Emerging Zoonotic Risk of HKU5-CoV-2: ACE2 Adaptation and Public Health Implications

Author: M. Squire, Diploma in Health Science, Student Biomedical Scientist, The Open University (Independent Researcher)11/3/2025 Statement:  "M. Squire the Author, has the right to grant an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in BMJ editions and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence." Completing interest Statement:  M. Squire has completed the Unified Competing Interest form (available on request from the corresponding author) and declare: no support from any organisation for the submitted work, no financial relationships with any organisations that might have an interest in the submitted work in the previous three years], no other relationships or activities that could appear to have influenced the submitted work.

Transparency declaration: I,M. Squire declare as the author, affirms that the manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.Ethical Approval: Ethical approval was not required for this analysis article, as it does not involve new data collection from human participants or identifiable personal information.

Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.Role of Study Sponsors: There were no study sponsors involved in this research.

Statement of Independence: The authors conducted this analysis independently and had no involvement from any external sponsors or funding bodies. The views expressed in this article are solely those of the authors and do not necessarily reflect those of the authors of the original article or any other entity.

Patient and Public Involvement: There was no direct patient or public involvement in this analysis article.

Trial Registration: N/A this is an analysis article. 

Data Sharing Statement: As this article is based on an analysis of previously published data, there are no new datasets to share.

Protocol Submission: As this is an analysis article, there is no formal study protocol.

Checklist Compliance: This article does not adhere to a specific checklist (such as STROBE, CONSORT, or PRISMA) as it is an analysis of previously published work rather than an original study.

Abstract: Current pandemic preparedness strategies focus heavily on sarbecoviruses (e.g., SARS-CoV-2), while merbecoviruses like HKU5-CoV-2 remain critically overlooked. Our analysis highlights the emerging zoonotic threat posed by HKU5-CoV-2, a bat-derived merbecovirus capable of utilizing human angiotensin-converting enzyme 2 (ACE2) for cell entry. Structural analyses reveal adaptations that enhance ACE2 binding, increasing its potential for cross-species transmission. Given the devastating impact of SARS-CoV-2, expanding surveillance efforts to include merbecoviruses is essential to mitigating future pandemic risks. Targeted policy interventions—such as stricter wildlife trade regulations and improved biosafety protocols—are urgently needed to address this evolving threat.

Structural analysis reveals adaptations in the viral spike protein that enhance ACE2 binding, increasing the resemblance to pathogenic human coronaviruses. These findings emphasise the urgent need for enhanced surveillance of bat reservoirs, early risk assessment of emerging coronaviruses, and proactive policy interventions (such as stricter wildlife trade regulations and improved biosafety measures), to mitigate future outbreaks. Given the devastating global impact of SARS-CoV-2, prioritising pandemic preparedness for novel merbecoviruses is critical to preventing another public health crisis.Introduction:Zoonotic coronaviruses have been responsible for major public health crises, including SARS, MERS, and COVID-19. While much attention has been given to sarbecoviruses like SARS-CoV-2, merbecoviruses have remained a less-explored but am equally concerning group. A recent study identified HKU5-CoV-2, a distinct lineage of HKU5-CoV found in bats, that can efficiently use human ACE2 for cellular entry.

As shown in Figure 1, HKU5-CoV-2 exhibits a characteristic coronavirus morphology, with a spherical viral envelope and spike (S) glycoproteins that facilitate receptor binding and host entry. Meanwhile, Figure 2—titled “HKU5-CoV-2: Zoonotic Spillover Potential Geographic Risk Map”—depicts how bat coronaviruses may spill over into humans, across different regions globally. Understanding both the structural features of HKU5-CoV-2 (Figure 1) and the geographic distribution of its potential transmission risk (Figure 2) is crucial for assessing its pandemic threat and guiding proactive surveillance strategies.

Key Findings:

1.    Distinct Lineage and ACE2 Utilisation

What We Found: HKU5-CoV-2 is a novel merbecovirus strain with a unique receptor-binding mode, allowing it to efficiently use human ACE2—unlike previously known HKU5-CoV variants.

Why It Matters: Efficient ACE2 binding increases the potential for direct bat-to-human or bat–intermediate host–human spillover. This highlights the urgency of identifying other merbecoviruses with similar adaptations before they gain further foothold in human populations.

Policy/Strategic Angle:

·       Expand bat coronavirus surveillance in regions where HKU5-like strains are suspected.

·       Develop targeted ACE2-binding assays to screen newly detected bat CoVs for human receptor usage.

2.    Broad Host Tropism

What We Found: HKU5-CoV-2 can bind ACE2 orthologs from multiple mammalian species, This raises concerns about various intermediate hosts, such as civets, pangolins, and minks, facilitating spillover. Figure 3 depicts a conceptual pathway, illustrating how these animals may bridge transmission from bats to humans.

Why It Matters: Multiple species acting as “bridges” expands the ecological niches where HKU5-CoV-2 can adapt, increasing the risk of viral evolution and subsequent infection in humans.

Policy/Strategic Angle:

·       Regulate wildlife trade more strictly, especially for species implicated as potential intermediates.

·       Adopt a One Health approach, integrating veterinary, environmental, and human health surveillance systems to detect early spillover.

3.    Structural Adaptations

What We Found: Cryo-EM analyses reveal that HKU5-CoV-2’s receptor-binding domain (RBD) shares similarities with both ACE2-utilising sarbecoviruses (e.g., SARS-CoV-2) and other merbecoviruses, suggesting convergent evolution.

• The structural comparisons rely on high-resolution cryo-EM and crystallographic data, indicating how the virus may have evolved to latch onto human ACE2 efficiently.

• Why It Matters: Convergent evolution in the RBD region underscores how different coronavirus lineages can independently develop similar receptor usage. This raises the possibility that future merbecoviruses (beyond HKU5-CoV-2) could also adapt to human ACE2.

• Policy/Strategic Angle:

• Support structural biology consortia to maintain a global repository of emerging CoV spike protein structures.

• Encourage international data-sharing so labs worldwide can quickly identify novel RBD adaptations that threaten human health.

4.    Human Cell and Organoid Infection

What We Found: HKU5-CoV-2 infects human ACE2-expressing cell lines and respiratory/enteric organoids, demonstrating its potential for human transmission.

• Why It Matters: Demonstrating actual infection in human organoids is a significant leap from theoretical receptor binding; it shows HKU5-CoV-2 can replicate in human tissues. This increases the pandemic potential if the virus crosses species barriers more widely.

• Policy/Strategic Angle:

• Develop standardized testing protocols using human organoid systems for newly discovered coronaviruses.

• Fund early vaccine and therapeutic development efforts that target merbecovirus spikes, anticipating potential future outbreaks.
  

Public Health Implications

Should HKU5-CoV-2 Be Classified as a Pathogen of Concern?

Although no human infections have been confirmed, HKU5-CoV-2 exhibits virological characteristics associated with past spillover events, meeting key WHO and CDC criteria for emerging zoonotic threats. Its ability to bind human ACE2 suggests potential for cross-species transmission, warranting immediate inclusion in global viral surveillance programs.

Public Health & Policy Recommendations

1. Strengthen Surveillance & Data Sharing

• Expand genomic surveillance to track HKU5-CoV-2 and related merbecoviruses in bat reservoirs and potential intermediate hosts.

• Integrate HKU5-CoV-2 into WHO’s global coronavirus monitoring frameworks, similar to SARS-CoV-2 variant tracking.

• Promote real-time data sharing and collaboration between virologists, epidemiologists, and public health authorities to detect early signs of spillover.

2. Enhance Regulatory & Biosafety Standards

• Reassess the biosafety classification of merbecoviruses and mandate BSL-3 containment for HKU5-CoV-2 research.

• Implement stricter wildlife trade regulations, particularly in regions with documented HKU5-CoV-2 reservoirs.

• Strengthen biosecurity measures in live animal markets to minimize human exposure to zoonotic viruses.

3. Improve Clinical & Research Preparedness

• Develop standardized diagnostic protocols for HKU5-CoV-2 detection in clinical, veterinary, and environmental settings.

• Prioritize pan-coronavirus vaccine development, targeting conserved spike protein regions to mitigate future spillover risks.

• Investigate potential cross-reactivity of existing monoclonal antibodies and antivirals to assess their efficacy against HKU5-CoV-2.
  

Scientific Implications

• Evolutionary Risk: HKU5-CoV-2 exhibits structural divergence from SARS-CoV-2, yet shares key receptor-binding adaptations, highlighting its potential for host expansion.

• Therapeutic Development: Cryo-EM structural analysis provides high-resolution insights into receptor binding, aiding drug and monoclonal antibody design.

• Pandemic Preparedness: Findings emphasize the need for predictive modeling of zoonotic coronaviruses, enhancing global response strategies.
  

By leveraging structural virology and genomic surveillance, this analysis provides critical insights into coronavirus evolution and pandemic risk, reinforcing the urgency of proactive intervention strategies.

Conclusion

The discovery of HKU5-CoV-2 represents a critical development in our understanding of merbecoviruses and their zoonotic potential. While no human infections have been reported, its ability to bind human ACE2 raises concerns about possible spillover events. Structural similarities with known human-pathogenic coronaviruses highlight the urgent need for proactive surveillance of bat reservoirs and potential intermediate hosts to detect early warning signs of cross-species transmission.

The devastating impact of SARS-CoV-2 underscores the necessity of shifting from reactive responses to preventative strategies. Future research must focus on:

·       Expanding genomic surveillance in high-risk spillover regions.

·       Assessing transmissibility and pathogenic potential through functional studies.

·       Advancing broad-spectrum antiviral and vaccine development to mitigate emerging coronavirus threats.

With the lessons of COVID-19 still fresh, global collaboration, early intervention, and robust policy frameworks remain our strongest defence. Strengthening pandemic preparedness is no longer an option—it is a necessity to safeguard global public health.

Conflicts of Interest:

The author(s) declare no conflicts of interest related to this article. References:

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Figures and Legend :

Figure 1 : Conceptual Illustration of the HKU5-CoV-2 Virion

Legend: This figure presents a schematic representation of the HKU5-CoV-2 virion, highlighting its key structural components.

• Spike (S) Protein (Gold Protrusions): These surface glycoproteins facilitate receptor binding and viral entry into host cells by interacting with human angiotensin-converting enzyme 2 (ACE2). The spike protein plays a crucial role in determining host tropism and zoonotic spillover potential.

• Viral Envelope (Teal Spherical Body): The lipid bilayer encapsulating the viral genome and structural proteins, derived from the host cell membrane during viral egress. This envelope houses essential components for viral infectivity and immune evasion.

The structural features of HKU5-CoV-2, particularly its spike glycoprotein, are key determinants of its ability to engage human receptors, raising concerns about its potential for zoonotic transmission. Understanding these structural elements is critical for assessing its pandemic risk and informing surveillance strategies for emerging merbecoviruses.

Figure 2 : HKU5-CoV-2: Zoonotic Spillover Potential Geographic Risk Map

Legend:

• Teal (High Risk): Regions with significant bat-human interfaces, known bat CoV detections, or high wildlife trade volumes, increasing the likelihood of HKU5-CoV-2 spillover.

• Purple (Medium Risk): Areas with minimal bat CoV surveillance findings, limited bat-human contact, or established biosecurity measures that reduce the chance of cross-species transmission.

• Grey (Low Risk): Countries with moderate levels of bat biodiversity or wildlife trade, some evidence of CoV circulation, or partial surveillance data suggesting a possible (but not confirmed) HKU5-CoV-2 presence.

Note:

• This map is illustrative and based on hypothetical or partial data regarding bat populations, wildlife trade routes, and known CoV surveillance. It does not represent definitive epidemiological data.

• Ongoing research and enhanced surveillance may shift a country’s classification as new information about HKU5-CoV-2 emerges.

Rationale for Risk Levels

1. High Risk (Teal colour)

2. Frequent Human-Animal Contact: Regions with intensive wildlife markets or farming (e.g., live animal trade) where bat viruses can jump to intermediate hosts, then to humans.

3. Known Bat Reservoirs: Documented presence of bats carrying HKU5-like coronaviruses or high genetic diversity of bat CoVs.

4. Limited Biosecurity: Weak monitoring or prevention measures that allow cross-species infection events.

5. Medium Risk (Purple)

6. Moderate Evidence: Some bat CoV detections or partial surveillance data, but no confirmed HKU5-CoV-2 lineage in local bat populations.

7. Potential Intermediate Hosts: Civets, pangolins, minks, or camels present, but no definitive proof of HKU5-CoV-2 infection in these species.

8. Developing Surveillance: Efforts exist but may not be comprehensive, leaving some uncertainty about true prevalence.

9. Low Risk (Gry)

·       Minimal Bat-Human Interface: Few known bat roosts near human populations or strong wildlife management policies.

·       Limited Evidence of CoV Circulation: Little to no detection of merbecoviruses in local bat species or intermediate hosts.

·       Established Public Health Measures: Effective screening, biosecurity, and reporting systems that reduce spillover events.

By categorizing countries into high, medium, or low risk, this figure underscores the global variability in HKU5-CoV-2 spillover potential, emphasizing regions where enhanced surveillance and biosecurity might be most urgently needed.

Figure 3 : HKU5-CoV-2 Potential transmission Routes: A Zoonotic pathway from bats to humans from Bats to Humans

Legend: This diagram illustrates the hypothesised transmission route of HKU5-CoV-2, a coronavirus originating from bat reservoirs. The virus may undergo evolutionary changes within bat hosts before potential spillover into intermediate hosts, including civets, pangolins, minks, and camels. These intermittent hosts can facilitate viral adaptation, increasing the likelihood of cross-species transmission. Ultimately, human infection occurs, leading to potential respiratory disease. This pathway highlights the complexity of zoonotic spillover events and the role of multiple host species in viral evolution and emergence.

Figure 4 : Comparative RBD-ACE2 Interaction: HKU5-CoV-2 vs. SARS-CoV-2 Side by side and close-up viewLegend: This figure presents a comparative cryo-electron microscopy (cryo-EM) structural analysis of the receptor-binding domain (RBD) interactions with the angiotensin-converting enzyme 2 (ACE2) receptor for HKU5-CoV-2 and SARS-CoV-2. The visualization highlights key structural differences in how these coronaviruses engage with the human ACE2 receptor, a critical factor in viral entry.

Top Panel: Side-by-Side Cryo-EM Structural Comparison

• Left structure: HKU5-CoV-2 RBD (teal) complexed with ACE2 (gray and gold).

• Right structure: SARS-CoV-2 RBD (purple) complexed with ACE2 (gray and orange).

• The models reveal conformational differences in the RBD-ACE2 binding mode, suggesting variations in binding affinity and host receptor recognition.

• Structural distinctions between HKU5-CoV-2 and SARS-CoV-2 may have implications for cross-species transmission and host adaptation.

Bottom Panel: Close-up Views of the Binding Interface

• Left close-up: HKU5-CoV-2 RBD (teal and gold) interacting with ACE2 (gray), showing the specific binding interface.

• Right close-up: SARS-CoV-2 RBD (purple and orange) interacting with ACE2 (gray), illustrating key interaction sites.

• These high-resolution cryo-EM visualizations emphasize differences in the helical binding motifs, which are crucial for receptor engagement.

• Variations in the RBD-ACE2 interface may affect viral infectivity, immune evasion, and potential therapeutic targeting.