Evolutionary Dynamics of Viral Attenuation

Abstract

The genetic trajectory leading to viral attenuation was studied in a canine parvovirus (CPV) strain grown on dog kidney cells for 115 transfers. Consensus sequences of viral populations at passages 0, 3, 30, 50, 80, and 115 were obtained from PCR products covering 86% of the genome; clones from each of the 80th and 115th passages were also sequenced, covering 69% of the genome. Sixteen changes were fixed in the 115th-passage virus sample. Levels of polymorphism were strikingly different over time, in part because of a plaque-cloning step at passage 112 that reduced variation: passage 80 had 19 variants common among the clones, but passage 115 had only a single common variant. Several mutations increased in the culture at the same time, with most reaching fixation only after the 80th passage. The pattern of evolution was consistent with recombination and not with separate selective sweeps of individual mutations. Thirteen of the changes observed were identical to or at the same positions as changes observed in other isolates of CPV or feline panleukopenia virus.The most successful viral vaccines use live attenuated virus strains. For more than a century, attenuation has involved propagation of a virus under novel conditions so that it becomes less pathogenic to its original host as it evolves under the new conditions (9, 10). Despite the antiquity of this method and its remarkable success in generating many vaccines that are widely used in humans and other animals, little is known about the process by which the attenuating mutations arise and evolve. In addition, there are some drawbacks to this method: the outcome of an attempted attenuation is largely unpredictable, and a successfully attenuated virus may revert to virulence, depending on the nature of the attenuating mutation(s). Although newer approaches show promise, many contemporary vaccines are still being developed by these classic methods.A better understanding of the classical attenuation process might thus suggest protocols to eliminate some disadvantages of these methods of attenuation. One of the obvious gaps in our understanding of attenuation is at the genetic level. Until recently, the molecular changes underlying the shift from virulence to attenuation were almost completely unknown for most attenuated vaccine viruses. Even now, the dynamics over an attenuation episode have yet to be described. To begin filling this gap, we examined the genetic evolution that occurred during the passaging of canine parvovirus (CPV) in tissue culture to give rise to a successfully attenuated vaccine that has been used effectively for 20 years.

Canine Parvovirus: Current Perspective

“…After a period of adaptation, the virus became highly infectious for dogs, resulting in the pandemic that became evident in 1978-1979. The rapid rate at which parvoviruses accumulate mutations in vivo similar to observations made in studies on CPV-2 vaccinal virus, where mutations were found to accumulate rapidly during passage in tissue culture [3,84].…”

“…Approximately 50% of the mass is protein, and the remainder is DNA. Because of the relatively high DNA-to-protein ratio, the buoyant density of the intact virion in cesium chloride (CsCl) is 1.39-1.42 g/cm 3 . Finally, the sedimentation coefficient of the virion in neutral sucrose gradients is 110-122 S [54,69].…”

Cloning and Characterization of a Bovine Adeno-Associated Virus

“…The high homology between the BAAV and AAV-5 Rep proteins, along with the biochemically distinct mechanisms of replication for these two viruses vis-à-vis other mammalian AAVs, suggest that BAAV and AAV-5 might form a distinct group within the Dependovirus genus. Thus, BAAV and human AAV-5 might be the result of a cross-species transmission such as has been proposed for the evolutionary history of parvoviruses (1).…”

Presence of Infectious RD-114 Virus in a Proportion of Canine Parvovirus Isolates

“…CPV2 strain Cornell-780916, which is the parental CPV2 in manufacturing several commercial live vaccines, was also obtained from ATCC (catalogue number: VR-953). This strain may have been propagated in CRFK cells during attenuation [1]; however, the detailed passage history is unknown. The stock viruses were stored at -80 ºC until use.…”

“…Given that these canine vaccines contained the original antigenic type of canine parvovirus (CPV) type 2 in common, and that many such CPVs have been isolated and attenuated using CRFK cells [1,5], we suspected that RD-114 virus may have contaminated the seed stock virus for the production of the vaccines [15]. In this study, we collected CPVs from a diagnostic laboratory and American Type Culture Collection (ATCC), and checked for the presence of infectious RD-114 virus.…”