Rabies serogroup viruses in neuroblastoma cells: propagation, "autointerference," and apparently random back-mutation of attenuated viruses to the virulent state

Abstract

Each of several strains of fixed rabies virus was found to replicate to high titers in C1300 mouse neuroblastoma (clone NA) cells, without adaptation. Rabies serogroup Lagos bat, Mokola, and Duvenhage viruses also replicated efficiently in NA cells. Kotonkan and Obodhiang viruses replicated efficiently after adaptation, to titers not previously obtained in vitro. Infection in NA cells was frequently more cytopathic than in BHK-21 cells, allowing titration of Kotonkan and Obodhiang viruses by plaque assay. Duvenhage virus caused syncytium formation. Serial propagation of rabies viruses at a high multiplicity of infection in NA cells led to a rapid decline in virus yields; similar "autointerference" has not previously been demonstrated with rabies virus in other cell systems. Rabies virus infection in NA cells exhibited extreme sensitivity to interference by experimentally added defective interfering virions. Although several strains of attenuated rabies virus consistently reverted rapidly to virulence after propagation in NA cells, other strains of attenuated rabies and rabies serogroup viruses acquired increased virulence at a more gradual rate or not at all, suggesting that diverse characters may control virulence. When attenuated Flury HEP rabies virus was serially propagated at a low multiplicity of infection in either NA cells or suckling mouse brain, virulence appeared at a very variable rate, indicating that these systems may selectively enhance replication of randomly occurring virulent virus mutants.

Virulence of Chick Embryo Fibroblast‐Passaged Flury HEP Rabies Virus and Its Revertants in Mice

“…Kaplan et al reported that all of the nude mice they infected with BHK-HEP virus died 12 to 18 days after injection and that the virus killed about 80% of ICR Comparison of these data with our results shown in Tables 1 and 2, indicates that CE-HEP virus, the vaccine strain in Japan, is a highly attenuated one. When CE-HEP virus was passaged in NA cells three or more times, it killed adult mice (4-week-old), and this was in agreement with the Clark's results (3,4). Unlike the CE-HEP virus passaged in brains of suckling mice, however, the virulent NA-HEP virus showed death patterns of the autointerfering type.…”

“…(b) Lanes 1 to 4 and 6 to 12 are the patterns of the passage 6 NA-HEP fractions Nos. 2,4,6,8,10,12,14,16,18,20, and 23 in Fig. 3, respectively.…”

“…In a comparative study of the less neurovirulent strains, HEP and ERA, and the highly neurovirulent strains, CVS and Pitman Moore, a pronounced difference between the two groups of the virus was found in the composition of the structural proteins, especially of the L and the G proteins (6). The HEP and several other strains of attenuated rabies virus lacking the ability to kill adult mice regained neurovirulence after a few passages in murine or human neuroblastoma cells (3,4). Clark et al and Wunner and Clark demonstrated that the virulence of rabies virus is not mediated by defective interfering (DI) particles and that back-mutation to virulence is a randomly occurring event (4,5,20).…”

“…The HEP and several other strains of attenuated rabies virus lacking the ability to kill adult mice regained neurovirulence after a few passages in murine or human neuroblastoma cells (3,4). Clark et al and Wunner and Clark demonstrated that the virulence of rabies virus is not mediated by defective interfering (DI) particles and that back-mutation to virulence is a randomly occurring event (4,5,20). Dietzschold et al found a single base change in the nucleotide sequences of the glycoprotein-genes of the avirulent variant and the parental virulent virus (7).…”

Characterization of Canine Distemper Viruses Adapted to Neural Cells and Their Neurovirulence in Mice

“…Mechanisms of the increase in neurovirulence after passage in neural cells are still speculative. Clark (2,3) reported rapid increases in neurovirulence of rabies virus by passage in mouse brain or in neuroblastoma cells in vitro, and suggested random mutation followed by selective replication of the mutant with neurotropic properties as a mechanism responsible for the increase in neurovirulence. Selective replication of virulent clones of CDV in the process of adaptation was also suggested by Appel (1).…”