In a recent study posted to the bioRxiv* preprint server, researchers used the CAST/EiJ mouse model to test whether genetic differences between clades 1, 2a, and 2b of the mpox virus contribute to changes in transmission and virulence of the disease.
Background
Mpox is a zoonotic disease caused by the mpox virus belonging to the Orthopoxvirus genus and Poxviridae family, similar to the smallpox-causing Variola virus. Until early 2022, mpox was endemic to Africa and is thought to have spread from rodents to non-human primates and humans.
Genome sequence-based studies have identified three major clades based on genetic differences. Clade 1, also referred to as the Congo Basin clade, caused close to 10% mortality, while clade 2a, also called the West African clade, with 95% nucleotide sequence similarity, caused less than 1% mortality. While both clades 1 and 2a were zoonotically transferred, Clade 2b, which caused the 2022 widespread outbreak, exhibits extensive transmission between humans and reduced mortality and is genetically similar to clade 2a. However, whether these genetic differences translate to changes in virulence and transmissibility is not known.
About the study
In the present study, the researchers identified a suitable small animal model that can be used to study the differences in virulence and transmission of mpox by screening 38 inbred mouse strains for vulnerability to the mpox virus. Identifying the appropriate animal model system presented a challenge since the animal must be inbred, susceptible to mpox, and can be bred in captivity.
The CAST/EiJ mouse model was found to be susceptible to the mpox virus. All infection experiments were conducted in biosafety level-3 facilities. Viral replication was analyzed by infecting African green monkey kidney epithelial cells (BS-C-1) with the mpox virus and using a plaque assay to determine the viral yield.
CAST/EiJ mice were intranasally and intraperitoneally inoculated with mpox viruses belonging to clades 1, 2a, and 2b. Viral titers of the inoculum were determined through plaque assays. Post-infection, the mice were euthanized, and viral titers were determined for infected organs. Furthermore, viral deoxyribonucleic acid (DNA) from the organs was quantitated using droplet digital polymerase chain reaction (ddPCR).
Results
The results reported that in CAST/EiJ mice, the virulence of the mpox virus from clade 1 was 1000 times more than that of the clade 2a mpox virus. Infections with 103 plaque-forming units (PFU) of clade 1 mpox virus resulted in 20% weight loss in all the mice and one death. When the dosage was increased to 104 and 105 PFU, all the mice showed more than 30% weight loss or died. For infections with clade 2a mpox virus, slightly higher doses were required to achieve comparable weight loss, and all mice died at 105 PFU. Furthermore, nasal turbinates, brain, lungs, and organs in the abdomen showed higher viral concentrations after infections with clade 1 virus, as compared to infections with clade 2a virus.
When the mice were inoculated intraperitoneally with clade 1 mpox virus, most mice died when infected with 1 and 10 PFU doses, and all mice died when infected with 100 and 100 PFU doses. However, 100 PFU doses of clade 2a mpox virus caused no deaths, and 1000 PFU doses resulted in 50% mortality. Viral titers in all organs after intraperitoneal infections were significantly higher for clade 1 mpox virus than for clade 2a.
Viral replication of clade 2a and clade 2b mpox viruses in BS-C-1 cells did not differ significantly. Severe disease, weight loss, and mortality were observed when CAST/EiJ mice were intranasally infected with 104 and 105 PFU doses of clade 2a virus. In contrast, similar doses of clade 2b viruses caused no weight loss or death. In the case of intraperitoneal inoculation, 103 and 104 PFU of clade 2a virus caused 100% mortality, while clade 2b virus did not cause weight loss or death even at 105 PFU dosage.
Viral titers in the nasal turbinates, lungs, and liver of mice intranasally infected with clade 2a mpox virus were greater than 105 PFU, 106 PFU, and 104 PFU, respectively. Infections with clade 2b virus resulted in only one mouse with detectable viral titers in the lungs, and the viral titers in nasal turbinates were lower than those in the case of clade 2a viral infections by three log units. The livers and spleens showed no viral titers. Intraperitoneal inoculations resulted in 100-fold higher viral titers for clade 2a mpox virus infections than clade 2 b viral infections. The results were also corroborated by ddPCR sequencing analysis, with lower virulence corresponding to lower viral replication.
Conclusions
Overall, the results indicated that the mpox virus belonging to clade 1 was the most virulent, followed by clade 2a virus. Clade 2b mpox virus, which caused the 2022 outbreak in almost 100 locations outside the endemic regions of Africa, was 100 times less virulent than the closely related clade 2a.
*Important notice
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
