The epitope that monoclonal CR3022 binds to represents a promising target for broad protection against a wide range of human and zoonotic coronaviruses. We develop a powerful model to evaluate antibody affinity maturation in vivo using immunoglobulin (Ig)-humanized mice that express the predicted germline heavy chain of antibody CR3022. Severe acute respiratory syndrome coronavirus (SARS-CoV)/SARS-CoV-2 sequential immunization leads to the convergent evolution of the germline CR3022 through somatic hypermutation (SHM), resembling the affinity-matured CR3022 from a human but now also adapting to key variants and divergent sarbecoviruses. While simple prime-boost strategies drive CR3022-epitope targeting, an intensive vaccination protocol elicits dominant responses to other epitopes. X-ray crystal structures reveal that SARS-CoV-2-neutralizing CR3022-like antibodies exhibit enhanced affinity by increasing polar and electrostatic interactions. Overall, these findings show that CR3022-like clones can be readily adapted through SHM to increase breadth and potency to sarbecoviruses by relatively minor shifts in affinity with appropriate vaccination strategies.

SARS-CoV-2 infection typically resolves within weeks, but rare cases of prolonged replication—sometimes exceeding a year—have been documented, particularly in immunocompromised individuals. These persistent infections pose health risks and may give rise to highly divergent variants, yet the underlying biology remains poorly understood. Here, we describe a model of SARS-CoV-2 persistence using transgenic Syrian hamsters (males) lacking the interleukin-2 receptor gamma subunit (IL2rg). Infection with the XBB.1.16 variant led to efficient viral replication in respiratory tissues by two weeks after infection, with dissemination to other sites, including the intestinal tract. Viral titers remained high in multiple tissues at 100 days after infection. Longitudinal oral swab sequencing revealed dynamic shifts in intrahost single-nucleotide variant (iSNV) frequencies, with constellations of iSNVs rising and falling together, consistent with strong genetic linkage. Synonymous and nonsynonymous mutations accumulated at similar rates, suggesting genetic drift as the dominant evolutionary force. Tissue- and swab-derived sequences revealed extensive within-host diversity and hinted at tissue-specific evolutionary trajectories. This model enables detailed investigation of SARS-CoV-2 persistence and within-host viral evolution and provides a controlled system to study how long-term replication in tissue reservoirs may contribute to viral diversification. SARS-CoV-2 persistence in IL2rg-deficient hamsters shows sustained replication, tissue dissemination, and dynamic intra-host evolution, establishing a model to define mechanisms driving long-term infection and its effects.

Ebola virus (EBOV) is likely a zoonotic and re-emerging virus that causes severe outbreaks of Ebola virus disease. The virus spreads to various tissues during the late stage of infection and has been detected in immune-privileged sites of survivors. However, the mechanism of how EBOV disseminates throughout the body is not completely elucidated. In this study, by using a biologically contained EBOVΔVP30 system, we demonstrate that a megakaryocytic-like MEG-01 cell line that stably expresses VP30 (MEG-01 VP30 cells) is susceptible to EBOVΔVP30 infection and that MEG-01 VP30 cells exposed to EBOVΔVP30 produce platelet-like particles (PLPs) that contain EBOV proteins and viral genetic material. We further found that the viral envelope glycoprotein is expressed on the surface of the produced PLPs and contributes to PLP internalization into recipient cells. In addition, viral mRNA and genome RNA are actively synthesized in these PLPs, which may lead to progeny EBOV production from recipient cells that internalize the PLPs. Taken together, our data provide new insights into the potential role of platelets in the widespread dissemination of EBOV and the pathogenesis of Ebola virus disease.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a global health concern due to a high fatality rate associated with human infections and no approved vaccines or therapeutics. While Syrian hamsters are a value animal model for coronavirus research, including SARS-CoV-2, MERS-CoV does not infect wild-type hamsters. Here, we generated transgenic Syrian hamsters expressing human dipeptidyl peptidase-4 (hDPP4), the cellular receptor for MERS-CoV., MERS-CoV replicated efficiently in the respiratory tract tissues of hDPP4 hamsters, causing lethal disease. Treatment with the 3CLpro inhibitor nirmatrelvir significantly reduced viral titers in the lower respiratory tract of infected hDPP4 hamsters. While airborne transmission was not observed, direct contact transmission was observed in all contact hDPP4 hamsters cohoused with infected cage mates. Immunization with purified MERS receptor-binding domain protein reduced virus replication and disease severity but did not prevent direct contact transmission. Collectively, our findings demonstrate that hDPP4 transgenic Syrian hamsters are useful for studying MERS-CoV pathogenesis, transmission, and countermeasure efficacy.

The outbreak of clade 2.3.4.4b H5N1 viruses among U.S. dairy cattle has raised concerns that sustained circulation among agricultural mammals could facilitate viral adaptation toward efficient human transmission. However, the evolutionary dynamics governing such adaptation remain poorly understood. Here we investigated the evolution of two bovine-derived H5N1 B3.13 genotype viruses during infection and airborne transmission in ferrets, building on prior characterization of their robust replication and inefficient airborne transmission. Within hosts, viral genetic diversity was limited and viruses were subject to genetic drift and weak purifying selection. Transmission, when it occurred, was characterized by stringent bottlenecks that sharply reduced viral genetic diversity. We found no evidence of mammalian adaptation during infection or transmission. Together, these findings indicate that bovine-derived H5N1 viruses face evolutionary constraints during acute mammalian infection and transmission, limiting movement toward enhanced airborne spread. These constraints may help explain why efficient mammalian replication does not necessarily coincide with efficient transmission. Continued circulation of HA clade 2.3.4.4b viruses nevertheless creates repeated opportunities for rare but consequential evolutionary events, underscoring the importance of sustained surveillance and risk mitigation.

The genomic flexibility of orthocoronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is showcased by the presence of accessory genes, which vary in number among virus species and strains. Given this flexibility, the viral coding capacity can be artificially expanded to express a heterologous gene from the viral genome, thereby enabling the development of a viral vector platform. Here, we systematically explored the extra gene coding capacity of SARS-CoV-2 by inserting an extra reporter gene at every intergenic region in its genome. We revealed the entire scheme of its extra gene expression and identified a genomic location that stably expresses reporter genes while maintaining the wild-type viral phenotype. Using this construct, we developed a set of fluorescent and luminescent reporter SARS-CoV-2 viruses available for in vivo flow cytometry and in vitro antiviral screening. Flow cytometric analysis with these reporter viruses revealed cell type-specific dynamics of SARS-CoV-2 infection in the lung tissue of K18-hACE2 mice. Our findings offer a platform for SARS-CoV-2 genome engineering, providing a set of reporter viruses for research applications.

Objectives: Clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) (HPAI H5N1) viruses are widespread globally and have transmitted from birds to dairy cattle at least four times in the United States, including once by a genotype B3.13 virus and three times by genotype D1.1 viruses. Despite their prevalence and known ability to infect humans, only a few studies have examined respiratory droplet transmission capabilities of clade 2.3.4.4b viruses in mammalian models of influenza infection. Here, we aimed to further evaluate this. Methods: We assessed respiratory droplet transmission of two recent human clade 2.3.4.4b HPAI H5N1 viruses—A/Michigan/90/2024 (‘MI90-H5N1’), a B3.13 isolate, and plaque-purified A/British Columbia/PHL2032/2024 (‘BC2032-H5N1’), a D1.1 isolate—in the ferret model. Results: We found that MI90-H5N1, in contrast to earlier findings, causes severe disease and partial lethality in ferrets, with virus spread to extra-respiratory organs and no respiratory droplet transmission. BC2032-H5N1 caused less severe disease with no lethality in ferrets and, consistent with a recent report, failed to transmit via respiratory droplets. Conclusion: Together with other reports, our results suggest that respiratory droplet transmissibility of clade 2.3.4.4b viruses is variable. Therefore, continued monitoring and risk assessment for emerging HPAI H5N1 viruses is essential to better understand their pandemic potential. Funding: This work was supported by the National Institute of Allergy and Infectious Diseases Centers of Excellence for Influenza Research and Response (contract 75N93021C00014) and by grants from the Japan Agency for Medical Research and Development (JP25wm0125002, JP253fa627001, and JP24fk0108626).

Recent discoveries of merbecoviruses such as HKU5, NeoCoV, and HKU5-CoV-2 that use angiotensin-converting enzyme 2 (ACE2) as their entry receptor underscore the potential for future spillover to humans. To combat these ACE2-utilizing merbecoviruses, we developed virus-like particle (VLP) vaccines displaying their receptor-binding domain and subdomain 1 (RBD-SD1) antigens fused to SpyTags. These antigens were then conjugated to SpyCatcher-mi3 nanoparticles. Immunization of mice with each RBD-SD1-mi3 vaccine produced robust IgG responses against homologous antigens and measurable but reduced binding to heterologous RBD-SD1 proteins. Antigenic cartography showed that antigenic relationships broadly reflected sequence identity with HKU5 positioned between NeoCoV and HKU5-CoV-2. Despite HKU5 and HKU5-CoV-2 sharing the greatest genetic similarity, cross-reactive titers between these two groups were not substantially higher than those observed with NeoCoV. In a surrogate virus neutralization test (sVNT), sera from mice vaccinated with NeoCoV RBD-SD1-mi3 or HKU5-CoV-2 RBD-SD1-mi3 showed strong inhibition of receptor binding by their respective Fc-fused RBD-SD1 proteins. Sera from mice vaccinated with HKU5 RBD-SD1-mi3 exhibited some cross-inhibition activity against both NeoCoV and HKU5-CoV-2 Fc-fused RBD-SD1. Together, these findings show that the RBD-SD1-displaying nanoparticle vaccine can elicit cross-reactive antibodies against ACE2-binding merbecoviruses and that antigenic relationships are shaped by more than sequence similarity.

While vaccines against Middle East respiratory syndrome coronavirus (MERS-CoV) have had substantial preclinical and clinical development, few vaccine candidates target other related viruses with potential for human spillover within the Merbecovirus subgenus, like NL140422 and HKU4. We designed nanoparticle vaccines displaying the spike ectodomains of MERS-CoV, NL140422, and HKU4 and evaluated their immunogenicity and protective efficacy in mice. All vaccines elicited high IgG antibody titers against the corresponding vaccine spike protein with moderate cross-binding to mis-matched spike proteins as well. Only the MERS-CoV spike protein induced detectable neutralizing antibodies against MERS-CoV. In human dipeptidyl peptidase 4 (hDPP4) transgenic mice, vaccination with MERS-CoV spike protein completely inhibited MERS-CoV replication in the lungs and nasal turbinates. Vaccination with NL140422 and HKU4 spike proteins provided partial but significant reductions of lung viral loads after MERS-CoV challenge, highlighting their utility as components of a future pan-Merbecovirus vaccine.

The emergence of SARS-CoV-2 in human circulation in 2019 led to a push for universal coronavirus vaccine development. While many vaccine candidates against SARS-like viruses focus on close relatives of SARS-CoV-1 and SARS-CoV-2, more distant viruses in Sarbecovirus clades 2 and 3 have received less attention in vaccine studies. Here, we evaluate in hamsters the immunogenicity of nanoparticle vaccines displaying the spike proteins from clade 2 viruses Yunnan2011, YN2013, and HKU3-8 and from the clade 3 virus BtKY72. We find that our nanoparticle vaccines elicit pan-sarbecovirus reactivity in vaccinated hamster sera, although this binding is clade-biased. Hamster antisera from BtKY72 VLP-S vaccination potently neutralizes authentic SHC014 bat coronavirus, but both clade 2 and clade 3 antisera fail to neutralize authentic WIV1 bat coronavirus or the SARS-CoV-2 XBB variant. All vaccine groups provide limited but significant protection against a challenge with XBB virus.

Ebolavirus disease (EVD) is caused by multiple species of orthoebolavirus. Monoclonal antibodies (mAbs) against the virus glycoprotein (GP) are the only class of therapeutic approved for treatment of EVD caused by Orthoebolavirus zairense (Ebola virus, EBOV). Therefore, mAbs targeting multiple orthoebolavirus species may represent the next generation of EVD therapeutics. Broadly reactive anti-GP mAbs were produced; among these, mAbs 11886 and 11883 were broadly neutralizing in vitro. A 3.0 Å cryo-electron microscopy structure of EBOV GP bound to both mAbs shows that 11886 binds a novel epitope bridging the glycan cap (GC), 310 pocket and GP2 N-terminus, whereas 11883 binds the receptor binding region (RBR) and GC. In vitro, 11886 synergized with a range of mAbs with epitope specificities spanning the RBR/GC, including 11883. Notably, 11886 increased the breadth of neutralization by partner mAbs against different orthoebolavirus species. These data provide a strategic route to design improved mAb-based next-generation EVD therapeutics.

Coronaviruses within the Merbecovirus subgenus, including Middle East respiratory syndrome coronavirus (MERS-CoV) and its dipeptidyl peptidase 4 (DPP4)-using relatives, pose a persistent zoonotic threat. Efforts to prepare for future Merbecovirus spillover events require vaccines that protect beyond a single virus strain. To evaluate antigenic conservation and cross-protective potential, SpyCatcher-mi3 nanoparticles displaying the receptor-binding domain and subdomain 1 (RBD-SD1) from three DPP4-using merbecoviruses, MERS-CoV, NL140422, and HKU4, were generated. Female mice immunized with these nanoparticle vaccines elicited robust IgG antibody endpoint binding titers and cross-reactive antibody responses against the three merbecoviruses. Only the MERS-CoV RBD-SD1 vaccine, however, elicited neutralizing antibodies against MERS-CoV. While vaccination with MERS-CoV RBD-SD1 reduced lung viral titers in MERS-CoV-challenged human DPP4 mice below the limit of detection, no significant reduction in virus titers was seen in NL140422- and HKU4-RBD-SD1-vaccine-immunized mice. These findings indicate that while the RBD-SD1 interface presents conserved antigenic features sufficient for serological cross-recognition, these epitopes may not be functionally immunodominant for cross-neutralization.

Endemic human coronaviruses OC43 and HKU1 cause widespread respiratory infections and can be associated with severe illness in immunocompromised and elderly individuals. Frequent adaptive evolution in the spike proteins of these embecoviruses and the potential for zoonotic transmission from a large animal reservoir necessitates the characterization of the immunogenic landscape of the spike proteins of embecoviruses. Here, we constructed nanoparticle vaccines displaying the spike antigens from OC43, HKU1 A, or HKU1 B, as well as a bivalent formulation incorporating spike antigens from OC43 and HKU1 A. Immunization of mice elicited spike-specific IgG antibody responses, with endpoint titers demonstrating cross-reactivity among clade-matched viruses. Notably, the bivalent formulation elicited antibody responses comparable to those of monovalent vaccines against matched antigens. These findings inform future design of vaccines against human-infecting embecoviruses and could serve as an important step toward a universal vaccine against common cold causing coronaviruses.

To address the need for broadly protective SARS-CoV-2 vaccines, we developed an attenuated a SARS-CoV-2 vaccine virus that lacks the open reading frames of two viral structural proteins: the envelope (E) and membrane (M) proteins. This vaccine virus (ΔEM) replicates in a cell line stably expressing E and M but not in wild-type cells. Vaccination with ΔEM elicits a CD8 T-cell response against the viral spike and nucleocapsid proteins. Two vaccinations with ΔEM provide better protection of the lower respiratory tissues than a single dose against the Delta and Omicron XBB variants in hamsters. Moreover, ΔEM is effective as a booster in hamsters previously vaccinated with an mRNA-based vaccine, providing higher levels of protection in both respiratory tissues compared to the mRNA vaccine booster. Collectively, our data demonstrate the feasibility of a SARS-CoV-2 ΔEM vaccine candidate virus as a vaccine platform.

ABSTRACT In general, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates well at 37°C, which is the temperature of the human lower respiratory tract, but it poorly at 30°C‒32°C, which is the temperature of the human upper respiratory tract. The replication efficiency of SARS-CoV-2 in the upper respiratory tract may directly affect its transmissibility. In this study, an XBB.1.5 isolate showed superior replicative ability at 32°C and 30°C, whereas most other Omicron sub-variant isolates showed limited growth. Deep sequencing analysis demonstrated that the frequencies of viruses possessing the NSP6-S163P and NSP13-P238S substitutions increased to more than 97% during propagation of the XBB.1.5 isolate at 32°C but did not reach 55% at 37°C. Reverse genetics revealed that these substitutions contributed to superior virus growth in vitro at these low temperatures by improving virus genome replication. Mutant virus possessing both substitutions showed slightly higher virus titers in the upper respiratory tract of hamsters compared to the parental virus; however, transmissibility between hamsters was similar for the mutant and parental viruses. Taken together, our findings indicate that NSP6-S163P and NSP13-P238S contribute to superior virus growth at low temperatures in vitro and in the upper respiratory tract of hamsters. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates efficiently at 37°C. However, the temperature of the human upper airway is 30°C–32°C. Therefore, the replicative ability of SARS-CoV-2 at low temperatures could influence virus replication in the upper airway and transmissibility. In this study, we assessed the growth of Omicron sub-variants at low temperatures and found that an XBB.1.5 isolate showed increased replicative ability. By deep sequencing analysis and reverse genetics, we found that amino acid changes in NSP6 and NSP13 contribute to the low-temperature growth; these changes improved RNA polymerase activity at low temperatures and enhanced virus replication in the upper airway of hamsters. Although these substitutions alone did not drastically affect virus transmissibility, in combination with other substitutions, they could affect virus replication in humans. Furthermore, since these substitutions enhance virus replication in cultured cells, they could be used to improve the production of inactivated or live attenuated vaccine virus.

The emergence of SARS-CoV-2 and its subsequent variants in addition to the previous SARS-CoV-1 outbreak indicates the importance of developing broadly protective sarbecovirus vaccines. To date, broadly protective vaccines have primarily focused on clade 1 sarbecoviruses including SARS-CoV-2 variants and SARS-like animal viruses. The discovery of clade 2 and clade 3 sarbecoviruses capable of infecting human cells highlights a need to preemptively develop vaccines that can protect against these viruses. Here, we develop stabilized multivalent subunit vaccines from clade 2 and clade 3 sarbecovirus S2 proteins and evaluate their immunogenicity. Clade 2 and clade 3 S2-subunit vaccines elicit cross-reactive antibodies in mice capable of binding to clade 1, 2, and 3 sarbecovirus antigens. Female mice immunized with these S2-based vaccines also provide protection against sarbecovirus challenges from clades 1a and 1b, including a mouse-adapted SARS-CoV-2 strain, XBB, and WIV1.

Since early 2024, highly pathogenic avian influenza H5N1 viruses have been causing outbreaks in dairy cattle in the United States. Here, we compared the replicative capacity of A/dairy cattle/Texas/24-008749-001/2024 (H5N1; Cow-H5N1) isolated from a dairy cow, A/chicken/Ghana/AVL-76321VIR7050-39/2021 (H5N1; Chicken-H5N1) isolated from a chicken, and a human H1N1 2009 pandemic virus in ex vivo explant cultures of mammary gland and teat from lactating cows. We also examined the expression of influenza virus receptors in these organs. We observed that human influenza virus receptors are widely distributed throughout the epithelium of alveoli, ducts, and gland cisterns within the mammary gland, and in the teat cistern epithelium of dairy cattle, whereas avian influenza virus receptors are distributed on the alveolar, ductal, and teat cistern epithelium. We also found that Cow-H5N1 virus replicates more efficiently than Chicken-H5N1 or human H1N1pdm viruses in the gland cistern epithelium of dairy cattle. Notably, bovine H5N1 viruses replicated efficiently in the epithelium of the bovine teat cistern. These findings suggest that H5N1 viruses invade the mammary gland through the teat canal, which is easily accessed by viruses.