Virulence differences of mpox (monkeypox) virus clades I, IIa, and IIb.1 in a small animal model

Abstract

Human mpox (monkeypox), a disease with similarities to smallpox, is endemic in Africa where it has persisted as a zoonosis with limited human-to-human spread. Unexpectedly, the disease expanded globally in 2022 driven by human-to-human transmission outside of Africa. It is not yet known whether the latter is due solely to behavioral and environmental factors or whether the mpox virus is adapting to a new host. Genome sequencing has revealed differences between the current outbreak strains, classified as clade IIb, and the prior clade IIa and clade I viruses, but whether these differences contribute to virulence or transmission has not been determined. We demonstrate that the wild-derived inbred castaneous mouse provides an exceptional animal model for investigating clade differences in mpox virus virulence and show that the order is clade I > clade IIa > clade IIb.1. The greatly reduced replication of the clade IIb.1 major outbreak strain in mice and absence of lethality at 100 times the lethal dose of a closely related clade IIa virus, despite similar multiplication in cell culture, suggest that clade IIb is evolving diminished virulence or adapting to other species.

Keywords: castaneous mouse; human monkeypox; monkeypox virus; orthopoxvirus; virus virulence.

Fig. 1.

MPXV Z-1979 and USA-2003: in vitro replication and African dormouse infection. (A) BS-C-1 cells were infected in triplicate with 0.05 PFU/cell of MPXV Z-1979 or USA-2003. At intervals, virus yields were determined by plaque assay on BS-C-1 cells. Bars indicate SD. (B) Each group (n = 3 to 5) of African dormice was mock infected or infected IN with 0.05 to 5,000 PFU of Z-1979 and survival plotted. (C) Same as panel B except animals were inoculated with 0.5 to 5,000 PFU of USA-2003.

Fig. 2.

Virulence of MPXV Z-1979 and USA-2003 upon IN infection of CAST mice. (A) CAST mice (groups of n = 4 to 5) were mock infected or infected IN with 10² to 10⁵ PFU of MPXV Z-1979 or USA-2003 and examined and weighed daily. The percentage of starting weights and survival are indicated. Bars indicate SD. (B) CAST mice (n = 4 to 5) were infected IN with 10² PFU of MPXV Z-1979 or USA-2003. On days 5 (d5) and 7 (d7), whole organs and organ pairs were removed, homogenized, and the total PFU determined by plaque assay. Bars represent geometric means. *P < 0.05 and **P < 0.01.

Fig. 3.

Virulence of MPXV Z-1979 and USA-2003 upon IP infection of CAST mice. (A) CAST mice (groups of n = 4 to 5) were mock infected or infected IP with 1 to 10³ PFU of MPXV Z-1979 or USA-2003 and examined and weighed daily. The percentage of starting weights and survival are indicated. Bars indicate SD. (B) CAST mice (n = 3 to 6) were infected IP with 10² PFU of MPXV Z-1979 or USA-2003. On day 6, whole organs and organ pairs were removed, homogenized, and the total PFU determined by plaque assay. Bars represent geometric means. *P < 0.05.

Fig. 4.

In vitro replication and virulence of USA-2003 and MA-2022. (A) One-step replication. BS-C-1 cells were infected in triplicate with 3 PFU/cell of USA-2003 or MA-2022 and harvested at the indicated times. Infectious virus in cell lysates was determined by plaque assay on BS-C-1 cells. Bars represent SDs. (B) Multistep replication. BS-C-1 cells were infected in triplicate with 0.05 PFU of USA-2003 or MA-2022. At the indicated times, the medium was removed, centrifuged, and supernatant collected. The pelleted cells were combined with the cells that adhered to the dish and lysed. Infectious virus in the lysates and supernatant was determined by plaque assay on BS-C-1 cells using a methylcellulose overlay. Bars indicate SDs. (C) Satellite (comet) plaque assay. BS-C-1 cell monolayers were infected with ~25 PFU of USA-2003 or MA-2022 for 1 h at 37 °C. Unadsorbed virus was removed, and the incubation was continued for 48 h without a methylcellulose overlay and then stained with crystal violet. (D) IN infection of CAST mice. CAST mice were inoculated with 10² PFU (n = 3), 10³ PFU (n = 4), 10⁴ PFU (n = 6 to 7), and 10⁵ PFU (n = 2) of USA-2003 and MA-2022. Daily weights and survival are plotted. (E) IP infection of CAST mice. CAST mice were inoculated with 10² PFU (n = 3), 10³ PFU (n = 4 to 7), and 10⁴ PFU (n = 6) of USA-2003 and MA-2022. Daily weights and survival are plotted.

Fig. 5.

Infectious virus and genome copies of USA-2003 and MA-2022 in organs of CAST mice. (A and B) CAST mice (groups of n = 3) were infected IN as in Fig. 5C, and infectious virus and MPXV genome copies determined. (C and D) CAST mice (groups of n = 3) were infected IP as in Fig. 5D, and infectious virus and MPXV genome copies determined.