Construction Of Infectious cDNA Clones Research Articles

Complete Genome Sequencing and Infectious cDNA Clone Construction of Soybean Mosaic Virus Isolated from Shanxi.

Soybean mosaic virus (SMV) is the predominant viral pathogen that affects the yield and quality of soybean. The natural host range for SMV is very narrow, and generally limited to Leguminosae. However, we found that SMV can naturally infect Pinellia ternata and Atractylodes macrocephala. In order to clarify the molecular mechanisms underlying the cross-family infection of SMV, we used double-stranded RNA extraction, rapid amplification of cDNA ends polymerase chain reaction and Gibson assembly techniques to carry out SMV full-length genome amplification from susceptible soybeans and constructed an infectious cDNA clone for SMV. The genome of the SMV Shanxi isolate (SMV-SX) consists of 9,587 nt and encodes a polyprotein consisting of 3,067 aa. SMV-SX and SMV-XFQ008 had the highest nucleotide and amino acid sequence identities of 97.03% and 98.50%, respectively. A phylogenetic tree indicated that SMV-SX and SMV-XFQ018 were clustered together, sharing the closest relationship. We then constructed a pSMV-SX infectious cDNA clone by Gibson assembly technology and used this clone to inoculate soybean and Ailanthus altissima; the symptoms of these hosts were similar to those caused by the virus isolated from natural infected plant tissue. This method of construction not only makes up for the time-consuming and laborious defect of traditional methods used to construct infectious cDNA clones, but also avoids the toxicity of the Potyvirus special sequence to Escherichia coli, thus providing a useful cloning strategy for the construction of infectious cDNA clones for other viruses and laying down a foundation for the further investigation of SMV cross-family infection mechanisms.

Construction of Full-length Infectious cDNA Clones of Apple chlorotic leaf spot virus and Their Agroinoculation to Woody Plants by a Novel Method of Vacuum Infiltration.

Construction and agroinoculation of full-length infectious cDNA clones of plant RNA viruses have been used in plant virology to prove Koch's postulates and for development of viruses as vectors for expressing foreign genes in plants. Four full-length cDNA clones (pIF3-12, pIF3-14, pIF3-15, and pIF3-19) of Apple chlorotic leaf spot virus (ACLSV) isolate 38/85 were produced. Two of the four full-length cDNA clones (pIF3-15 and pIF3-19) proved to be infectious on Nicotiana occidentalis 37B test plants by agroinoculation and were then mechanically transmissible to healthy N. occidentalis 37B. The genomic cDNAs of ACLSV pIF3-15 and pIF3-19 shared nucleotide identity of 77.5%, demonstrating mixed infections of multiple strains of ACLSV in the source tree of isolate 38/85. The two full-length cDNA clones were agroinoculated to apple seedlings by a newly developed vacuum infiltration method. The success rate of agroinoculation was greater than 78%, defined as the number of PCR positive seedlings to the number of apple seedlings that survived. ACLSV was transmissible from agroinoculated seedlings by cleft grafting. The results of this study will be useful for construction of infectious cDNA clones of plant viruses from full-length PCR fragments and agroinoculating woody host plants using the vacuum infiltration method outlined here.

Reverse Genetics Approaches for Chikungunya Virus.

Reverse genetic systems based on an infectious cDNA clone, a double-stranded copy of the viral genome carried on a plasmid vector, have greatly enhanced the understanding of RNA virus biology by facilitating genetic manipulation of viral RNA genomes. To date, infectious cDNA clones of Chikungunya virus (CHIKV) have been constructed using different combinations of plasmid vectors and/or bacterial host strains. Here, we describe our approaches for the construction of infectious cDNA clones of CHIKV and the protocol for genetic manipulation of the clones by site-directed mutagenesis.

Construction of infectious cDNA clones derived from the potyviruses clover yellow vein virus and bean yellow mosaic virus.

Infectious cDNA clones are now indispensible tools for the genetic analysis of viral factors involved in viral virulence and host resistance. In addition, infectious cDNA-derived virus vectors that express foreign genes in infected plants enable the production of useful proteins at low cost and can confer novel crop traits. We constructed infectious cDNA clones derived from two potyviruses, Clover yellow vein virus and Bean yellow mosaic virus, which infect legume plants and cause disease. Here, we present our procedure for constructing these potyvirus infectious clones.

Construction of infectious cDNA clones of PRRSV: Separation of coding regions for nonstructural and structural proteins

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the causative agent of the ongoing "porcine high fever syndrome" in China, is capable of genetic and antigenic mutations at high frequency. How to design vaccine rationally to keep up with the ever-changing prevalent PRRSV variant is of great interest. We developed an infectious cDNA clone of an attenuated strain of Type II PRRSV, and further manipulated the infectious cDNA clone by inserting polylinker between ORF1 and ORF2, encoding for nonstructural-or structural-protein, respectively. The cDNA was generated from the cell-attenuated virus strain, APRRS, via RT-PCR, and followed by nucleotide sequencing and molecular cloning. The full-length of the APRRS genomic RNA was determined as 15521 nucleotides in length excluding poly(A) tail, which has a 99.7% nucleotide identity with that of PRRSV Nsp strain, also a vaccine strain. Based on the nucleotide sequencing results, the full-length cDNA clone was assembled in pBlueScript vector, under the control of T7 promoter at the immediate 5' terminus of genome. To discern the rescued viruses from that of parental virus, a Mlu I restriction site was engineered into ORF5 coding region. Upon transfection of the in vitro transcripts of both the original and Mlu I-tagged cDNAs into MA-104 cells, typical PRRSV cytopathic effects were observed. The rescued viruses from the full-length cDNA clones displayed the same virological and molecular properties. Subsequently, PCR-based mutagenesis was conducted to separate the coding regions between PRRSV nonstructural genes, ORF1, and structural proteins, ORF2-ORF7. The synthetic RNA of such mutant clone, pCSA, was infectious and the rescued virus shared similar properties with that of the parental virus. This study provided a valuable tool for development of chimeric PRRSV as vaccine candidate offering cross-protection to various genetically diversified PRRSV strains, and a platform for further development of PRRSV as a gene expression vector for recombinant vaccines against other significant swine diseases.

Cloning and characterization of a complete open reading frame of the hepatitis C virus genome in only two cDNA fragments.

The synthesis of long cDNA molecules encoding the complete genome of RNA viruses has recently been demonstrated; this major improvement has numerous practical applications such as construction of infectious cDNA clones or study of sequence variability at the level of a single RNA molecule. Using hepatitis C virus (HCV) as a model, we established an RT-PCR technique for amplification of cDNA fragments with a length of about 5 kb. The RT reaction was carried out with a Moloney murine leukaemia virus reverse transcriptase lacking detectable RNase H activity. For PCR reactions an enzyme mix containing Taq and Pwo DNA polymerases was used. Hot start and addition of 5% DMSO were also important to efficiently achieve long PCR products. About 10(6) HCV genome equivalents/ml in serum were needed in order to amplify the HCV genome in only two cDNA fragments covering about 98% of the complete genome. Analysis of the HCV quasi-species is also possible by this method as shown by sequencing of the hypervariable region 1 (HVR1) after cloning of cDNAs. The integrity of the long cDNA clones was proven by (1) restriction analyses, (2) partial sequencing and (3) expression of respective gene products. In vitro transcribed cDNAs were translated in rabbit reticulocyte lysate. Structural and nonstructural HCV proteins were identified by immunoprecipitation using patient serum. These results suggest that the two cDNA clones encode a complete and functional open reading frame of HCV.

Construction of Infectious cDNA Clones for Dengue 2 Virus: Strain 16681 and Its Attenuated Vaccine Derivative, Strain PDK-53

We identified nine nucleotide differences between the genomes of dengue-2 (DEN-2) 16681 virus and its vaccine derivative, strain PDK-53. These included a C-to-T (16681-to-PDK-53) mutation at nucleotide position 57 of the 5′-untranslated region, three silent mutations, and substitutions prM-29 Asp to Val, NS1-53 Gly to Asp, NS2A-181 Leu to Phe, NS3-250 Glu to Val, and NS4A-75 Gly to Ala. Unpassaged PDK-53 vaccine contained two genetic variants as a result of partial mutation at NS3-250. We constructed infectious cDNA clones for 16681 virus and each of the two PDK-53 variants. DEN-2 16681 clone-derived viruses were identical to the 16681 virus in plaque size and replication in LLC-MK2cells, replication in C6/36 cells, E and prM epitopes, and neurovirulence for suckling mice. PDK-53 virus and both clone-derived PDK-53 variants were attenuated in mice. However, the variant containing NS3-250-Glu was less temperature sensitive and replicated better in C6/36 cells than did PDK-53 virus. The variant containing NS3-250-Val had smaller, more diffuse plaques, decreased replication, and increased temperature sensitivity in LLC-MK2cells relative to PDK-53 virus. Both PDK-53 virus and the NS3-250-Val variant replicated poorly in C6/36 cells relative to 16681 virus. Unpassaged PDK-53 vaccine virus and the virus passaged once in LLC-MK2cells had genomes of identical sequence, including the mixed NS3-250-Glu/Val locus. Although the NS3-250-Val mutation clearly affected virus replicationin vitro,it was not a major determinant of attenuation for PDK-53 virus in suckling mice.