Recombinant virus

An important safety issue of viral vectors is whether or not the recombinant viruses are able to replicate in the infected cells. Replication of viral vectors is unwanted in most gene-therapy approaches. Therefore, replication-defective vectors have been designed, which are able to perform only one initial infectious cycle within the target cell. In addition, replication-competent vectors have been designed, which are able to productively infect the target cell and to spread in the target tissue. 

Whitcomb JM, Huang W, Fransen S, Limoli K, Toma J, Wiin T, Chappey C, Kiss LD, Paxinos EE, Petropoulos CJ (2007) Development and characterization of a novel single-cycle recombinant-virus assay to determine human immunodeficiency virus type 1 coreceptor tropism, Antimicrob Agents Chemother 51 566-575... 

Physiological properties of hypothalamic MCH neurons identified with selective expression of reporter gene after recombinant virus infection. Neuron 42, 635-52. 

Phenotypic resistance assays directly measure the ability of HlV-1 to replicate in a cell culture in the presence of different antiretroviral drug concentrations. This process is similar to that used to determine antibiotic resistance and is, therefore, more familiar to most clinicians. The recombinant virus, composed of a virus s reverse transcripfase and protease genes, is inserted into a standard reference strain of virus. The recombinant virus is then tested in vitro for fhe amount of drug needed to inhibit virus replication by 50%, relative to the amount of drug needed to inhibit a reference strain of virus. Phenotypic resistance testing is limited by the fact that it is conducted in vitro and not in vivo. 

The plasmid DNA used for cotransfection should be as pure as possible. Insect cells are sensitive to impurities in plasmid samples and may lyse before the recombinant virus is regenerated, resulting in very low viral titres. For good cotransfection experiments, monolayers of healthy cells with an initial confluency of 60-70% are required. 

Protocol 1.7) and normally generate high-titre stocks. The viral titre is determined by plaque assay (Protocol 1.8) so that known amounts of the recombinant virus are used in subsequent virus amplification experiments (Protocol 1.9) to produce large viral stocks. 

After 5 days transfer the supernatant to sterile centrifuge tubes. The supernatant of plate number 2 contains the recombinant virus. 

Protocol 1.8 Purification of recombinant virus and determination of viral titre by plaque assay... 

Add 100 pil of the recombinant virus stock, keeping the multiplicity of infection below one. 

Calculate the volume of recombinant virus required to infect at a multiplicity of infection of 3-10 by the equation ... 

It is thought likely that up to 30 extra genes can be incorporated into vaccinia. The upper capacity has not been determined, but is likely to exceed 50 kb. This facilitates the development of a multivalent vaccine via expression of several pathogen-derived genes in the recombinant virus. 

Luskin, M.B., Pearlman, A.L., and Sanes, J.R. (1988) Cell lineage in the cerebral cortex of the mouse studied in vivo and in vitro with a recombinant virus. Neuron 1 635-647. 

Paul A. K., Martin D. A., and Robert D. P. (1990) Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors. Nucleic Acids Kes. 18, 5667-5672. 

The lack of human pathogenicity of plant viruses rules out the risks of human infection by exposure in the field or in food products to a plant virus. However, biological containment of the virus expression vector remains a primary safety concern as it can be considered a risk to the environment. This includes the spread of recombinant viruses to weeds... 

Eukaryotic Animal (recombinant) virus vectors Transient or lytic infection Infection in susceptible cells All essential viral genes strong promoter/enhancers polyadenylation signal intron sequences... 

RNA copies of recombinant viruses are produced in cells containing a helper virus and packaged into viral particles. 

Recombinant virus particles infect a target cell. 

Viral genome can be inserted with a large DNA sequence by homologous recombination, and the recombinant virus, which is defective in replication, can be plaque purified by using transcomplementing cells. However, this viral vector has several disadvantages it is difficult to obtain preparations that are completely defective in replication, the modified vector generates immune response and antibodies specific for HSV-1 are present. 

Bonnet, M. C., Tartaglia, J., Verdier, R, Kourilsky, P, Lindberg, A., Klein, M., and Moingeon, P. 2001. Recombinant viruses as a tool for therapeutic vaccination against human cancers. Immunol. Letters 74 11-25. 

Recombinant Viruses or Virus-Like Particles for Nucleic Acid Delivery... 

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