic wasting syndrome in pigs. Cloning of the PCV2 genome in a plasmid allows the construction of infectious clones. Our objective was to clone single PCV2 genomes from an isolate containing a mixture of strains, in a plasmid in order to obtain pure PCV2 strains. Methods: PCR amplification of PCV2 genomes and cloning followed by restriction enzyme analysis and sequencing. Transfection and PCV2 titration on PK-15 cells. Results: Single-copy PCV2 genomes from three Belgian PCV2 strains were cloned. Unexpectedly, agarose gel analysis revealed that additional circular DNA species were generated in Escherichia coli. Restriction enzyme analysis and sequencing showed that the circular DNA species were truncated and derived from the plasmid containing the PCV2 genome. Mutagenesis of the PCV2 replicase gene abolished the formation of these DNA species. The infectious clones were transfected in PK-15 cells and pure PCV2 viral strains were obtained. Conclusion: Infectious clones were obtained that can be used for antigenic mapping and mutagenesis. In addition, our findings suggest that the replicase protein was expressed in E. coli and involved in the generation of the truncated DNA species.

One-step cloning approach for construction of agroinfectious begomovirus clones

Most begomoviruses have a bipartite genome containing two circular ssDNA segments (DNA-A and DNA-B). Routine infectious clone construction relies upon cloning of the whole genome, which is then subcloned as a tandem one-and-half or two genome- (containing two replication origins) cassette into a vector prior to agro-inoculation. The construction of cassettes containing two replication origins is, however, a time-consuming process. Here an improved method for rapid construction of agroinfectious begomovirus clones is described. Total DNA was extracted from a tomato plant infected with Tomato golden vein virus and viral ssDNA molecules were amplified using phi-29 bacteriophage polymerase. High molecular weight DNA was partially digested with a single cutting restriction endonuclease (BamHI) and DNA fragments containing genome dimers were ligated into pCAMBIA0380, and used to transform Escherichia coli cells. This transformation yielded clones containing either DNA-A or DNA-B dimers. One clone each was used to transform Agrobacterium tumefaciens cells. DNA-A and DNA-B transformants were mixed and inoculated into test plants. All inoculated plants (tomato and Nicotiana benthamiana) became infected, confirming the infectivity of the clones. This approach was proven to be extremely fast and useful for the production of infectious clones.

Complete genome characterization of Rocio virus (Flavivirus: Flaviviridae), a Brazilian flavivirus isolated from a fatal case of encephalitis during an epidemic in São Paulo state

The flaviviruses of major medical importance in South American countries are yellow fever, dengue, Saint Louis encephalitis, West Nile and Rocio viruses. Rocio virus (ROCV) has been responsible for epidemics of severe encephalitis in Brazil with a case-fatality rate of 10 % and development of sequelae in 20 % of the survivors. We have sequenced and characterized the entire genome of ROCV for the first time, by determining the general traits of the open reading frame and the characteristics of viral genes including the potential cleavage sites, conserved or unique motifs, cysteine residues and potential glycosylation sites. The conserved sequences in the 3'-non-coding region were identified, and the predicted secondary structures during cyclization between 5'- and 3'-non-coding regions were studied. Multiple protein and phylogenetic analyses based on antigenically important and phylogenetically informative genes confirmed a close relationship between ROCV and Ilheus virus (ILHV), together constituting a unique and distinct phylogenetic subgroup as well as the genetic relationship of ROCV with several members of the Japanese encephalitis group. Although ROCV is phylogenetically related to ILHV, our study shows that it is still a virus distinct from the latter virus. This is the first flavivirus uniquely indigenous to Brazil that has been sequenced completely and the genome characterized. The data should be useful for further studies at the molecular level, including construction of infectious clone, identification of gene function, improved disease surveillance based on molecular diagnostic tools and vaccine development.

Infectious clone construction of dengue virus type 2, strain Jamaican 1409, and characterization of a conditional E6 mutation

A full-length infectious cDNA clone (ic) was constructed from the genome of the dengue virus type 2 (DENV-2) Jamaica83 1409 strain, pBAC1409ic, by using a bacterial artifical chromosome plasmid system. Infectious virus was generated and characterized for growth in cell culture and for infection in Aedes aegypti mosquitoes. During construction, an isoleucine to methionine (Ile-->Met) change was found at position 6 in the envelope glycoprotein sequence between low- and high-passage DENV-2 1409 strains. In vitro-transcribed genomic RNA of 1409ic with E6-Ile produced infectious virions following electroporation in mosquito cells, but not mammalian cells, while 1409ic RNA with an E6-Met mutation produced virus in both cell types. Moreover, DENV-2 1409 with the E6-Ile residue produced syncytia in C6/36 cell culture, whereas viruses with E6-Met did not. However, in vitro cell culture-derived growth-curve data and in vivo mosquito-infection rates revealed that none of the analysed DENV-2 strains differed from each other.

Amplification of the full-length hepatitis A virus genome by long reverse transcription-PCR and transcription of infectious RNA directly from the amplicon.

The genetic study of RNA viruses is greatly facilitated by the availability of infectious cDNA clones. However, their construction has often been difficult. While exploring ways to simplify the construction of infectious clones, we have successfully modified and applied the newly described technique of "long PCR" to the synthesis of a full-length DNA amplicon from the RNA of a cytopathogenic mutant (HM 175/24a) of the hepatitis A virus (HAV). Primers were synthesized to match the two extremities of the HAV genome. The antisense primer, homologous to the 3' end, was used in both the reverse transcription (RT) and the PCR steps. With these primers we reproducibly obtained a full-length amplicon of approximately 7.5 kb. Further, since we engineered a T7 promoter in the sense primer, RNA could be transcribed directly from the amplicon with T7 RNA polymerase. Following transfection of cultured fetal rhesus kidney cells with the transcription mixture containing both the HAV cDNA and the transcribed RNA, replicating HAV was detected by immunofluorescence microscopy and, following passage to other cell cultures, by focus formation. The recovered virus displayed the cytopathic effect and large plaque phenotype typical of the original virus; this result highlights the fidelity of the modified long reverse transcription-PCR procedure and demonstrates the potential of this method for providing cDNAs of viral genomes and simplifying the construction of infectious clones.

324 The effect of oral zinc supplementation on the amyloid precursor protein of Alzheimer's disease in human plasma and rat brain

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 (COVID-19). Although multiple mutations have been observed in SARS-CoV-2, functional analysis of each mutation of SARS-CoV-2 has been limited by the lack of convenient mutagenesis methods. In this study, we establish a PCR-based, bacterium-free method to generate SARS-CoV-2 infectious clones. Recombinant SARS-CoV-2 could be rescued at high titer with high accuracy after assembling 10 SARS-CoV-2 cDNA fragments by circular polymerase extension reaction (CPER) and transfection of the resulting circular genome into susceptible cells. The construction of infectious clones for reporter viruses and mutant viruses could be completed in two simple steps: introduction of reporter genes or mutations into the desirable DNA fragments (∼5,000 base pairs) by PCR and assembly of the DNA fragments by CPER. This reverse genetics system may potentially advance further understanding of SARS-CoV-2.

Infectious Transcripts of Tick-Borne Encephalitis Virus, Generated in Days by RT-PCR

Construction of infectious clones of flaviviruses can be problematic owing to instability, toxicity, and recombination events occurring while cloning cDNA in the bacterial vectors. To overcome these difficulties we have devised a rapid and simple method for producing an infectious genetically engineered tick-borne encephalitis virus in less than 10 days using viral RNA from an unpurified virus suspension. The experimental protocol utilized the high fidelity reverse transcription-polymerase chain reaction to produce two long (5.7 and 5.2 kb) overlapping cDNA segments. To produce full-length cDNA the two overlapping segments were either ligated or fused by polymerase chain reaction. The cDNA was then transcribed and the derived full-length RNA was injected intracerebrally into young mice which reproduced the infectious virus within 8–20 days. To differentiate the engineered virus from parent virus, aSunI restriction site was introduced by substituting nucleotides at positions 5688 and 5691 of the viral genome. This restriction site was present in the engineered virus recovered from infected mice. Antigenic and electrophoretic analysis of the proteins recovered from the engineered virus confirmed that it was indistinguishable from parent virus. In addition to its applicability as a rapid method of producing infectious engineered virus, this protocol offers the opportunity to introduce changes by site-directed mutagenesis without needing to clone the viral DNA. The method should be applicable to most viruses possessing an infectious RNA molecule and reduces the time required to produce a genetically engineered virus from years to days. When appropriate, the choice of mice for transfection of RNA has the advantage of being extremely simple, very sensitive, and producing high titers of stable virus.