A Single Amino Acid Change in the Marburg Virus Glycoprotein Arises during Serial Cell Culture Passages and Attenuates the Virus in a Macaque Model of Disease

Abstract

Marburg virus (MARV) causes disease with high case fatality rates, and there are no approved vaccines or therapies. Licensing of MARV countermeasures will likely require approval via the FDA's Animal Efficacy Rule, which requires wellcharacterized animal models that recapitulate human disease. This includes selection of the virus used for exposure and ensuring that it retains the properties of the original isolate. The consequences of amplification of MARV for challenge studies are unknown. Here, we serially passaged and characterized MARV through 13 passes from the original isolate. Surprisingly, the viral genome was very stable, except for a single nucleotide change that resulted in an amino acid substitution in the hydrophobic region of the signal peptide of the glycoprotein (GP). The particle/PFU ratio also decreased following passages, suggesting a role for the amino acid in viral infectivity. To determine if amplification introduces a phenotype in an animal model, cynomolgus macaques were exposed to either 100 or 0.01 PFU of low-and highpassage-number MARV. All animals succumbed when exposed to 100 PFU of either passage 3 or 13 viruses, although animals exposed to the high-passage-number virus survived longer. However, none of the passage 13 MARV-exposed animals succumbed to 0.01-PFU exposure compared to 75% of passage 3-exposed animals. This is consistent with other filovirus studies that show some particles that are unable to yield a plaque in cell culture can cause lethal disease in vivo. These results have important consequences for the design of experiments that investigate MARV pathogenesis and that test the efficacy of MARV countermeasures.IMPORTANCE Marburg virus (MARV) causes disease with a high case fatality rate, and there are no approved vaccines or therapies. Serial amplification of viruses in cell culture often results in accumulation of mutations, but the effect of such cell culture passage on MARV is unclear. Serial passages of MARV resulted in a single mutation in the region encoding the glycoprotein (GP). This is a region where mutations can have important consequences on outbreaks and human disease [S. Mahanty and M. Bray, Lancet Infect Dis 4:487-498, 2004, https://doi.org/10.1016/S1473 -3099(04)01103-X]. We thus investigated whether this mutation impacted disease by using a cynomolgus macaque model of MARV infection. Monkeys exposed to virus containing the mutation had better clinical outcomes than monkeys exposed to virus without the mutation. We also observed that a remarkably low number of MARV particles was sufficient to cause death. Our results could have a significant impact on how future studies are designed to model MARV disease and test vaccines and therapeutics.KEYWORDS Marburg virus, adaptation, attenuation, glycoprotein, monkey model, pathogenesis, signal peptide

Intramuscular Exposure of Macaca fascicularis to Low Doses of Low Passage- or Cell Culture-Adapted Sudan Virus or Ebola Virus

“…The methylcellulose and crystal violet assay was used to determine the titer of the NHP exposure dose for the 8U EBOV dose study (as previously described in [ 15 ]), while the agarose and neutral red assay was used for all other viral titrations. As expected, all of the virus exposure doses were below the assay limit of detection, consistent with previous work showing that some particles do not yield plaques in cell culture, but that they can nonetheless cause lethal disease in NHPs [ 15 , 16 ]. When the exposure doses were prepared, calculations were performed using viral titers determined previously (approximately one year prior to the animal exposures for all stocks except the 8U EBOV).…”

“…Nonhuman primates were observed at least twice daily for up to 21 days post-exposure, at which time any survivors were euthanized. Clinical scores were reported to the responsible veterinarian, and euthanasia was approved when scores indicated that an NHP was terminally ill. A brief description of the clinical scoring system (described previously in [ 16 ]) is as follows. Animals received one point each for reductions in feed or fluid intake, dehydration, no stool production, rough hair coat, nasal discharge, or bleeding at the blood collection site.…”

“…There does not appear to be a difference in survival when animals are exposed to a virus stock with a predominantly 7U genotype (7U virus stock) versus a virus stock with a predominantly 8U genotype (8U virus stock) EBOV or SUDV, but disease course may be impacted [ 13 , 14 ]. Furthermore, another characteristic of cell culture-passaged viruses is altered particle-to-plaque forming unit (PFU) ratio; filovirus particle-to-PFU ratios have been shown to decrease after cell culture passage [ 15 , 16 ]. Previous studies have also shown that using a lower exposure dose can help to uncover differences between different virus stocks [ 15 , 16 ].…”

“…Furthermore, another characteristic of cell culture-passaged viruses is altered particle-to-plaque forming unit (PFU) ratio; filovirus particle-to-PFU ratios have been shown to decrease after cell culture passage [ 15 , 16 ]. Previous studies have also shown that using a lower exposure dose can help to uncover differences between different virus stocks [ 15 , 16 ].…”

Cell culture propagation of foot-and-mouth disease virus: adaptive amino acid substitutions in structural proteins and their functional implications

“…Commonly, these FMD vaccines are produced by propagation of FMDV in BHK-21 cells [7]. It is known for many viruses, however, that cultivation outside the natural host, for instance during the course of adaptation to a permanent cell line, can result in alterations to the amino acid sequence of viral proteins [9][10][11][12][13]. Especially RNA viruses, such as FMDV, consist of viral populations without a defined genomic sequence but of distributions of related but non-identical genomes (quasispecies).…”