Engineering Novel Viruses Before and After Recombinant DNA

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As mentioned earlier, viruses in nature, depending upon their replication strategy and host range, have many ways to evolve through mutation, recombination, reassortment, and selection. These innate properties have been used as tools in virology for decades. Examples include cold-adapted and attenuated live vaccine strains of influenza, cross-species rotavirus reassortants as vaccine candidates, and the attenuated yellow fever virus 17D vaccine strain that was derived by serial passaging in cell culture. Over the last 30 years, the recombinant DNA revolution has changed virology forever. Besides its impact on our ability to study specific viral nucleic acids and proteins, recombinant DNA technology (and PCR) sped up our ability to determine complete viral genomic sequences and create specific viral variants. The ability of scientists to engineer novel viruses varies greatly. Small positive-strand RNA viruses of bacteria, plants, and animals provided some of the first examples where infectious virus was produced via recombinant DNA. More recently, it has also become possible to engineer the >29-kb RNA genome of coronaviruses and both monopartite and segmented negative-strand RNA viruses. Viruses in the family Reoviridae, with double-stranded RNA genomes, have proven more difficult. For herpesviruses, whose genomic DNA is infectious, the same technologies used for cloning large pieces of chromosomal DNA have been applied successfully to propagate overlapping fragments of entire herpesvirus genomes. For the poxviruses, whose genomic DNA is not infectious, recombinant viruses are generated within infected cells by homologous DNA recombination between a plasmid containing the engineered segment and an infecting parental virus.

Synthetic Poliovirus

Typically, recombinant DNA manipulations of viruses begin with viral nucleic acid (RNA or DNA) that is then cloned and amplified in bacteria using plasmid vectors or in vitro using PCR. Once the genome sequence of a virus is known, however, it becomes possible to create this sequence artificially using overlapping synthetic oligonucleotides and gene synthesis techniques that have been available for many years. An example of this, which received a great deal of attention from the media and the scientific community, was recently published for an attenuated form of poliovirus.53 While the poliovirus case came as no surprise to virologists and molecular biologists, it did bring several issues to the forefront. If an infectious virus can be created by synthetic methods, can it ever really be eradicated? In light of potential bioterrorist or biowarfare agents, should there be a restriction on making the genome sequence available in public databases? Should research on these agents be banned, classified, or otherwise regulated? These and other questions will continue to be debated in the years to come.

Mousepox

The ability to engineer viruses raises the concern about modifying existing viruses to make them more virulent. The bulk of examples in virology run counter to this idea. Viruses are usually highly adapted to a particular niche, and most mutations are deleterious. Propagation of pathogenic viruses in cell culture often leads to adaptation to that environment and attenuation in their animal host. Recombinants or chimeras between even closely related viruses are usually impaired relative to either parent. We do see examples, however, of viruses that are benign in one animal host but highly pathogenic in another species. This is often the case in epizootic emerging viruses. An extreme example of host species-specific pathogenesis is the myxoma poxvirus, which causes a benign cutaneous fibroma in wild rabbits of the Americas but a highly lethal disease in the European rabbit. This virus was used for biological control of feral European rabbits in Europe and Australia. In 2001, an Australian group published a paper describing the construction of recombinant mousepox virus expression interleukin-4 (IL-4) and its pathogenesis in mice.54 This study provides a striking and sobering example of an engineered virus with enhanced pathogenicity. IL-4 is a cytokine that regulates the immune response at various levels. The recombinant mousepox virus suppressed both innate and adaptive immune responses that normally control infection and was lethal for otherwise genetically resistant mice. Moreover, even animals that had been previously vaccinated and protected from virulent mousepox were susceptible to lethal infection by the mousepox-IL-4 recombinant virus. This study raises obvious concerns about the efficacy of current vaccination against modified versions of the smallpox virus.

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