Junior Ayuk Enow

Myxoma virus (MYXV), a member of the Leporipoxvirus genus (species Leporipoxvirus myxoma; family Poxviridae), causes a highly lethal disease known as myxomatosis in European rabbits. In late 2018, a new natural MYXV isolate, MYXV-Tol (a.k.a. hare MYXV; ha-MYXV), emerged and caused myxomatosis-like disease with high mortality in Iberian hares, European brown hares, and European rabbits. This variant contains an approximately 2.8-kb insertion of a recombination cassette within the M009L gene encoding four additional genes, including the C7-like host range gene, M159L. M159 is essential for replication of MYXV-Tol in hare cells and is likely a key determinant of its pathogenicity in both hares and rabbits. Here, we compared the pathogenicity of wild-type MYXV-Tol (vMyx-Tol), an M159 deletion strain (vMyx-Tol-M159KO), and the classical MYXV-Lau strain (vMyx-Lau) in European rabbits. All three viruses caused systemic disease; however, vMyx-Tol and vMyx-Tol-M159KO produced clinical signs distinct from classical myxomatosis. Infection with vMyx-Tol and vMyx-Tol-M159KO was characterized by the absence of the typical primary and secondary nodular lesions, and caused severe edema, marked fluid accumulation, lymphocyte infection, and significantly reduced or no virus-neutralizing antibody responses. The disease caused by both vMyx-Tol and vMyx-Tol-M159KO progressed rapidly within 9-11 days, resulting in animals reaching humane euthanasia endpoints like vMyx-Lau. Deletion of M159 did not significantly alter MYXV-Tol pathogenicity in rabbits. Collectively, these findings demonstrate that MYXV-Tol has evolved to cause an atypical, amyxomatous-like acute to hyperacute disease in European rabbits and likely in hares.

Poxviruses encode a plethora of proteins adapted to diverse cellular responses against viruses. The poxvirus-encoded E3-like proteins are multifunctional, regulating diverse cellular antiviral responses. The canonical Vaccinia E3-like proteins have two domains: an N-terminal Z-form nucleic acid-binding domain (Zα-BD) and a C-terminal double-stranded RNA-binding domain (dsRNA-BD). Using protein sequence and structural homology modeling, we identified dsRNA-BD-fold-containing proteins in all the poxviruses except avipoxviruses, salmon poxvirus, and entomopoxviruses. Furthermore, we show that the acquisition of these proteins likely happened under three distinct events and can be classified into three categories: (i) the E3-like proteins with dsRNA-BD with the presence or absence of the N-terminal domain; (ii) unconventional/putative dsRNA-BD-fold-like proteins, present in macropoxvirus and molluscipoxvirus, and (iii) the dsRNA-BD-containing protein present in crocodile poxvirus. Members of leporipoxvirus, waddenpoxvirus, cetaceanpoxvirus, and selected members of orthopoxvirus contain E3-like proteins missing the N-terminal Zα-BD required for necroptosis inhibition. Using AlphaFold, we also show that the Zα-BD of chordopoxviruses, E3-like proteins, is structurally more variable than the dsRNA-binding domain. Compared to orthopoxviruses that inhibit necroptosis and contain an N-terminus Zα-BD, our results show that leporipoxviruses induce receptor-interacting protein kinase (RIPK) RIPK1- and RIPK3-mediated necroptosis in human and mouse necroptosis-competent cell lines. These data suggest that leporipoxviruses lack countermeasures against necroptosis, unlike orthopoxviruses, which encode multiple key regulators of necroptosis, possibly due to a lack of selective pressure in their lagomorph host species. IMPORTANCE: The evolutionary arms race between viruses and their hosts has led to the viral acquisition of genes that antagonize the host’s antiviral defenses. However, related viral and host proteins have evolved under positive, purifying, and neutral selection. Poxvirus-encoded E3-like proteins play a pivotal role in regulating the host’s cellular antiviral and apoptotic responses. The two domains of canonical E3-like proteins, an N-terminal Z-form nucleic acid-binding domain (Zα-BD) and a C-terminal double-stranded RNA-binding domain (dsRNA-BD), have evolved under varying host immune selection pressures. Here, we demonstrate that the dsRNA-BD is structurally conserved among E3 orthologs, whereas the Zα-BD, involved in regulating necroptosis, exhibits structural diversity and is absent in some poxviruses. Leporipoxviruses, which lack Zα-BD-containing proteins and evolved in species without functional necroptosis, do activate necroptosis in necroptotic-competent cells. Our study thus highlights that a lack of selective pressure from the host can shape viral countermeasures and viral divergence.

Poxviruses (PXV) are large double-stranded DNA viruses (dsDNA) that infect and cause disease in a wide variety of hosts, including humans.. They are studied as models to understand host-specific disease and they have a wide variety of applications in biotechnology, including vaccines, oncolytic virotherapy, and immunotherapy. Despite a nearly fourfold variation in genome sizes across the Poxviridae family, the basic genome architectural features driving their evolution remain poorly understood. In this study of the summated poxvirus genome sequence database, we report that while only a weak negative correlation exist between viral coding densities and genome sizes, we detected a strong positive correlation between the number of assigned open reading frames (ORF) and genome sizes. We observe an inflection point at a genome size of 229 kb, above or below with the percentage of genes encoding secreted proteins increasing with genome size, and the percentage of predicted cytoplasmic proteins decreasing with genome size. Additionally, we observed a weak positive correlation between the potential of transcript overlap that would be expected to generate dsRNA and genome size. Unexpectedly, we observed a significant positive correlation between coding density and GC-content in mpox virus isolates ( R ² = 0.799). Further analysis illustrated a rise in both GC-content and coding density of mpox isolates between 2022 and 2024. Codon usage bias was observed to be clustered by virus families, with tryptophan being the least utilized amino acid across all poxviruses. Protein distribution analysis revealed a right-skewed distribution of poxviral predicted ORFs, with the 150 - 250 amino acid cohort containing the highest number of viral proteins. Finally, we observe a significant positive correlation between ITR sizes and duplicate gene clusters ( R ² = 0.679). Together, these architectural findings outline the constraints and adaptive pathways governing poxviral evolution and biology. Importance: This study establishes a family genome design rulebook for poxviruses and illustrates how these rules might be linked with their biology and evolution. We demonstrate that genome growth is accompanied by a modest increase in noncoding DNA and an increase in extracellular proteome investment. We further demonstrate that ITR expansions correlate with duplicate gene clusters, rather than genome size, indicating that ITRs serve as hubs of innovation. Our results provide a standard for generating hypotheses and experiments with poxviruses. This work also introduces matrices for within-family viral comparisons.

Myxoma virus (MYXV) is a leporipoxvirus that causes lethal disease in Leporids. Hares and rabbits belong to the Leporidae family and are believed to have had a common ancestor 12 million years ago. After seventy years of contact with European hares without causing mortalities or disease manifestation, a recombinant MYXV infected Iberian hares (Lepus granatensis) causing high mortalities. Like all poxviruses, MYXV encodes a wealth of immune modulators required for successful virulence that also mediate host species jumping, for example, into hares. Here, we summarize the data of known MYXV immune modulators, their cellular functions, and their effects on European rabbits. Additionally, we suggest that the critical restrictions MYXV would encounter in colonizing a potentially new host species stem from their interactions with the host’s innate immune environment. Lastly, we synthesize our understanding of some poxvirus genome architectural features that might have facilitated the host species jump of MYXV into hares from rabbits.

Oncolytic viruses (OVs) have emerged as one of the most promising cancer immunotherapy agents that selectively target and kill cancer cells while sparing normal cells. OVs are from diverse families of viruses and can possess either a DNA or an RNA genome. These viruses also have either a natural or engineered tropism for cancer cells. Oncolytic DNA viruses have the additional advantage of a stable genome and multiple-transgene insertion capability without compromising infection or replication. Herpes simplex virus 1 (HSV-1), a member of the oncolytic DNA viruses, has been approved for the treatment of cancers. This success with HSV-1 was achievable by introducing multiple genetic modifications within the virus to enhance cancer selectivity and reduce the toxicity to healthy cells. Here, we review the natural characteristics of and genetically engineered changes in selected DNA viruses that enhance the tumor tropism of these oncolytic viruses.

Combination of selinexor and oncolytic MYXV significantly reduced viability of human cancer cells. PANC-1 (A–D) and Colo205 (E–H) cells in 96-well plates were left untreated (mock) or treated with different concentration of selinexor, or infected with different MOI of MYXV, or treated with a combination of selinexor and MYXV for indicated timepoints. Cell viability was measured using the MTS assay reagents. Data represent mean ± SD and n = 4.

Selinexor significantly enhanced MYXV infection and replication in 3D culture of human cancer cell lines. PANC-1 (A), MDA-MB435 (C), and HT29 (E) cells were treated with different concentration of selinexor for 1 hour and then infected with vMyx-GFP-TdTomato in the presence of selinexor. Fluorescence images were taken 72 or 96 hpi. Quantification of the level of GFP fluorescence as relative fluorescence units (RFU) from PANC-1 (B), MDA-MB435 (D), and HT29 (F) cells are shown. Data represent mean ± SD and n = 3. Statistically significant differences in comparison with infection without selinexor are indicated. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

CRM1 knockdown significantly enhanced MYXV replication in human cancer cells. PANC-1 cells were transiently transfected with CRM1 siRNA or control NT siRNA. After 48 hours, the cells were infected with vMyx-GFP at MOI of 0.5 or 5.0 for 1 hour and replaced with fresh media. A, Images showing expression of GFP after 48 and 72 hpi. The cells were harvested at 48 and 72 hpi to determine progeny virus formation by titration assay on permissive RK13 cells. Virus titers after infection with an MOI of 0.5 (B) or 5.0 (C). Data represent mean ± SD and n = 3. Statistically significant differences in comparison with infection with virus alone are indicated. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. D, A549 cells were transfected with CRM1 siRNA and infected with vMyx-GFP [MOI = 0.1 (low MOI) or 3.0 (high MOI)]. After 24 hpi, the cells were fixed and stained with antibodies against DHX9. Nuclei were stained with DAPI and imaged by fluorescence microscope. E, Western blot analysis of CRM1 protein level in A549 cells after transfection of siRNAs for 48 hours; actin as loading control. Assays were performed in triplicate and a representative is shown.

Prolonged replication of MYXV in the tumor bed of the Selinexor treated mice.

Selinexor significantly enhances MYXV replication in PANC-1 xenograft tumors in NSG mice, reduces tumor burden and prolong survival. A, Diagram of experimental setup. NSG mice were inoculated with PANC-1 cells at day 0 via subcutaneous injection on both flanks. Animals received four doses of selinexor by oral gavage and three intratumoral injection of vMyx-FLuc on the right flank. Mice were imaged for luciferase activity 24 and 48 hours after first virus injection. B, Luminescence images taken using IVIS imaging system 24 and 72 hours after the first injection. C, Quantification of luminescence signals as relative light units (RLU) from images taken 24 and 72 hours after first injection. D–E, Tumor volumes on the left and the right flanks were measured at different days after the start of treatments. Results are presented as mean ± SEM. F, Kaplan–Meier survival curves comparing animals with different treatment groups. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.