Precision Virology: The Shift From Serial Passaging to Reverse Genetics

Since the establishment of virology over a century ago, serial passaging of virus stocks has been used to study viruses and develop products such as vaccines for medical use. However, in recent decades, the development of reverse genetics-based virus production methodologies has transformed the study of viruses. These methods allow scientists to engineer and reconstitute virus stocks directly from genetic sequences. Consequently, the field of virology is decisively shifting, where possible, towards using reverse genetics for the fundamental task of creating virus stocks for all downstream uses such as R&D, product manufacturing and QC testing.

The Legacy of Serial Passaging 

Dating back to the 1880s with Louis Pasteur’s work on rabies, serial passaging was the bedrock of early virology. For example, the first vaccines were created by passaging a human virus through non-human cells or at suboptimal temperatures for replication to attenuate the virus. The resulting weakened virus stimulated a protective immune response without causing severe disease. Notable successes of this method of serial passaging based vaccine development include generation of the yellow fever 17D vaccine strain and the chickenpox vaccine.

However, serial passaging is an inherently unpredictable process. Passaging a virus often results in the generation of stochastic mutations with unpredictable effects on viral replication. Generating genotypically and phenotypically reproducible virus stocks is constrained by the inherent error rates of viral polymerases that have evolved to support low fidelity viral genomic replication as a survival strategy. While useful, the limits of serial passaging methodologies spurred scientists to develop alternative methods of virus rescue to better recapitulate the phenotypes of circulating virus strains for R&D and product development.

The Development of Reverse Genetics

Reverse genetics in virology emerged in the late 20th century from the maturation of molecular cloning and recombinant DNA technologies, thereby enabling scientists to generate reproducible infectious virus stocks entirely from cloned viral genomes. Early breakthroughs targeted positive-sense RNA viruses and were followed by the development of more complex systems for negative-sense RNA viruses and viruses with segmented genomes. Reverse genetics methodologies transformed virology by allowing precise and targeted manipulation of viral genomes, thereby making it possible to link specific viral genes to pathogenicity. Today, reverse genetics is a foundational tool for studying viral replication, virus-host interactions, vaccine design, and antiviral development.

An Example of an Ongoing Regulatory Shift: the FDA Animal Rule and Viral Characterization

The transition from serial passaging based methodologies to greater use of reverse genetics is not merely due to technological advances; it is increasingly a regulatory necessity. As an example, the FDA Animal Rule (21 CFR 314.610) permits the approval of certain drugs and biologics based on animal efficacy studies when human clinical trials are neither ethical nor feasible. This regulatory mechanism is commonly used for the development of countermeasures against highly pathogenic viral agents such as Smallpox and Ebola.

A cornerstone of the Product Development Under the Animal Rule guidance is the requirement for highly characterized virus stocks. The rule requires thorough genetic and phenotypic characterization of the virus stocks used in animal studies (e.g., challenge virus) to ensure that results from the animal studies are reproducible and predictive of human outcomes.

Why Characterization Matters

For regulatory acceptance of data under the Animal Rule, virus stocks used in animal studies must be well characterized in terms of:

• Genetic sequence
• Stability over time
• Phenotypic properties (e.g., virulence)
• Passage history
  
  

Serially passaged virus stocks often contain heterogeneous populations with multiple mutations. This can complicate the data analysis from animal studies and raise questions about the reproducibility of the results. Conversely, the increased genetic fidelity and reduced batch-to-batch variability of virus stocks generated using reverse genetics approaches increases the reliability of data generated from animal studies. Thus, vaccines or therapeutics tested under the Animal Rule are more likely to be supported by data generated by using virus stocks created from reverse genetics methods.

Using high-quality virus stocks strengthens the trustworthiness of the animal efficacy data, which is a key consideration when human trials are not conducted.

How Advanced Virology Inc. Enhances Product Development

Advanced Virology Inc.’s custom services can support FDA Animal Rule applications by providing well-characterized, reproducible challenge virus stocks that align with regulatory expectations for nonclinical efficacy studies. By supplying high-fidelity virus stocks, we ensure consistency across all steps of R&D and product development, including in vitro and animal studies.

Using our virus stocks strengthens data quality, integrity, and reproducibility, which facilitates accurate interpretation of dose–response and mechanism-of-action results. Our services help streamline interactions with regulators and reduce risk during review by demonstrating that study outcomes are based on reproducible and well-characterized biological reagents.