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PubMed2000

Infectious cDNA clone of the RA27/3 vaccine strain of Rubella virus.

Rubella virus (RUB), a small plus-strand RNA virus, is a significant human pathogen. The RA27/3 vaccine strain of RUB is one of the most successful live attenuated vaccines developed. In this article, we report the construction of an RA27/3 infectious clone, a complete cDNA copy of the RA27/3 genome that can be transcribed in vitro to generate infectious RNA molecules. Virus generated from such in vitro transcripts was phenotypically similar to RA27/3 virus. To investigate the attenuation of the RA27/3 strain, a series of chimeras was made by the insertion of different fragments of the RA27/3 genome into an infectious clone based on the Therien wild-type strain of RUB. Analysis of the resulting chimeric viruses revealed that the pattern of RA27/3 attenuation in cell culture is complex: attenuating elements in the RA27/3 genome were found in the 5' untranslated region (UTR), a region of the nonstructural proteins containing the protease motif and the capsid gene. Within the 5' UTR, the attenuation determinant was mapped to nt 7. Surprisingly, these analyses also revealed a potentiating mutation at nt 164 of the RA27/3 genome. Although this determinant was within the coding sequences of the nonstructural proteins, the encoded amino acid had no effect on cell culture phenotype and thus the determinant may operate at the level of RNA structure. In addition to investigation of the mechanisms of RA27/3 attenuation, the availability of the RA27/3 infectious clone offers the opportunity for strict genetic control over RUB vaccine manufacturing, for development of novel DNA-based vaccines against RUB, and for development of recombinant RUB vaccines that also target other diseases.

5' Untranslated Regions↗

PubMed2006

Infectious clone-derived viruses from virulent and vaccine strains of porcine reproductive and respiratory syndrome virus mimic biological properties of their parental viruses in a pregnant sow model.

Understanding of the molecular basis of virulence and attenuation of porcine reproductive and respiratory syndrome virus (PRRSV) is important for the development of a safe and efficacious vaccine. Prime Pac (PP) is an attenuated vaccine strain of PRRSV which is being used in our laboratories as a source of gene(s) for the generation of chimeric constructs in the background of a highly virulent PRRSV derived from an infectious clone (FL12) to examine the molecular determinants of virulence and attenuation. To facilitate these studies, we generated a full-length cDNA clone of the PP vaccine strain by serially replacing the genomic fragments of the FL12 with the corresponding regions from the PP strain. The virus rescued from this newly assembled cDNA clone (PP18) exhibited in vitro growth properties and in vivo apathogenic characteristics of the parental PP virus. Using pregnant sows as the experimental model of reproductive pathogenesis, we have been able to unequivocally demonstrate the clearly contrasting phenotypes of the virulent and the attenuated viruses derived from the infectious clones (FL12 and PP18). The development of an infectious clone derived from a bona fide attenuated PRRSV vaccine strain should significantly facilitate ongoing studies to determine the molecular basis of virulence and attenuation.

Animals↗

PubMed2002

Infectious cDNA clone of the epidemic west nile virus from New York City.

We report the first full-length infectious clone of the current epidemic, lineage I, strain of West Nile virus (WNV). The full-length cDNA was constructed from reverse transcription-PCR products of viral RNA from an isolate collected during the year 2000 outbreak in New York City. It was cloned into plasmid pBR322 under the control of a T7 promoter and stably amplified in Escherichia coli HB101. RNA transcribed from the full-length cDNA clone was highly infectious upon transfection into BHK-21 cells, resulting in progeny virus with titers of 1 x 10(9) to 5 x 10(9) PFU/ml. The cDNA clone was engineered to contain three silent nucleotide changes to create a StyI site (C to A and A to G at nucleotides [nt] 8859 and 8862, respectively) and to knock out an EcoRI site (A to G at nt 8880). These genetic markers were retained in the recovered progeny virus. Deletion of the 3'-terminal 199 nt of the cDNA transcript abolished the infectivity of the RNA. The plaque morphology, in vitro growth characteristics in mammalian and insect cells, and virulence in adult mice were indistinguishable for the parental and recombinant viruses. The stable infectious cDNA clone of the epidemic lineage I strain will provide a valuable experimental system to study the pathogenesis and replication of WNV.

Animals↗

PubMed1999

Hepatitis C virus: an infectious molecular clone of a second major genotype (2a) and lack of viability of intertypic 1a and 2a chimeras.

Of the six major genotypes of hepatitis C virus (HCV), infectious cDNA clones of only genotype 1 have been reported. Here, we report the construction of an infectious cDNA clone representing a second major HCV genotype, genotype 2. This infectious clone (pJ6CF) encodes the consensus polyprotein of strain HC-J6(CH), genotype 2a. Its encoded polyprotein differs from those of the infectious clones of genotypes 1a and 1b by approximately 30%. Intertypic chimeric cDNA clones constructed from infectious clones of genotypes 1a and 2a of HCV were not infectious. RNA transcripts of four chimeras containing the 2a structural genes (C, E1, and E2) in the backbone of an infectious genotype 1a clone (pCV-H77C) were not viable in a chimpanzee regardless of whether p7 was from the 1a or 2a clone. However, the chimpanzee was subsequently infected with RNA transcripts of each of the two infectious parent clones, indicating that the inability of the chimeras to replicate was intrinsic to the clones and not the result of preexisting protective immune responses.

3' Untranslated Regions↗

PubMed2000

Studies of the immortalizing activity of HTLV type 1 Tax, using an infectious molecular clone and transgenic mice.

Expression of Tax in the mature lymphoid compartment of transgenic mice resulted in a lymphoproliferative malignancy of natural killer cells and cytotoxic T lymphocytes. Transgenic mouse tumors exhibited mutations in the p53 tumor suppressor gene, and functional inactivation of wild-type p53 protein. Tax transgenic mice heterozygous for the p53 gene exhibited more rapid tumor dissemination and accelerated mortality. Studies of Tax trans-activation in an infectious clone of HTLV-1 demonstrated a critical role for nuclear factor B activation in lymphocyte immortalization. A mutant disrupting Tax activation of the cAMP response element binding (CREB) protein resulted in preferential immortalization of CD8(+) lymphocytes, rather than preferential immortalization of CD4(+) lymphocytes seen with the wild-type infectious clone. A mutation disrupting Tax interaction with CREB-binding protein, CBP, did not affect lymphocyte immortalization by the infectious molecular clone. These models provide new insights into the molecular details of HTLV-1 leukemogenesis.

Animals↗

PubMed2004

Characterization of the neutralization determinants of equine arteritis virus using recombinant chimeric viruses and site-specific mutagenesis of an infectious cDNA clone.

We have used an infectious cDNA clone of equine arteritis virus (EAV) and reverse genetics technology to further characterize the neutralization determinants in the GP5 envelope glycoprotein of the virus. We generated a panel of 20 recombinant viruses, including 10 chimeric viruses that each contained the ORF5 (which encodes GP5) of different laboratory, field, and vaccine strains of EAV, a chimeric virus containing the N-terminal ectodomain of GP5 of a European strain of porcine reproductive and respiratory syndrome virus, and 9 mutant viruses with site-specific substitutions in their GP5 proteins. The neutralization phenotype of each recombinant chimeric/mutant strain of EAV was determined with EAV-specific monoclonal antibodies and EAV strain-specific polyclonal equine antisera and compared to that of their parental viruses from which the substituted ORF5 was derived. The data unequivocally confirm that the GP5 ectodomain contains critical determinants of EAV neutralization. Furthermore, individual neutralization sites are conformationally interactive, and the interaction of GP5 with the unglycosylated membrane protein M is likely critical to expression of individual epitopes in neutralizing conformation. Substitution of individual amino acids within the GP5 ectodomain usually resulted in differences in neutralization phenotype of the recombinant viruses, analogous to differences in the neutralization phenotype of field strains of EAV and variants generated during persistent infection of EAV carrier stallions.

Amino Acid Sequence↗

PubMed2001

Construction and initial characterization of an infectious plasmid clone of a newly identified hamster parvovirus.

The construction and characterization of a full-length infectious plasmid clone of the newly identified hamster parvovirus (HaPV) are described. Following transfection of hamster BHK cells with the infectious clone, pHaPV, the specific intracellular DNA replicative forms, RNA transcripts and viral proteins that were expected for this rodent parvovirus were generated. Infected cells were lysed and progeny virus was produced, demonstrating that pHaPV could generate a productive virus infection. The complete sequences of both hairpin termini, which had not been previously determined, were obtained. Preliminary host-range studies, which compared virus production and macromolecular synthesis in various cell lines following either HaPV infection or pHaPV transfection, demonstrated an early block of infection of HaPV in both monkey COS-1 and murine A9 cells. The availability of an HaPV infectious clone will facilitate its genetic analysis and allow the elucidation of the determinants important in host range, tissue tropism and pathogenicity of this newly identified rodent parvovirus.

Animals↗

PubMed1991

Isolation and characterization of an infectious molecular clone of the MN strain of HIV-1.

Infectious molecular clones of the human immunodeficiency virus (HIV) have been very important tools for the analysis of regulatory gene functions and the study of differential cell tropism. We have cloned and characterized a proviral sequence of HIVmn from mn strain infected H9 cells. This clone, called KP1, was found to be infectious for different cell lines and human peripheral blood lymphocytes (PBL). KP1 proviral DNA was detected in HUT-78 cells and human PBL by polymerase chain reaction (PCR) analysis after infection of these cells with cell-free supernatants from KP1 transfected human rhabdomyosarcoma (RD) cells. To the best of our knowledge, this is the first report of an infectious molecular clone of HIVmn which is a representative of one of the most prevalent strains of HIV-1 in North America and Europe. Biologically active clones of a broadly antigenic strain such as HIVmn will be extremely useful in therapeutic approaches for AIDS.

Base Sequence↗

PubMed1998

An infectious cDNA clone of porcine reproductive and respiratory syndrome virus.

A plasmid containing a full-length cDNA copy of the Lelystad virus isolate (LV) of porcine reproductive and respiratory syndrome virus was constructed. When RNA that was transcribed in vitro from this full-length cDNA clone was transfected to BHK-21 cells, infectious LV was produced and secreted. The virus was rescued by passage to porcine alveolar lung macrophages or CL2621 cells. When infectious transcripts were transfected to porcine alveolar lung macrophages or CL2621 cells no infectious virus was produced due to the poor transfection efficiency of these cells. The growth properties of the viruses produced by BHK-21 cells transfected with infectious transcripts of LV cDNA resembled the growth properties of the parental virus from which the cDNA was derived. The infectious clone of LV enables us to mutagenize the viral genome at specific sites and thus will be useful for detailed molecular characterization of the virus, as well as for the development of a safe and effective live vaccine for use in pigs.

Animals↗

PubMed2004

Infectious cDNA clones of porcine reproductive and respiratory syndrome virus and their potential as vaccine vectors.

Full-length infectious cDNA clones have recently become available for both European and North American genotypes of porcine reproductive and respiratory syndrome virus (PRRSV), and it is now possible to alter the PRRSV genome and create genetically defined mutant viruses. Among many possible applications of the PRRSV infectious cDNA clones, development of genetically modified vaccines is of particular interest. Using infectious clones, the PRRSV genome has been manipulated by changing individual amino acids, deleting coding regions, inserting foreign sequences, and generating arterivirus chimeras. The limited available data suggest that all structural proteins of PRRSV are essential for replication of the virus, and that PRRSV infectivity is relatively intolerant of subtle changes within the structural proteins. The major tasks in PRRSV research are to identify virulence factors and pathogenic mechanisms, and to understand the structure-function relationships of individual viral proteins. Utilizing these infectious clones as tools, a new generation of safe and efficacious PRRS vaccines may be constructed.

Animals↗

PubMed1989

Use of infectious molecular clones of simian immunodeficiency virus for pathogenesis studies.

Three infectious molecular clones of SIVmac and one of HIV-2 exhibit remarkable variation in their biological properties despite similarities in genome organization and sequence relatedness. Cloned viruses differed in their ability to grow in various cultured cells, in their ability to infect macaques, and in the location of the env stop codon. Sequences from the 3' end predict that at least three of the four clones do not have an intact, functional nef gene. All four cloned viruses yield infectious virus in HUT-78 and all four cloned viruses are cytopathic.

Animals↗

PubMed1993

Rapid production of full-length, infectious geminivirus clones by abutting primer PCR (AbP-PCR).

The application of the polymerase chain reaction (PCR) method of DNA amplification for the isolation of full-length, infectious clones of geminiviruses is described. Non-overlapping, abutting 20-mer oligonucleotide primers were used to produce a linear product from the circular geminivirus genomic template. Clones of African cassava mosaic virus (ACMV) DNA A, obtained by this method, were infectious following mechanical inoculation (in the presence of ACMV DNA B) onto Nicotiana benthamiana. Normal ACMV symptoms resulted and typical geminate viral particles were detected by electron microscopy. The use of PCR for the detection and production of full-length, infectious geminivirus clones is discussed.

Amino Acid Sequence↗

PubMed1999

An infectious molecular clone of a Japanese genotype 1b hepatitis C virus.

We describe an infectious molecular clone of a Japanese genotype 1b strain of hepatitis C virus (HCV-N). The molecularly cloned sequence of HCV-N was compared with alignments of other HCV sequences, leading to the identification of 15 unique, nonconservative amino acid substitutions within the HCV-N open reading frame (ORF). These were repaired to the consensus genotype 1b residue, and the infectivity of RNA transcribed from the repaired clone was assessed by intrahepatic inoculation of a chimpanzee. Viral RNA was first detected in the serum of this chimpanzee 3 weeks following inoculation, and was intermittently present over the next 14 weeks. A strong and persistent anti-HCV serological response developed 13 weeks following inoculation, with seroconversion in the recombinant immunoblot assay (RIBA). A weaker, transient serological response, characterized by seroconversion in a third-generation enzyme-linked immunosorbent assay (ELISA) but not RIBA, occurred between weeks 1 and 5. This may have represented an anamnestic response to HCV antigens translated directly from the intrahepatically inoculated RNA, because the animal previously had undergone 2 unsuccessful attempts at rescue of HCV by intrahepatic RNA inoculation. There was neither biochemical nor histological evidence of liver disease. Although this is within the range of expected outcomes in an HCV-naive chimpanzee, prior immunologic priming may have modified the infection in this animal. The HCV-N clone is the first infectious molecular clone of HCV that is comprised entirely of genotype 1b sequence, and it contains an ORF sequence that is significantly divergent from that of a previously described genotype 1a/1b chimera.

3' Untranslated Regions↗

PubMed2006

Recovery of infectious rabbit hemorrhagic disease virus from rabbits after direct inoculation with in vitro-transcribed RNA.

We report the first full-length infectious clone of strain JX/CHA/97 of rabbit hemorrhagic disease virus (RHDV). The transcripts from the full-length cDNA clones were infectious when they were directly injected into rabbits. The sequence of the virus recovered from the rabbits was identical to that of the injected RNA transcripts. The cDNA clone was engineered to contain one silent nucleotide change to create an EcoRV site (A to T at nucleotide 2908). The genetic marker was retained in the recovered progeny virus. The transfection of RNA transcripts into RK-13 cells resulted in the synthesis of viral antigens, indicating that the cDNA clones were replication competent. This stable infectious molecular clone should be an important tool for developing a better understanding of the molecular biology and pathogenesis of RHDV.

Animals↗

PubMed2003

Immunogenicity and pathogenicity of chimeric infectious DNA clones of pathogenic porcine circovirus type 2 (PCV2) and nonpathogenic PCV1 in weanling pigs.

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), whereas the ubiquitous porcine circovirus type 1 (PCV1) is nonpathogenic for pigs. We report here the construction and characterization of two chimeric infectious DNA clones of PCV1 and PCV2. The chimeric PCV1-2 clone contains the PCV2 capsid gene cloned in the backbone of the nonpathogenic PCV1 genome. A reciprocal chimeric PCV2-1 DNA clone was also constructed by replacing the PCV2 capsid gene with that of PCV1 in the backbone of the PCV2 genome. The PCV1, PCV2, and chimeric PCV1-2 and PCV2-1 DNA clones were all shown to be infectious in PK-15 cells, and their growth characteristics in vitro were determined and compared. To evaluate the immunogenicity and pathogenicity of the chimeric infectious DNA clones, 40 specific-pathogen-free (SPF) pigs were randomly assigned into five groups of eight pigs each. Group 1 pigs received phosphate-buffered saline as the negative control. Group 2 pigs were each injected in the superficial inguinal lymph nodes with 200 micro g of the PCV1 infectious DNA clone. Group 3 pigs were each similarly injected with 200 micro g of the PCV2 infectious DNA clone, group 4 pigs were each injected with 200 micro g of the chimeric PCV1-2 infectious DNA clone, and group 5 pigs were each injected with 200 micro g of the reciprocal chimeric PCV2-1 infectious DNA clone. As expected, seroconversion to antibodies to the PCV2 capsid antigen was detected in group 3 and group 4 pigs. Group 2 and 5 pigs all seroconverted to PCV1 antibody. Gross and microscopic lesions in various tissues of animals inoculated with the PCV2 infectious DNA clone were significantly more severe than those found in pigs inoculated with PCV1, chimeric PCV1-2, and reciprocal chimeric PCV2-1 infectious DNA clones. These data indicated that the chimeric PCV1-2 virus with the immunogenic ORF2 capsid gene of pathogenic PCV2 cloned into the nonpathogenic PCV1 genomic backbone induces a specific antibody response to the pathogenic PCV2 capsid antigen but is attenuated in pigs. Future studies are warranted to evaluate the usefulness of the chimeric PCV1-2 infectious DNA clone as a genetically engineered live-attenuated vaccine against PCV2 infection and PMWS.

Animals↗

PubMed1984

Nucleotide sequence of an infectious molecularly cloned genome of ground squirrel hepatitis virus.

We have determined the complete nucleotide sequence of an infectious cloned genome of ground squirrel hepatitis virus (GSHV), a nonpathogenic member of the hepadnavirus group. The genome is 3,311 base pairs long and contains the major open reading frames described for the related human and woodchuck hepatitis B viruses (HBV and WHV, respectively). These reading frames include genes for the major structural proteins (the surface and core antigens), unassigned open reading frames (A and B), the longer of which is presumed to encode the viral DNA polymerase, and an open reading frame preceding and continuous with the surface antigen gene. The arrangement of these open reading frames is similar to that encountered in the genomes of HBV and WHV: all of the reading frames are encoded on the same strand, they are positioned in the same fashion with respect to each other, and a large portion (at least 51%) of the genome can be translated in two reading frames. Comparisons of the predicted translational products of the three mammalian hepadnaviruses reveal 78% amino acid homology between the proteins of GSHV and WHV and 43% homology between those of GSHV and HBV. In addition, a perfect direct repeat of 10 to 11 base pairs, separated by ca. 46 to 223 base pairs, is present in the three mammalian viruses and in duck hepatitis B virus; the position of the repeats near the 5' termini of the two strands of virion DNA suggests a role in viral replication.

Amino Acid Sequence↗

PubMed2006

Construction of a severe acute respiratory syndrome coronavirus infectious cDNA clone and a replicon to study coronavirus RNA synthesis.

The engineering of a full-length infectious cDNA clone and a functional replicon of the severe acute respiratory syndrome coronavirus (SARS-CoV) Urbani strain as bacterial artificial chromosomes (BACs) is described in this study. In this system, the viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and further amplified in the cytoplasm by the viral replicase. Both the infectious clone and the replicon were fully stable in Escherichia coli. Using the SARS-CoV replicon, we have shown that the recently described RNA-processing enzymes exoribonuclease, endoribonuclease, and 2'-O-ribose methyltransferase were essential for efficient coronavirus RNA synthesis. The SARS reverse genetic system developed as a BAC constitutes a useful tool for the study of fundamental viral processes and also for developing genetically defined vaccines.

Animals↗

PubMed1988

Differences in cytopathogenicity and host cell range among infectious molecular clones of human immunodeficiency virus type 1 simultaneously isolated from an individual.

A cytopathic human immunodeficiency virus type 1 (HIV-1) isolate containing multiple virus genotypes was molecularly cloned, and the biological activity of six randomly selected clones was assessed by transfection into human lymphoid or glial cell lines. Five infectious clones of HIV-1, termed N1T-A through -E, were isolated in this manner. Clones N1T-A, -B, -C, and -E could be distinguished by restriction endonuclease mapping whereas clones N1T-B and -D had identical maps with the enzymes used. Each clone exhibited a distinct host cell range as well as markedly different infection kinetics and cytopathogenic properties when tested in human cell lines of T-lymphocytic, monocytic, and astrocytic origin. In particular, infection with HIV-1 clone N1T-E was characterized by slow kinetics and lack of significant cytopathic effects in acutely and chronically infected cells. Clone N1T-A, similar to the parental isolate N1T, exhibited a wide host cell range, fast kinetics of infection, and high cytopathogenicity. These data indicate that HIV-infected individuals may carry multiple HIV-1 genotypes with distinct cytopathogenic potential and cell tropism. Analysis of virus isolates must take into account the contribution, or masking, of individual virus clones.

Acquired Immunodeficiency Syndrome↗