Study: Virulence Differences of Monkeypox Virus Clades 1, 2a and 2b.1 in a Small Animal Model. Image Credit: MIA Studio/Shutterstock

Clade 2b Mpox virus is the least virulent strain according to a new study

In a recent study published on bioRxiv* preprint server, the 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 disease transmission and virulence.

Study: Differences in virulence of monkeypox virus clades 1, 2a and 2b.1 in a small animal model.  Image Credit: Studio MIA/Shutterstock
Study: Differences in virulence of monkeypox virus clades 1, 2a and 2b.1 in a small animal model. Image Credit: Studio MIA/Shutterstock


Mpox is a zoonotic disease caused by the mpox virus belonging to the Orthopoxvirus gender and Poxviridae family, similar to that which causes smallpox Smallpox 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 called the Congo Basin clade, caused nearly 10% mortality, while clade 2a, also called the West African clade, with 95% nucleotide sequence similarity, caused less than 1% of mortality. While both clades 1 and 2a were transferred by zoonosis, clade 2b, which caused the widespread epidemic of 2022, exhibits widespread human-to-human transmission and reduced mortality and is genetically similar to clade 2a. However, it is not known whether these genetic differences translate into changes in virulence and transmissibility.

About the study

In the present study, researchers identified a suitable small animal model that can be used to study differences in virulence and transmission of mpox by screening 38 inbred mouse strains for susceptibility to mpox virus. Identifying the appropriate animal model system has 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 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 mpox virus and using a plaque assay to determine viral yield.

CAST/EiJ mice were inoculated intranasally and intraperitoneally with mpox viruses belonging to clades 1, 2a and 2b. Viral titers of the inoculum were determined by plaque assays. After infection, mice were euthanized and viral titers were determined for infected organs. In addition, viral deoxyribonucleic acid (DNA) of organs was quantified using droplet digital polymerase chain reaction (ddPCR).


The results reported that in CAST/EiJ mice, the virulence of clade 1 mpox virus was 1000 times greater than that of clade 2a mpox virus. Infections at 103 clade 1 mpox virus plaque-forming units (PFU) caused 20% weight loss in all mice and one death. When the dose was increased to 104 and 105 PFU, all mice showed greater than 30% weight loss or died. For clade 2a mpox virus infections, slightly higher doses were required to achieve comparable weight loss, and all mice died at 105 PFU. In addition, nasal turbinates, brain, lungs, and abdominal organs showed higher viral concentrations after infections with clade 1 virus, compared to infections with clade 2a virus.

When mice were inoculated intraperitoneally with clade 1 mpox virus, most mice died when infected with doses of 1 and 10 PFU, and all mice died when infected with doses of 100 and 100 PFU. However, 100 doses of PFU of clade 2a mpox virus caused no deaths and 1000 doses of PFU 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 infected intranasally with 104 and 105 PFU doses of clade 2a virus. In contrast, similar doses of clade 2b virus did not cause weight loss or death. In 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 10 years5 PFU dosage.

Viral titers in 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. Clade 2b virus infections resulted in only one mouse with detectable viral titers in the lungs, and viral titers in the nasal turbinates were three log units lower than clade 2a virus infections. Livers and spleens showed no viral titer. Intraperitoneal inoculations resulted in 100-fold higher viral titers for mpox clade 2a viral infections than for clade 2b viral infections. The results were also corroborated by ddPCR sequencing analysis, with lower virulence corresponding to lower viral replication.


Overall, the results indicated that mpox virus belonging to clade 1 was the most virulent, followed by clade 2a virus. The clade 2b mpox virus, which caused the 2022 outbreak in nearly 100 sites outside 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 considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.

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