Mechanistic insights into bone remodelling dysregulation by human viral pathogens

Abstract

Bone-related diseases (osteopathologies) associated with human virus infections have increased around the globe. Recent findings have highlighted the intricate interplay between viral infection, the host immune system and the bone remodelling process. Viral infections can disrupt bone homeostasis, contributing to conditions such as arthritis and soft tissue calcifications. Osteopathologies can occur after arbovirus infections such as chikungunya virus, dengue virus and Zika virus, as well as respiratory viruses, such as severe acute respiratory syndrome coronavirus 2 and enteroviruses such as Coxsackievirus B. Here we explore how human viruses dysregulate bone homeostasis, detailing viral factors, molecular mechanisms, host immune response changes and bone remodelling that ultimately result in osteopathologies. We highlight model systems and technologies to advance mechanistic understanding of viral-mediated bone alterations. Finally, we propose potential prophylactic and therapeutic strategies, introduce ‘osteovirology’ as a research field highlighting the underestimated roles of viruses in bone-related diseases, and discuss research avenues for further investigation.

Approximately 15% of total body mass consists of skeletal bones, providing crucial structural integrity and support for soft tissues. The intricate interplay between bone metabolism and immunology, termed osteoimmunology¹, is vital for maintaining bone health. Dysregulation of osteoimmunology can lead to inflammatory bone loss or soft tissue calcifications, mimicking bone formation² (Box 1 and Figs. 1 and 2). Beyond spontaneous autoimmunity, bone cancers and age-associated degeneration, microbial infections can trigger bone diseases and tissue calcifications, giving rise to the emerging field of osteomicrobiology³.

BOX 1 Bone homeostasis and pathogenesis.

Bone remodelling is a continuous metabolic process that mediates the breakdown and synthesis of bone matrix throughout life. This process is facilitated by three cell types, which reside in the bone microenvironment, namely the bone-forming osteoblasts (OBs), bone-resorbing osteoclasts (OCs) and bone-matrix-residing osteocytes (Ocys), as reviewed elsewhere¹¹⁸ (Fig. 1).

Briefly, OCs are multinucleated giant cells derived from the fusion of myeloid precursor cells (for example, myeloid/monocyte), known as osteoclastogenesis (Fig. 1). They mediate bone resorption, a physiological process that involves the breakdown of bone tissues through the secretion of acids and proteases¹¹⁹. This process is predominantly mediated by OCs and/or immune cells (for example, T cells and B cells) in bone tissues¹²⁰, with the local presence of pro-osteoclastogenic signalling molecules: (1) macrophage colony stimulating factor (M-CSF/CSF1) and (2) RANKL (ref. 119). Conversely, OBs are differentiated from MSC-derived osteoprogenitor cells in a process called osteoblastogenesis¹²¹ (Fig. 1). They regulate bone-matrix formation through Wnt/β-catenin, BMP-SMAD, Hedgehog and Notch signalling cascades¹²². Subsequently, OBs cease to synthesize bone matrix and terminally differentiate into Ocys, a mature form of OBs that coordinate bone remodelling through the lacuna-canalicular system in bone¹²³. In addition, OBs express the RANKL-binding ‘decoy’ receptor, OPG, which competes with RANK for RANKL and thus inhibits osteoclastogenesis¹²⁴. Hence, the RANKL–RANK–OPG axis plays a central role in regulating bone homeostasis (Fig. 1).

Altered functions of bone-resorbing OCs and bone-forming OBs can result in pathogenic bone loss or calcification (Fig. 2a,b). During homeostasis, specific bone sites are resorbed by OCs for bone matrix maintenance, but imbalance of osteoclastic coupling factors (for example, RANKL) results in excessive OC formation and pathological bone-loss disorders, such as osteoporosis and periodontal disease (reviewed previously¹²⁵). OCs play a crucial role in pathologic bone loss by producing high levels of cathepsin K and collagenase, enzymes that breaks down the bone matrix (Fig. 2a). Several underlying molecular mechanisms have been proposed, including: (1) disruption in bone remodelling with accelerated bone resorption or attenuated bone formation; (2) direct hormonal actions on OC differentiation; (3) inflammatory factors such as TNF-α, IL-1β, IL-6, IL-17 and CCL2 (reviewed previously¹²⁶; Fig. 2a). Furthermore, calcification (both micro and macro) is a highly regulated physiological process in bone formation, but a pathologic process in soft tissues (for example, brain, placenta, joints and blood vessels; Fig. 2b). Several mechanisms known to be involved in the onset of pathologic calcification¹²⁷ include: (1) abnormal differentiation of osteogenic-potential cells, such as smooth muscle cells, pericytes and fibro/adipogenic progenitors, into OB-like cells^128,129 (Fig. 2b); (2) local increase in osteogenic morphogens; (3) dysregulated calcium and phosphate ion levels that are elevated above physiological levels; (4) inflammation; (5) cell damage, apoptosis and necrosis; and (6) imbalance or loss of calcification inhibitors (reviewed previously⁸¹; Fig. 2b).

Fig. 1 |. The cross-talk of bone remodelling.

The bone remodelling process involves bone resorption (osteoclastogenesis) and bone formation (osteoblastogenesis). Osteoclastogenesis (left) starts with RANKL production by immune cells, such as T and B lymphocytes, and OBs. The binding of RANKL to the RANK expressed on monocytic CSF1R⁺ OC precursor cells drives differentiation into bone-resorptive OCs. Bone resorption can be negatively regulated though the production of OPG by OBs, which compete with RANK for RANKL, blocking osteoclastogenesis. Osteoblastogenesis (right) starts with the differentiation of osteoprogenitor cells into pre-OBs, regulated by transcription factors RUNX2 and OSX. Upon maturation to OBs, the cells promote calcification and can subsequently differentiate into Ocys which help maintain the equilibrium between OBs and OCs.

Fig. 2 |. Pathogenic bone loss and calcifications.

a, Pathogenic bone loss is typically driven by inflammation. Immune cells produce RANKL and inflammatory mediators (for example, TNF, IL-1B and IL-6) leading to the differentiation of monocytic cells into OCs producing cathepsin K (CTSK) and collagenase that breaks down the bone matrix giving rise to bone loss. b, Pathogenic calcification is abnormal osteogenesis driven by non-OB cells with osteogenic potential such as smooth muscle cells (SMCs), pericytes and fibro/adipogenic progenitors (FAPs). These can cause vascular, brain and muscle calcifications, respectively. Non-OB osteogenic cells differentiate into OB-like cells under conditions such as high phosphate (P) or inflammatory-driven overt tissue-nonspecific alkaline phosphatase (TNAP) production (convert pyrophosphate; PPi to P). Subsequently, these cells undergo apoptosis, leading to soft-tissue calcifications beginning with small calcium deposits (microcalcifications) that can grow into large calcification sites (macrocalcifications).

The coronavirus disease 2019 (COVID-19) pandemic has brought attention to virus research. Although the pathogenesis of COVID-19 is still being unravelled, emerging evidence suggests an association with arthritis^4–6. Even before the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), viruses such as chikungunya (CHIKV) and dengue (DENV) have long been associated with arthritis and bone loss⁷. Additionally, Zika virus (ZIKV) and Coxsackievirus B (CVB) have been associated with abnormal soft-tissue calcifications^8,9. In this Perspective, we delve into the host–virus mechanisms underlying CHIKV, DENV and SARS-CoV-2 infections that result in viral arthritis, as well as ZIKV and CVB that induce pathological soft-tissue calcifications. Potential technologies for virus-induced bone loss and calcifications are discussed (Box 2), presenting innovative approaches to address these osteopathologies. We propose the term ‘osteovirology’ to emphasize the underappreciated roles of viruses in mediating pathological bone loss and soft-tissue calcifications.

BOX 2 Technological advances for osteovirology.

Two-dimensional co-cultures of multiple cell types in vitro have the ability to mimic a tissue microenvironment, thus being a useful experimental tool for osteovirology. Co-cultures in trans-well systems can be used to investigate the exchange of secreted factors relevant for bone formation. A 3D co-culture model involving primary human Ocys collagen-embedded on the apical side with OBs on the basal side of a porous membrane closely mimics bone morphology and demonstrates the expression of osteosclerostin and RANKL (ref. 143).

The use of ex vivo models, such as primary bone explants, preserve multicellular 3D environments allowing the analysis of osteogenesis. Alternatively, 3D models based on co-culturing human pluripotent stem cells with primary cells in a physiological dense matrix can generate cultures with cellular morphology, architecture and transcriptomic profile similar to those found in brain, heart and bone^144–146. Recently, a bone organoid has been developed by demineralization and micro-slicing of primary bone samples, then co-culturing the samples with murine OBs and bone marrow mononuclear cells, creating a microenvironment for studying the bone remodelling cycle¹⁴⁶.

Single-cell RNA sequencing (scRNAseq) is a valuable tool for characterizing cellular heterogeneity, identifying new cell subtypes and subclusters, understanding potential molecular mechanisms, and unravelling cell‒cell interactions¹⁴⁷. In response to the challenges posed by the hardness of bone tissue, standardized protocols have been devised to enhance sample preparation, with a primary focus on the generation of a viable cell suspension from skeletal cells. For example, the processing of mouse femur fragments entails a series of steps, including collagenase P digestion, DNase treatment, and subsequent filtration¹⁴⁸. Recognizing that scRNAseq methodologies lack the spatial context of cells within their tissue environment, spatial transcriptomics has emerged as a potent complementary technique. To facilitate spatial transcriptomics analysis of bone tissues, specimens can be prepared by fixing and decalcifying them using EDTA before micro-sectioning¹⁴⁹. Additionally, it is feasible to quantify virus transcripts through the application of virus-inclusive scRNAseq (viscRNA-Seq), thereby facilitating the integration of host and virus assessments¹⁵⁰. Most scRNAseq methods are carried out by 3′ poly-A mRNA capture, hence, viruses which generate polyadenylated transcripts, such as alphaviruses and SARS-CoV-2, are able to be analysed by these methods. However, non-polyadenylated viruses, such as flaviviruses, can be analysed by a capture strategy based on oligo(dT) with virus-specific primers¹⁵⁰. Spatial transcriptomics can be achieved either by laser capture microdissection, or by annealing fixed tissue directly to barcoded primers attached to microscope slides, with a resolution of 10–30 μm. In the context of basic bone biology, these technologies have successfully aided in identification of novel OC recycling mechanisms, suggesting their promise in osteovirology^146,150.

Virus-mediated bone loss and arthritis

Pathogenic bone loss and arthritis are conditions characterized by the destruction of bone tissue and joint inflammation, often resulting in pain, limited mobility and decreased quality of life (Box 1). Several clinically relevant RNA viruses (CHIKV, DENV and SARS-CoV-2) have been reported to be involved in osteoclast (OC)-mediated bone disease. Here, we discuss and speculate on potential mechanisms of virus-induced arthritis and related bone pathology. These mechanisms shed light on how viruses can disrupt the delicate balance of bone remodelling and immune response, leading to a better understanding of the pathogenesis of these conditions and the development of potential therapeutic strategies.

Chikungunya virus

Arthritogenic alphaviruses including CHIKV, Ross River virus (RRV), Barmah Forest virus, o’nyong-nyong virus, Mayaro virus, Sindbis virus and Semliki Forest virus pose significant public health threats worldwide. CHIKV, in particular, causes chronic viral arthritis outbreaks in Africa and Southeast Asia, and has emerged in South America¹⁰. In Makonde, chikungunya means ‘bending over in pain’, capturing the stooped posture resulting from severe musculoskeletal pain observed in CHIKV-infected patients¹¹. Following the acute phase of symptomatic CHIKV infection, characterized by high fever, rash, headache, fatigue, muscle pain (myalgia) and joint pain (arthralgia), over 40% of CHIKV patients, predominantly women¹², develop persistent rheumatic manifestations. These chronic manifestations include inflammation of tendons, ligaments and synovial tissues, along with bone loss^13,14, lasting for months to years after initial infection. However, patients with CHIKV experiencing chronic arthralgia are tested negative for rheumatoid arthritis (RA) biomarkers like rheumatoid factor and anti-cyclic citrullinated peptide antibodies¹⁵. Additionally, there is no association with predisposing RA risk alleles, such as HLA class II histocompatibility antigen, DRB1 beta chain (HLA-DRB1)¹⁶. Ironically, symptoms of chronic CHIKV infection fulfil diagnostic criteria for RA¹⁴, which indicates that chronic alphavirus-induced polyarthritis mimics RA-like bone disease.

Despite CHIKV being studied for the past 70 years, the mechanisms behind its long-term rheumatic-like bone sequelae remain largely unknown. Several risk factors have been implicated in chronic CHIKV disease including comorbidities (pre-existing inflammatory joint disease, heart failure and diabetes), older age (35–45 years old or older) and high viraemia during acute CHIKV disease. In CHIKV-infected, aged rhesus macaques compared with adult macaques, a low level of plasma interferon (IFN) is associated with higher and persistent viral replication. This suggests that an aging immune system is important for CHIKV persistence and increased disease severity¹⁷. Although persistent CHIKV joint-related symptoms are present across all ages and in both sexes; older individuals, particularly women, with underlying comorbidities such as osteoarthritis tend to have higher risk of developing chronic CHIKV disease¹⁸.

The impact of host response to CHIKV infection on bone pathologies.

Inflammation is associated with the onset of alphaviral polyarthritis, a condition characterized by inflammation of multiple joints. Several pro-inflammatory cytokines and chemokines including C–C motif chemokine ligand 2 (CCL2), interleukin-6 (IL-6), IL-1β, tumour necrosis factor alpha (TNF-α) and IL-17 are shown to be highly elevated in the plasma of CHIKV-infected patients, in both acute and chronic phases of disease¹⁹. Indeed, IL-6 is one of the predominant mediators of CHIKV-induced arthralgia¹⁹ and this cytokine is known to stimulate RANKL (receptor activator of nuclear factor-κB ligand)-dependent osteoclastogenesis, resulting in high bone resorption activity²⁰. In both CHIKV- and RRV-infected patients elevated levels of RANKL were detected in serum and synovial fluid samples. This suggests that alphavirus infection triggered osteoclastogenesis within joint tissues (Fig. 3)^21,22. Furthermore, in vitro infection of OB with alphaviruses such as CHIKV and RRV results in elevated RANKL secretion, activation of osteoclastogenesis and bone resorption^21,22. The direct effect of alphavirus infection on bone tissue has been demonstrated using animal models of CHIKV and RRV infection^21–23 (Box 3), where elevated RANKL expression resulted in severe periarticular bone loss in mouse tibia during peak alphavirus disease. In an RRV mouse model, a disrupted RANKL/OPG (osteoprotegerin) ratio and its associated bone loss was reversed by administering IL-6 specific antibodies, suggesting that bone loss after infection is regulated through IL-6/RANKL-dependent osteoclastogenesis²¹ (Fig. 3). Infiltration of mononuclear cells, such as monocytes and macrophages, seem critical for infection-induced bone disease^24,25. Human monocytes are susceptible to CHIKV infection²⁶ and non-human primate macrophages serve as a reservoir for persistent CHIKV infection²⁷. Recruitment of monocytes to the site of alphavirus infection is mediated by chemotactic cytokines such as CCL2, which is also a potent osteoclastogenic factor in bone²⁸. Inhibition of CCL2 with the anti-inflammatory small molecule bindarit significantly reduced OC activation in joint tissue of CHIKV-infected mice, and thus ameliorated CHIKV-induced osteoclastogenesis²².

Fig. 3 |. Inflammatory bone loss induced by arthritogenic alphaviruses.

The activation of the NLRP3 inflammasome significantly contributes to inflammation induced by alphaviruses, ultimately disrupting bone remodelling in the context of virus-induced arthritis. The activation of the NLRP3 inflammasome promotes the expression of IL-1β, IL-18, IL-6, MCP-1/CCL2 and IFN-γ, further intensify the inflammatory response. The resulting inflammation creates a pro-inflammatory signalling loop that significantly contributes to the pathophysiology of bone resorption. OBs infected with alphaviruses produce elevated levels of RANKL, while showing decreased OPG expression. This disruption in the RANKL/OPG ratio within the synovial fluid plays a pivotal role in the maturation and activation of OCs, which are central to virus-induced bone resorption through RANKL-mediated osteoclastogenesis. Furthermore, alphavirus-infected OBs produce elevated levels of IL-1β, IL-6, TNF, IFN-γ, and MCP-1/CCL2, which promote the recruitment of OC precursors to the infection site. This cascade of events leads to heightened OC activity and subsequent bone loss.

BOX 3 Experimental animal models for virus-induced bone pathologies.

CHIKV arthritis models

The primary animal models used to study the pathogenesis of CHIKV are mice and nonhuman primates (NHPs). Immunocompetent WT adolescent C57BL/6 (3–4 weeks old²²) or adult C57BL/6 (>6 weeks old²⁴) mice have been used to recapitulate CHIKV arthritis, myositis and bone pathology. Being the natural host of CHIKV sylvatic cycle, cynomolgus macaques exhibit typical acute CHIKV disease, followed by macrophage activation and virus persistence in synovial and musculoskeletal tissues for up to 44 days after infection²⁷. CHIKV-infected, aged rhesus macaques are associated with increased and persistent virus replication along with lower levels of plasma IFN when compared to adult macaques¹⁷. Hence, NHPs provide a more comprehensive animal model for studying CHIKV musculoskeletal disease due to their closer physiological resemblance to humans in immune system function, joint structure and bone remodelling^27,130.

DENV disease models

Several mouse models have been established to study DENV pathogenesis, including immunocompetent WT, immunocompromised AG129 mice (IFN-α/β-receptor and IFN-γ-receptor double knockout), humanized and transgenic mice¹³¹. Immunocompetent WT mice generally exhibit resistance to infection and develop mild disease or neuropathogenesis. By contrast, immunocompromised AG129 mice are highly susceptible to DENV infection and are commonly used to evaluate antivirus therapies, vaccine effectiveness and pathogenesis. Additionally, STAT1-deficient mice have been utilized to demonstrate osteolytic activity and inflammation in bone tissue upon DENV infection⁴⁴. Humanized DENV mouse models are ideal for understanding human immune responses towards infection but do not recapitulate severe DENV manifestations¹³¹. NHPs such as rhesus macaques, cynomolgus macaques and marmosets are susceptible to DENV infection, develop viraemia and display an antibody response together with clinical signs recapitulating DENV disease manifestations in humans¹³².

SARS-CoV-2 bone-loss models

To date, several COVID-19 animal models have been utilized to investigate SARS-CoV-2-associated skeletal complications. Within two weeks of infection, 19-week-old K18 human ACE2 (hACE2) transgenic mice infected with SARS-CoV-2 exhibits bone resorption and bone loss¹³³. Other COVID-19 animal models include naturally infected, non-transgenic ferrets, hamsters and NHPs that exhibit mild-to-moderate clinical disease¹³⁴. SARS-CoV-2 infection of golden Syrian hamsters displayed extrapulmonary manifestations and inflammatory bone loss even up to 60 days post-infection⁶³. The NHP macaque model has been utilized to evaluate the severity of disease and age-related pathogenesis due to the virus’s pervasiveness in several tissue types, serving as an ideal model for studying long-term complications of post-acute COVID-19 (reviewed previously¹³⁵).

ZIKV brain calcification models

Immunocompetent human STAT2 knock-in (hSTAT2 KI) pregnant dam on C57BL/6 background is susceptible to ZIKV and exhibits prenatal infection with vertical transmission across the placenta, infecting the fetal brain¹³⁶. Pups born to ZIKV-infected pregnant hSTAT2 KI mice display abnormal intracranial calcification, recapitulating human fetal brain calcifications⁷⁴. Dystrophic subcortical brain calcifications and behavioural deficits were observed at 12 days post-infection in Swiss mice infected with ZIKV at postnatal day three¹³⁷. NHP ZIKV prenatal infection models including rhesus macaques, pigtail macaques and squirrel monkeys develop brain calcification in surviving infants¹³⁸. In particular, rhesus macaques infected with ZIKV early in pregnancy exhibit brain calcifications and neuropathology at birth¹³⁹.

CVB disease models

Several CVB-susceptible infection models were established to mimic human infection specifically focusing on virus myocardial injury, including adolescent (3-week-old) and adult (6–7-week-old) BALB/c, A/J or C57BL/6 mice¹⁴⁰. Following acute myocarditis, CVB3-infected mice developed multi-organ virus tropism (for example, lung and pancreas) along with cardiac fibrosis and ectopic calcification⁹⁴. Acute myocarditis and high mortality are observed in BALB/c, A/J or C57BL/6 mice infected with in-vitro-passaged CVB. However, use of heart-passaged CVB in BALB/c and A/J mice resulted in chronic myocarditis and fibrosis¹⁴¹. Although NHPs have not been widely used CVB animal models, rhesus macaques have proven valuable for studying CVB-induced myocarditis and vaccines¹⁴².

CHIKV infection can trigger NLRP3 (nucleotide binding oligomerization domain (NOD)-like receptor pyrin domain containing 3) inflammasome-dependent bone loss via IL-1β activation (Fig. 3). Blocking NLRP3 activation with the small-molecule NLRP3 inhibitor MCC950²³, or IL-1 activity with an IL-1 receptor antagonist anakinra in mice, ameliorated osteoclastogenic bone loss and myositis after infection²⁹. More recently, several groups showed the importance of T cell responses and IL-17-producing cells (that is, Th17 CD4 T cells, Tc17 CD8 T cells, and neutrophils) as drivers of an inflammatory response against arthritogenic alphavirus infection³⁰. Overall, these studies indicate that CHIKV infection can induce high levels of IL-1β, IL-6 and CCL2, which trigger an osteoclastogenic bone microenvironment leading to bone defects and pathologies (Fig. 3). However, mechanisms behind chronic CHIKV bone pathologies remain largely unknown and warrant further investigation.

The CHIKV genome consists of ~12 kb open reading frames (ORFs) that encode for five structural (envelope glycoproteins, capsid and 6K viroporin) and four nonstructural (nsP1–4) proteins. Previously, it has been demonstrated that an R532H mutation in nsP1 reduced severe joint inflammation by lowering IL-1β production in infected mice. This suggests activation of the NLRP3–IL-1β inflammasome pathway by nsP1 (ref. 31). However, infection in mice indicated attenuation of the mutant virus compared to infection with wild-type (WT) CHIKV, suggesting that reduced NLRP3 activation could be a result of attenuated viral replication. Thus, it remains unclear how CHIKV proteins can activate the NLRP3 inflammasome leading to osteoclastogenesis.

In physiological bone resorption, high levels of lysosomal papain-like cysteine proteases, such as cathepsin K, are expressed by OCs for cleaving bone collagen into collagen fragments. By-products of this collagen-degradation process, specifically hydroxyproline, were detected at significant levels in the urine of CHIKV-infected patients³², suggesting that bone collagen degradation occurs during alphavirus infection. The C-terminal domain of nsP2 functions as a cysteine protease with a conserved Cys–His catalytic dyad, similar to cathepsins³³. Therefore, it is possible that nsP2 mimics host cathepsins to enhance collagen degradation and bone disease. Alternatively, nsP2 could contribute to post-translational processing of pro-cathepsins into mature, active cathepsins. However, whether CHIKV nsP2 has a role in triggering bone pathologies requires investigation and validation.

Taken together, these studies suggest that CHIKV infection activates host inflammatory signalling and disrupts the balance between bone-forming osteoblasts (OBs) and bone-resorbing OCs, resulting in bone pathologies and arthritis^22,23,29,30. Further elucidation of the molecular mechanisms behind CHIKV and other alphaviruses altering bone metabolism and the resulting long-term persistent arthralgia will be important for the development of future therapies.

Dengue virus

DENV belongs to the Flaviviridae family and is classified into four serotypes (DENV-1 to DENV-4). With approximately 50 to 100 million symptomatic DENV cases reported annually³⁴, it is a major cause of illness worldwide. The outcomes of this disease range from asymptomatic to ‘severe dengue’, which typically occurs upon repeated infection³⁴. The hallmarks of severe dengue are capillary leakage and low blood platelet count (thrombocytopenia), which can lead to organ failure and death³⁵. Although approximately 5% of patients with DENV develop severe illness, 25% develop self-limiting symptomatic illness. This condition is commonly referred to as ‘breakbone fever’, characterized by symptoms such as high fever and excruciating inflammatory muscle, bone and joint pain³⁶. DENV infects people of all ages, but infants born to seropositive mothers, and older individuals tend to incur more severe disease complications. In addition, persistent inflammation and severe joint pain is reported to be more common in women³⁷. To date, the exact pathophysiology of DENV-induced arthralgia and the virus involvement in bone remodelling remains largely unknown.

Mechanisms of DENV-induced arthralgias.

Severe dengue has been associated with an exacerbated host immune response and overwhelming inflammation³⁵. Key inflammatory markers (that is, IL-6, TNF, IL-1β, IL-17 and CCL2) have been shown to be upregulated in peripheral blood mononuclear cells of patients with symptomatic dengue fever³⁸. Infection of a CD16+ monocyte subset with DENV, resulted in production of IL-6, TNF, IL-1β and CCL2 (ref. 39). These factors are relevant during CHIKV infection and OC-mediated bone resorption associated with inflammatory bone diseases⁴⁰. This suggests that DENV infection could lead to a dysregulation of bone homeostasis. In vitro, DENV infection of monocytic cells (THP-1) led to upregulated osteopontin (OPN) levels⁴¹, which is a bone remodelling factor. OPN induces OC differentiation and blocks bone mineral deposition during inflammatory processes⁴². DENV could thus promote RANKL-mediated osteoclastogenesis and osteoclastic activity, which is observed during CHIKV infection.

Inflammatory bone pain is often associated with high osteoclastogenesis due to an acidic microenvironment created by OCs during bone resorption⁴³. DENV-2 infection of human OCs in vitro, resulted in an enhanced NFATc1-mediated OC activation, which also involved C-type lectin member 5A (CLEC5A). NFATc1 is speculated to be a contributor of severe bone pain in patients with dengue⁴⁴. Furthermore, DENV infection in mice resulted in bone inflammation and increased osteoclastic-mediated trabecular bone loss, whereas CLEC5A-deficient mice were protected from osteolytic activity (Box 3). Additionally, DENV envelope protein was shown to interact with CLEC5A and activate the NLRP3 inflammasome, resulting in secretion of IL-1β, IL-18 and TNF-α (ref. 45), which are known players in osteolytic bone diseases and vascular calcification (reviewed previously⁴⁶).

Aside from host-mediated inflammatory responses during DENV arthralgia, viral factors that cause DENV-infection-related pain have not yet been described. Chondroitin sulfate (CS) is a glycosaminoglycan that serves as a major component in the extracellular matrix of joint tissue⁴⁷. DENV non-structural protein 1 (NS1) was shown to interact with heparan sulfate and CS on the cell surface of epithelial cells and mesenchymal stem cell (MSCs)⁴⁸. In patients with acute dengue, secreted NS1 can be detected in the serum⁴⁹ with high levels being associated with severe disease. Binding of NS1 to endothelial cells was shown to induce vascular permeability and leakage⁵⁰. It is possible that secreted NS1 binds to CS that is localized in all joints. DENV NS1 was shown to induce production of inflammatory cytokines through the assembly of NS1 high-density lipoprotein complexes in monocytes and macrophages⁵¹. Hence, localization of NS1 at CS-rich areas in joints could promote proinflammatory cytokine production by monocytes or macrophages, favouring OC differentiation and bone resorption. Additionally, elevated levels of prostaglandins (proinflammatory mediators) were detected in synovial fluids of patients with arthritis⁵², and NS1 treatment of monocytes stimulated production of prostaglandins. Therefore, NS1 could further contribute to the inflammatory process in joints. The role of DENV NS1 in dengue-related arthralgias is an interesting area for exploration and warrants experimental investigations of the hypotheses mentioned above.

Severe acute respiratory syndrome coronavirus 2.

The betacoronavirus SARS-CoV-2 is the causative agent of COVID-19. Although acute COVID-19 can be asymptomatic, symptomatic patients often present non-specific and broad spectrum symptoms such as fever, cough, myalgia, fatigue, acute respiratory distress syndrome or pneumonia⁵³. Some SARS-CoV-2-infected individuals have reported persistent chronic manifestations affecting normal function of the lungs, brain, heart, kidneys, liver and/or musculoskeletal bone joints (reviewed previously⁵⁴). Musculoskeletal sequelae (such as joint pain) is a common symptom, persisting up to two years post-infection⁵⁵. Additionally, patients with SARS-CoV-2 and no prior joint disorder reported morning stiffness and polyarthralgia of the knees, hands or ankles^4,5. However, many symptoms are self-reported and well curated studies of articular manifestations, including details of standardized musculoskeletal examination on a systematic basis, are lacking. Diagnostic imaging from isolated cases have described ankle joint bone erosion resembling rheumatoid nodules in hospitalized patients with COVID-19⁵⁶. Indeed, bone loss has been identified as a prognostic symptom of severe clinical outcomes and future mortality in hospitalized patients with COVID-19⁵⁷.

Sex-related (abundance of viral entry receptors, hormones) and age-related (comorbidities, immune responses) factors may impact pathogenesis of COVID-19. Older individuals and men have an increased risk of developing severe COVID-19, whereas women are at increased risk of long COVID and musculoskeletal pain⁵⁸. Severity of COVID-19 in pregnant women is correlated with an increased level of serum bone morphogenetic proteins (BMP), such as BMP4, in infants from severe cases⁵⁹. Additionally, SARS-CoV-2 infection has been linked to reactive arthritis in children⁶. Adult patients with pre-existing rheumatic diseases also reported increased arthritic ‘flares’ upon SARS-CoV-2 infection, who typically have a higher risk of severe inflammation, long-term complications, hospitalization and mortality.

Mechanisms of SARS-CoV-2-associated arthralgias.

Insights into the cause of COVID-19-associated joint pain are limited. Angiotensin converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) have been identified as entry receptor and factor for SARS-CoV-2⁶⁰. Apart from lung tissue, both ACE2 and TMPRSS2 are expressed in human cortical and trabecular bone, chondrocytes and OB-enriched synovium tissue⁶¹. This suggests that bone may be a potential site for viral replication. However, clinical and experimental findings have clearly demonstrated the absence of detectable SARS-CoV-2 in bone tissues of patients with COVID-19⁶² or in COVID-19 animal models⁶³ (Box 3). Hence, this implies that alternative mechanisms may be involved in driving SARS-CoV-2-associated arthralgias in patients with acute and long COVID-19.

The cytokine storm is a characteristic of COVID-19, driving hyper-inflammation and further aggravating pre-existing respiratory syndromes⁶⁴. Coincidentally, SARS-CoV-2 induced pro-inflammatory cytokines are also known osteoclastogenic immune mediators, including IL-6, IL-17, C-X-C motif chemokine ligand 10 (CXCL10), TNF-α and vascular endothelial growth factor A (VEGF-A)⁶⁵. These cytokines could be contributing to an OC-skewed bone resorption process. Further, SARS-CoV-2 infection has been shown to induce robust inflammasome activation, particularly the activation of NLRP3 inflammasome, which is associated with severity of COVID-19 disease⁶⁶.

Although it is likely that SARS-CoV-2-mediated inflammation may contribute to bone loss⁶⁷, the exact viral mechanism underlying SARS-CoV-2-induced bone loss remains ill-defined. To date, several SARS-CoV-2 proteins have been identified to be directly involved in triggering host inflammatory responses, (1) structural proteins—envelope (E)⁶⁸, spike (S)⁶⁹, nucleocapsid (N)⁷⁰, and (2) accessory proteins—ORF3a (ref. 71), ORF7a and ORF8 (ref. 72). Specifically, E and S proteins have been shown to induce pro-inflammatory cytokines through toll-like receptor (TLR) 2 signalling pathway^68,69. SARS-CoV-2 N and ORF3a proteins induced NLRP3 activation by direct interaction with NLRP3, promoting the assembly of the NLRP3 inflammasome⁷⁰. Another study showed ORF3a serving as a virus ion channel protein, viroporin, facilitating the efflux of potassium ions and mitochondrial reactive oxygen species production, which triggers NLRP3 inflammasome activation⁷¹. Finally, SARS-CoV-2 ORF7a and ORF8 have been shown to bind to IL-17 receptors (IL-17RA and IL17-RC) for induction of IL-17 signalling⁷². Nevertheless, the role of these viral proteins in SARS-CoV-2-associated arthralgias has not been investigated.

Virus-mediated calcifications

Congenital viral infections can have profound and long-lasting effects on a developing fetus, often leading to a spectrum of health complications. Interestingly, they can induce pathogenic calcification, characterized by the abnormal accumulation of calcium phosphate in soft tissues, often linked to cellular injury or death following viral infection⁷³ (Box 1). Calcification observed in the brain shares similarities with physiological processes in bone and cartilage formation⁷⁴. These are driven by osteogenic signalling pathways activated by morphogens from the transforming growth factor-β (TGF-β) family, such as BMPs. Although over 20 BMPs have been identified to date⁷⁵, BMP2, BMP4, BMP6, BMP7 and BMP9 stand out for their strong osteogenic properties. In this section, we delve into recent advancements on host osteogenic activation and the occurrence of pathogenic calcification in response to congenital viral infections. Our discussion encompasses clinical observations and sheds light on the known molecular mechanisms underpinning soft tissue calcifications triggered by these viral infections.

Zika virus

ZIKV is a neurotropic flavivirus mainly spread by an infectious Aedes mosquito bite⁷⁶. Contemporary ZIKV strains, compared with ancestral strains, have acquired mutations that enhance neurovirulence, leading to anomalies within the human fetal brain⁷⁷. Placental ZIKV transmission is associated with infantile complications such as microcephaly (abnormally small head), intracranial calcification (calcium deposits in brain) and arthrogryposis (stiffness in multiple joints), which limits movement. These complications are collectively known as congenital ZIKV syndrome (CZS). Although prenatal ZIKV infection in any trimester may result in CZS, the risk is greatest during the first trimester⁷⁶. Although children born with CZS have a high likelihood of mortality, surviving children may exhibit several structural (craniofacial disproportion), functional (hearing loss, dystonia) and/or neurologic impairments (seizures, epilepsy) up to three years following birth⁷⁸. In 2016, among 400,000 reported ZIKV cases in Brazil, 42% of babies born to ZIKV-positive mothers had congenital craniofacial deformities⁷⁶.

ZIKV-associated calcification of brain regions (such as corticol–subcorticol white matter junctions, basal ganglia and thalamus) is one of the most common clinical findings of ZIKV-infected infants⁷⁹. Typically, brain calcification is associated with aging. But abnormal calcium deposition in the brains of children infected with ZIKV before birth, remains poorly studied. While calcium deposition induced by ZIKV infection is often partially or completely resolved in infants by one year of age⁸⁰, however clearance does not mitigate motor skill disorders (epilepsy andbbc feeding problems). This suggests that calcification causes permanent brain tissue damage and neurological impairment in the developing child⁸¹. Exemplifying this, ZIKV-exposed children in a prospective cohort study in Rio de Janeiro, Brazil displayed severely delayed neurodevelopment⁸².

Mechanisms of ZIKV-induced fetal brain calcification.

Acute ZIKV infection disrupts neurogenesis by killing neural-lineage cells, such as neural progenitor⁸³ and glial cells⁸⁴. Pericytes (specialized cells located around blood vessels) were found to play a role during early stages of ZIKV brain infection⁸⁵ and are thought to be involved in the overall process of ZIKV-induced fetal brain calcification⁷⁴ (Fig. 4). ZIKV-infected human pericytes express high levels of BMP2/4, resulting in osteogenic activation and calcium deposition⁷⁴. BMP2 is also affiliated with osteoblastic differentiation of MSCs in vascular calcification⁸⁶, suggesting that perivascular cells may function as osteogenic precursors in ZIKV brain calcification. Furthermore, ZIKV-infected human pericytes were able to activate master osteogenic regulators (for example, RUNX2 and OSX/SP7), differentiate into osteogenic cells, and activate mineralization genes (for example TNAP, DMP1 and E11/PDPN) that initiated calcium deposition (Fig. 4)⁷⁴. However, it remains unclear how ZIKV infection triggers calcification within the fetal brain on a molecular level.

Fig. 4 |. Mechanisms of ectopic calcification induced by ZIKV.

Intra-uterine exposure to ZIKV culminates with fetal brain calcification, mainly localized at the cortical–subcortical white matter junctions, basal ganglia and thalamus. This process is triggered by ZIKV infection of pericytes, which initiates a process similar to physiological osteogenesis. Upon virus replication, the ZIKV NS3 protease domain cleaves the pro-BMP2/4, generating mature forms of BMP2/4. In turn, BMP2/4 heterodimers transduce signals by binding to type I and II serine/threonine kinase receptors which induce the phosphorylation of SMAD1/5/9 and interaction with SMAD4, forming an osteogenic transcription factor. Its translocation to the nucleus activates the expression of master osteogenic regulators (RUNX2 and OSX/SP7), culminating with the upregulation of osteogenic genes (TNAP, E11/PDPN, DMP1 and BMP2) and brain calcification.

Structural and non-structural viral proteins may influence host cellular responses and subsequent pathogenesis⁸⁷. For example, unbiased virus protein screens performed in human brain pericytes showed that ZIKV NS3 serine protease could effectively process the mature form of BMP2/4, resulting in SMAD1/5/9 phosphorylation and in vitro calcium deposition⁷⁴ (Fig. 4). Interestingly, both African and Asian/American ZIKV strains activate early osteogenic signalling, but cellular calcification is limited to Asian/American ZIKV strains and not seen in ancestral African ZIKV strains. This is likely due to the rapid cell death induced by African ZIKV strains during replication⁸⁸, and this cytopathic effect masks the onset of cell-mediated calcium deposition⁷⁴. Taken together, these findings highlight the importance of perivascular cells in promoting osteogenic differentiation into osteoblastic-like cells and their association in the process of ZIKV-related brain calcification.

Coxsackievirus B

Coxsackie B viruses are a collection of serotypes under the Enterovirus genus known for their ability to cause a broad spectrum of diseases. Outbreaks have been recorded globally and recent retrospective studies attempt to better understand CVB epidemiology⁸⁹. Depending on the virus subtype and the affected tissue, CVB can cause vomiting, diarrhoea, severe pain in chest or abdomen (pleurodynia), as well as inflammation of meninges, brain and pancreas⁹⁰. CVB is considered the predominant cause of cardiomyopathy (up to 25–40% of myocarditis cases) and myocardial calcifications in neonates and young children⁹¹. Irreversible myocardial damage caused after neonatal CVB infection is associated with severe disease and high mortality in young children⁹², however there are currently no specific treatment options available or commercial vaccines for CVB.

Most of our understanding of CVB myocardial calcification and its pathogenesis is based on in vivo animal studies⁹⁰ (Box 3). Cardiac fibroblasts and myocytes are highly susceptible to CVB infection⁹³ resulting in myocardial inflammation, necrotic cell death, fibrosis and dystrophic calcification⁹⁴. Previously, inflammatory infiltrates were shown to be absent from focal calcified lesions during the early viraemic phase. This suggests that cardiac myocyte injury was due to direct myocyte infection and virus-mediated cell death. In the sub-acute phase of infection, extensive CVB replication in heart tissue⁹⁴ triggers migration of activated innate immune cells such as macrophages, neutrophils and natural killer cells, to the infection site. This results in the release of cytokines (for example, TNF, IFN-γ, IL-1β, IL-2)⁹⁵ and a secondary wave of adaptive immune responses aimed at clearing the virus⁹⁶. However, persistent immune responses were reported to exacerbate myocardial damage⁹⁷. Chronic CVB-induced myocarditis results when viral replication has halted, but the viral genome persists in heart tissue. This persistence potentially triggers severe myocardial calcification and dilated cardiomyopathy⁹⁸.

While there is some understanding of the association between CVB infection and myocardial calcification, the specific pathways and factors involved in this process are complex and not fully elucidated. The exact mechanisms by which persisting virus genomes trigger calcification in the heart are not entirely clear and remain a subject of ongoing research. Exploring various factors, including the role of persistent inflammation, immune responses and potential interactions between viral components and host cellular pathways, in driving the calcification process is warranted, as targeted therapies and preventive measures for associated heart conditions are still sought after.

Mechanisms of CVB myocardial calcification.

Although CVB pathogenesis has been well characterized, current research focuses on elucidating both direct and indirect mechanisms of CVB-induced myocardial calcification.

The CVB3 protease 2A was previously shown to cleave dystrophin, a major constituent of muscle structural integrity. Furthermore, dystrophin cleavage by CVB3 protease 2A was shown to exacerbate virus release⁹⁹ and myocyte death¹⁰⁰. Significantly less cardiac calcification was observed in CVB3-infected mice carrying cleavage-resistant dystrophin¹⁰⁰. Similar to ZIKV-induced brain calcification⁷⁴, CVB3 infection in the heart results in an upregulation of osteogenic signalling (for example, BMP2, RUNX2, OSX/SP7) and osteoblast-like differentiation, leading to abnormal calcification¹⁰¹. High serum concentrations of inorganic phosphate, were detected in CVB3-infected mice. Thus, calcification of heart tissue during CVB infection could be caused by phosphate dysregulation⁹⁴.

Although some studies have suggested a connection between CVB infection and myocardial calcification, the exact nature of this relationship is not fully understood, and opposing effects complicate the interpretation. Unexpectedly, tibias of CVB3-infected mice demonstrated severe bone loss mediated through increased RANKL-dependent osteoclastogenesis, similar to bone loss during alphavirus infecition^21,22. Interestingly, blocking the RANKL–RANK interaction in CVB-infected mice ameliorated bone loss and inhibited heart calcification, suggesting that RANKL could contribute to CVB-induced cardiac calcification⁹⁴. These opposing effects during CVB infection and calcification could be explained by immune responses triggered after CVB infection causing acute or chronic inflammation. Also, RANKL–RANK signalling or calcium-regulating parathyroid hormones, could be responses that actively counteract the calcification processes¹⁰².

Although CVB3 protease 2A has been linked to myocardial calcification, open questions on the mechanisms of CVB-associated myocardial calcifications remain. (1) How does CVB infection specifically regulate RANKL–RANK signalling during calcification? Are there viral factors involved and if so, how? (2) What are the host factors leading to calcification related heart conditions after CVB infection? Are there genetic factors determining susceptibility? (3) What is the timeline of RANKL–RANK involvement in CVB-related calcification? Does it occur primarily during the acute phase of infection or persist into chronic stages? (4) Could RANKL–RANK signalling be targeted for therapies against CVB-related calcification and associated heart conditions?

Therapeutic strategies for virus-related bone diseases

Although joint inflammation, bone loss and soft-tissue calcification have been connected to viral infection, disease-specific treatment options are lacking. Repurposing of approved drugs that treat osteoporosis (low bone mineral density and mass) could be a feasible therapeutic approach. Specifically, treatment using antiresorptive bisphosphonates (such as, etidronate, clodronate or alendronate), hormones (like oestrogen or parathyroid hormone with calcitonin) or monoclonal antibodies targeting RANK (denosumab) or sclerostin (romosozumab) have proven effective in ameliorating osteoporosis. Additionally, arthralgias after viral infection may be alleviated with anti-inflammatory drugs (for example, ibuprofen, diclofenac, dipyrone) and anti-rheumatics (for example, methotrexate, hydroxychloroquine)¹⁰³. Site-specific calcium chelation therapy with ethylenediaminetetraacetic acid (EDTA), and targeting RUNX2 activity by small-molecule inhibitors could be used for preventing virus-induced calcification¹⁰⁴. These treatments function largely by immunosuppressive means or general symptomatic relief. However, there is a need to develop treatment options that target virus factors that drive bone diseases. Hence, we explore potential therapeutic targets and drugs against bone loss and calcifications (Table 1) after viral infection, in the following section.

Table 1 |.

Therapeutic strategies for virus bone loss and calcification

Target (virus/protein)	Therapeutic	Category	Mechanisms	Reference
Bone loss
Alphavirus/NSP2	E64d	Peptide-like protease inhibitor	Binding of alphavirus NSP2 SAM MTase and protease domains	106
	Riboflavin	B vitamin (B2)	Inhibitory effect on alphavirus NSP2 protease domain	107
DENV/E	DET4, MLH40	Synthetic peptides	Inhibitory effect on envelope domain III	108
	BP34610	Small molecule	Inhibitor of DENV-1–4 E	108
DENV/NS1	1G5.3	Monoclonal antibody	Neutralization of DENV and ZIKV NS1	109
Calcification
ZIKV/NS3	Niclosamide nitazoxanide	Protease inhibitor	Inhibitory effect on NS3 2B53 pockets (NS2B–NS3 binding site)	110
	Bromocriptine	Protease inhibitor	Inhibitory effect on active residues of the NS2B–NS3 proteolytic cavity	110
	Small molecules NSC10580, ZINC01706300 and NSC45741, ZINC263598830	Enzyme inhibitor	Inhibitory effect on NS3 helicase domain	111
CVB/VP1	Pirodavir	Small molecule	Inhibitory effect on picornavirus capsid	114
CVB/NS2	DENSpm	Polyamine analogue	Mutagenesis of NS2	115

As mentioned previously, CHIKV nsP2 protease can trigger arthritis making it a potential target for therapy¹⁰⁵. The protease inhibitor E64d was shown to bind between the interface the SAM MTase and protease domains of nsP2, and inhibit alphavirus replication in vitro¹⁰⁶. Similarly, riboflavin (vitamin B2) was shown to inhibit CHIKV protease activity and replication in vitro¹⁰⁷.

DENV E protein is needed for virus attachment and fusion, thus it is a promising drug target for treating dengue disease¹⁰⁸. Synthetic peptides such as DET4 and MLH40 target the E protein domain III and inhibit virus entry, as well as replication in vitro. The small molecule BP34610 was identified as an inhibitor of DENV-1–4 E protein, displaying synergistic antivirus activity with ribavirin in vitro¹⁰⁸.

Secreted NS1 during flavivirus infection can trigger proinflammatory signals in human primary macrophages, making NS1 another attractive target⁵¹. A pan-flavivirus NS1-specific antibody termed 1G5.3, blocks NS1 resulting in reduced viraemia while improving survival in DENV murine models¹⁰⁹.

ZIKV NS3 is a major drug target for calcification after infection, because it enhances osteogenic factors BMPs and OPG⁷⁴. In vitro, niclosamide and nitazoxanide were shown to block NS2B–NS3 interaction, which is needed for efficient virus replication¹¹⁰. In addition, the NS3 helicase domain could be targeted with small molecules to block viral replication¹¹¹. Furthermore, ZIKV NS3-based vaccines could be promising, as blocking vertical transmission from mother to child could prevent fetal brain calcification¹¹².

The capsid protein VP1 and non-structural protein NS2A have been shown to contribute to heart calcification after CVB infection¹¹³. The small molecule pirodavir was shown to inhibit replication CVB1, CVB3 and CVB4 by binding to VP1 in vitro¹¹⁴. In vitro and in vivo studies showed that polyamines are essential for virus replication of picornaviruses, including CVB. As such, the polyamine analogue DENSpm can block CVB3 replication by impairing genome packaging and NS2A protease activity¹¹⁵, and thus could prevent myocardial calcification. However, many of these candidates need to be evaluated in pre-clinical settings using established animal models described in Box 3.

Conclusion and future perspectives

Various viral infections have the propensity to induce osteogenic pathways of bone resorption and bone formation, resulting in chronic debilitating bone diseases. Despite the broad range of viruses describe here and the potential socio-economic burden of virus-related bone disease, research focusing on the underlying molecular mechanisms is limited. While arthritis-like symptoms caused by alphaviruses may not be fatal, their impact on quality of life is considerable, often leading to symptoms that persist for years after infection. Furthermore, prenatal CVB infections can lead to heart calcification with subsequent life-threatening cardiac failure during early childhood. In addition, prenatal infections with ZIKV result in fetal brain calcifications following neurodevelopmental delays later in childhood.

Here, we have discussed clinically relevant viruses, including CHIKV, DENV, SARS-CoV-2, ZIKV and CVB, as well as their bone-related diseases and underlying mechanisms. We have presented potential drugs for repurposing that could relieve symptoms or target host immune responses, and proposed drugs that could target the causative virus. It is important to acknowledge that our understanding of SARS-CoV-2 and its potential impact on bone loss remains limited, making it challenging to speculate about potential therapies. Nevertheless, we can leverage knowledge gained from studying the effects of other viruses on bone health as we move forward in our research and exploration of this area.

Going further, congenital pathogens known as TORCH pathogens (Toxoplasma gondii; ‘other’ including syphilis, varicella-zoster virus, parvovirus B19, and human immunodeficiency virus; rubella; cytomegalovirus; herpes simplex virus) frequently cause calcifications in the brain. But the mechanisms remain largely unclear. TORCH pathogens could produce factors increasing BMPs, or brain calcification could be triggered by host-mediated pathways such as inflammatory responses.

Animal models are crucial for identifying cell targets, disease mechanisms involved in virus-induced bone pathology, as well as pre-clinical evaluation of treatments (Box 3). Mice has the advantage of being a comparatively established model, with available tools and numerous genetically modified strains that enable investigation of specific target genes¹¹⁶. Collaborative Cross mice¹¹⁷ could provide an avenue for mimicking divergent host genetics and clinical disease. Although mice would be suited to study acute viral infection, non-human primates would be more fitting for investigating chronic viral infections and disease manifestations, due to a closer physiological resemblance to humans. Combining animal models with technologies such as those described in Box 2, is needed to address questions in this field. Our current understanding is that virus-induced bone pathologies stem from the host’s inflammatory signalling, which disrupts the balance between bone-forming OBs and bone-resorbing OCs. The timing and genetic factors related to virus-induced bone loss are poorly defined for most viruses. Ex vivo bone cultures, organoid or in vivo models that replicate human genetic diversity would be valuable for unravelling host–pathogen interactions in virus-induced bone disease.

In summary, to advance the field of osteovirology and to improve patient outcomes, we propose prioritizing the following research: (1) deciphering virus–host protein interactions, and immune responses in the bone microenvironment to ascertain molecular mechanisms behind virus-associated bone pathologies; (2) understanding the long-term effects of viral infections on bone health, structure and metabolism; (3) determine physiologically relevant animal models of infection (Box 3) to aid understanding of other potential soft tissue sites also affected by osteogenic dysregulation (for example, liver, placenta or lung); (4) utilizing recent advancements in multi-cellular culture model systems and multi-omics technologies to gain molecular insight into virus-mediated bone pathologies (Box 2); and (5) evaluation of pre-existing co-morbidities (for example, autoimmune diseases) in relation to viral bone pathology severity. Overall, the identification of virus determinants and comprehending the underlying mechanisms ultimately responsible for bone diseases will facilitate the development of virus-specific therapeutic interventions.