Substantial viral diversity in bats and rodents from East Africa: insights into evolution, recombination, and cocirculation.

BACKGROUND: Zoonotic viruses cause substantial public health and socioeconomic problems worldwide. Understanding how viruses evolve and spread within and among wildlife species is a critical step when aiming for proactive identification of viral threats to prevent future pandemics. Despite the many proposed factors influencing viral diversity, the genomic diversity and structure of viral communities in East Africa are largely unknown. RESULTS: Using 38.3 Tb of metatranscriptomic data obtained via ultradeep sequencing, we screened vertebrate-associated viromes from 844 bats and 250 rodents from Kenya and Uganda collected from the wild. The 251 vertebrate-associated viral genomes of bats (212) and rodents (39) revealed the vast diversity, host-related variability, and high geographic specificity of viruses in East Africa. Among the surveyed viral families, Coronaviridae and Circoviridae showed low host specificity, high conservation of replication-associated proteins, high divergence among viral entry proteins, and frequent recombination. Despite major dispersal limitations, recurrent mutations, cocirculation, and occasional gene flow contribute to the high local diversity of viral genomes. CONCLUSIONS: The present study not only shows the landscape of bat and rodent viromes in this zoonotic hotspot but also reveals genomic signatures driven by the evolution and dispersal of the viral community, laying solid groundwork for future proactive surveillance of emerging zoonotic pathogens in wildlife. Video Abstract.

Deep spatial proteomics reveals region-specific features of severe COVID-19-related pulmonary injury.

As a primary target of severe acute respiratory syndrome coronavirus 2, lung exhibits heterogeneous histopathological changes following infection. However, comprehensive insight into their protein basis with spatial resolution remains deficient, which hinders further understanding of coronavirus disease 2019 (COVID-19)-related pulmonary injury. Here, we generate a region-resolved proteomic atlas of hallmark pathological pulmonary structures by integrating histological examination, laser microdissection, and ultrasensitive proteomics. Over 10,000 proteins are quantified across 71 post-mortem specimens. We identify a spectrum of pathway dysregulations in alveolar epithelium, bronchial epithelium, and blood vessels compared with non-COVID-19 controls, providing evidence for transitional-state pneumocyte hyperplasia. Additionally, our data reveal the region-specific enrichment of functional markers in bronchiole mucus plugs, pulmonary fibrosis, airspace inflammation, and alveolar type 2 cells, uncovering their distinctive features. Furthermore, we detect increased protein expression associated with viral entry and inflammatory response across multiple regions, suggesting potential therapeutic targets. Collectively, this study provides a distinct perspective for deciphering COVID-19-caused pulmonary dysfunction by spatial proteomics.

Isolation of ACE2-dependent and -independent sarbecoviruses from Chinese horseshoe bats.

While the spike proteins from severe acute respiratory syndrome coronaviruses-1 and 2 (SARS-CoV and SARS-CoV-2) bind to host angiotensin-converting enzyme 2 (ACE2) to infect cells, the majority of bat sarbecoviruses cannot use ACE2 from any species. Despite their discovery almost 20 years ago, ACE2-independent sarbecoviruses have never been isolated from field samples, leading to the assumption these viruses pose little risk to humans. We have previously shown how spike proteins from a small group of ACE2-independent bat sarbecoviruses may possess the ability to infect human cells in the presence of exogenous trypsin. Here, we adapted our earlier findings into a virus isolation protocol and recovered two new ACE2-dependent viruses, RsYN2012 and RsYN2016A, as well as an ACE2-independent virus, RsHuB2019A. Although our stocks of RsHuB2019A rapidly acquired a tissue-culture adaption that rendered the spike protein resistant to trypsin, trypsin was still required for viral entry, suggesting limitations on the exogenous entry factors that support bat sarbecoviruses. Electron microscopy revealed that ACE2-independent sarbecoviruses have a prominent spike corona and share similar morphology to other coronaviruses. Our findings demonstrate a broader zoonotic threat posed by sarbecoviruses and shed light on the intricacies of coronavirus isolation and propagation in vitro. IMPORTANCE Several coronaviruses have been transmitted from animals to people, and 20 years of virus discovery studies have uncovered thousands of new coronavirus sequences in nature. Most of the animal-derived sarbecoviruses have never been isolated in culture due to cell incompatibilities and a poor understanding of the in vitro requirements for their propagation. Here, we built on our growing body of work characterizing viral entry mechanisms of bat sarbecoviruses in human cells and have developed a virus isolation protocol that allows for the exploration of these understudied viruses. Our protocol is robust and practical, leading to successful isolation of more sarbecoviruses than previous approaches and from field samples that had been collected over a 10-year longitudinal study.

N-linked glycoproteins and host proteases are involved in swine acute diarrhea syndrome coronavirus entry.

IMPORTANCE: Gaining insight into the cell-entry mechanisms of swine acute diarrhea syndrome coronavirus (SADS-CoV) is critical for investigating potential cross-species infections. Here, we demonstrated that pretreatment of host cells with tunicamycin decreased SADS-CoV attachment efficiency, indicating that N-linked glycosylation of host cells was involved in SADS-CoV entry. Common N-linked sugars Neu5Gc and Neu5Ac did not interact with the SADS-CoV S1 protein, suggesting that these molecules were not involved in SADS-CoV entry. Additionally, various host proteases participated in SADS-CoV entry into diverse cells with different efficiencies. Our findings suggested that SADS-CoV may exploit multiple pathways to enter cells, providing insights into intervention strategies targeting the cell entry of this virus.

Identification of TMEM53 as a novel SADS-CoV restriction factor that targets viral RNA-dependent RNA polymerase.

ABSTRACTZoonotic transmission of coronaviruses (CoVs) poses a serious public health threat. Swine acute diarrhea syndrome coronavirus (SADS-CoV), originating from a bat HKU2-related CoV, causes devastating swine diseases and poses a high risk of spillover to humans. Currently, licensed therapeutics that can prevent potential human outbreaks are unavailable. Identifying the cellular proteins that restrict viral infection is imperative for developing effective interventions and therapeutics. We utilized a large-scale human cDNA screening and identified transmembrane protein 53 (TMEM53) as a novel cell-intrinsic SADS-CoV restriction factor. The inhibitory effect of TMEM53 on SADS-CoV infection was found to be independent of canonical type I interferon responses. Instead, TMEM53 interacts with non-structural protein 12 (NSP12) and disrupts viral RNA-dependent RNA polymerase (RdRp) complex assembly by interrupting NSP8-NSP12 interaction, thus suppressing viral RdRp activity and RNA synthesis. Deleting the transmembrane domain of TMEM53 resulted in the abrogation of TMEM53-NSP12 interaction and TMEM53 antiviral activity. Importantly, TMEM53 exhibited broad antiviral activity against multiple HKU2-related CoVs. Our findings reveal a novel role of TMEM53 in SADS-CoV restriction and pave the way to host-directed therapeutics against HKU2-related CoV infection.

Characterization of a mouse-adapted strain of bat severe acute respiratory syndrome-related coronavirus.

Bats carry genetically diverse severe acute respiratory syndrome-related coronaviruses (SARSr-CoVs). Some of them utilize human angiotensin-converting enzyme 2 (hACE2) as a receptor and cannot efficiently replicate in wild-type mice. Our previous study demonstrated that the bat SARSr-CoV rRsSHC014S induces respiratory infection and lung damage in hACE2 transgenic mice but not wild-type mice. In this study, we generated a mouse-adapted strain of rRsSHC014S, which we named SMA1901, by serial passaging of wild-type virus in BALB/c mice. SMA1901 showed increased infectivity in mouse lungs and induced interstitial lung pneumonia in both young and aged mice after intranasal inoculation. Genome sequencing revealed mutations in not only the spike protein but the whole genome, which may be responsible for the enhanced pathogenicity of SMA1901 in wild-type BALB/c mice. SMA1901 induced age-related mortality similar to that observed in SARS and COVID-19. Drug testing using antibodies and antiviral molecules indicated that this mouse-adapted virus strain can be used to test prophylactic and therapeutic drug candidates against SARSr-CoVs. IMPORTANCE The genetic diversity of SARSr-CoVs in wildlife and their potential risk of cross-species infection highlights the importance of developing a powerful animal model to evaluate the antibodies and antiviral drugs. We acquired the mouse-adapted strain of a bat-origin coronavirus named SMA1901 by natural serial passaging of rRsSHC014S in BALB/c mice. The SMA1901 infection caused interstitial pneumonia and inflammatory immune responses in both young and aged BALB/c mice after intranasal inoculation. Our model exhibited age-related mortality similar to SARS and COVID-19. Therefore, our model will be of high value for investigating the pathogenesis of bat SARSr-CoVs and could serve as a prospective test platform for prophylactic and therapeutic candidates.

Assessment and sero-diagnosis for coronaviruses with risk of human spillover.

Zoonotic coronaviruses (CoVs) caused major human outbreaks in the last two decades. One of the biggest challenges during future CoV disease is ensuring rapid detection and diagnosis at the early phase of a zoonotic event, and active surveillance to the zoonotic high-risk CoVs appears the best way at the present time to provide early warnings. However, there is neither an evaluation of spillover potential nor diagnosis tools for the majority of CoVs. Here, we analyzed the viral traits, including population, genetic diversity, receptor and host species for all 40 alpha- and beta-CoV species, where the human-infecting CoVs are from. Our analysis proposed 20 high-risk CoV species, including 6 of which jumped to human, 3 with evidence of spillover but not to human and 11 without evidence of spillover yet, which prediction were further supported by an analysis of the history of CoV zoonosis. We also found three major zoonotic sources: multiple bat-origin CoV species, the rodent-origin sub-genus Embecovirus and the CoV species AlphaCoV1. Moreover, the Rhinolophidae and Hipposideridae bats harbour a significantly higher proportion of human-threatening CoV species, whereas camel, civet, swine and pangolin could be important intermediate hosts during CoV zoonotic transmission. Finally, we established quick and sensitive serologic tools for a list of proposed high-risk CoVs and validated the methods in serum cross-reaction assays using hyper-immune rabbit sera or clinical samples. By comprehensive risk assessment of the potential human-infecting CoVs, our work provides a theoretical or practical basis for future CoV disease preparedness.

Entangling of Peptide Nanofibers Reduces the Invasiveness of SARS-CoV-2.

The viral spike (S) protein on the surface of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to angiotensin-converting enzyme 2 (ACE2) receptors on the host cells, facilitating its entry and infection. Here, functionalized nanofibers targeting the S protein with peptide sequences of IRQFFKK, WVHFYHK and NSGGSVH, which are screened from a high-throughput one-bead one-compound screening strategy, are designed and prepared. The flexible nanofibers support multiple binding sites and efficiently entangle SARS-CoV-2, forming a nanofibrous network that blocks the interaction between the S protein of SARS-CoV-2 and the ACE2 on host cells, and efficiently reduce the invasiveness of SARS-CoV-2. In summary, nanofibers entangling represents a smart nanomedicine for the prevention of SARS-CoV-2.

A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry.

It is unknown whether pangolins, the most trafficked mammals, play a role in the zoonotic transmission of bat coronaviruses. We report the circulation of a novel MERS-like coronavirus in Malayan pangolins, named Manis javanica HKU4-related coronavirus (MjHKU4r-CoV). Among 86 animals, four tested positive by pan-CoV PCR, and seven tested seropositive (11 and 12.8%). Four nearly identical (99.9%) genome sequences were obtained, and one virus was isolated (MjHKU4r-CoV-1). This virus utilizes human dipeptidyl peptidase-4 (hDPP4) as a receptor and host proteases for cell infection, which is enhanced by a furin cleavage site that is absent in all known bat HKU4r-CoVs. The MjHKU4r-CoV-1 spike shows higher binding affinity for hDPP4, and MjHKU4r-CoV-1 has a wider host range than bat HKU4-CoV. MjHKU4r-CoV-1 is infectious and pathogenic in human airways and intestinal organs and in hDPP4-transgenic mice. Our study highlights the importance of pangolins as reservoir hosts of coronaviruses poised for human disease emergence.

Strategy To Assess Zoonotic Potential Reveals Low Risk Posed by SARS-Related Coronaviruses from Bat and Pangolin.

In the last 2 decades, pathogens originating in animals may have triggered three coronavirus pandemics, including the coronavirus disease 2019 pandemic. Thus, evaluation of the spillover risk of animal severe acute respiratory syndrome (SARS)-related coronavirus (SARSr-CoV) is important in the context of future disease preparedness. However, there is no analytical framework to assess the spillover risk of SARSr-CoVs, which cannot be determined by sequence analysis alone. Here, we established an integrity framework to evaluate the spillover risk of an animal SARSr-CoV by testing how viruses break through key human immune barriers, including viral cell tropism, replication dynamics, interferon signaling, inflammation, and adaptive immune barriers, using human ex vivo lung tissues, human airway and nasal organoids, and human lung cells. Using this framework, we showed that the two pre-emergent animal SARSr-CoVs, bat BtCoV-WIV1 and pangolin PCoV-GX, shared similar cell tropism but exhibited less replicative fitness in the human nasal cavity or airway than did SARS-CoV-2. Furthermore, these viruses triggered fewer proinflammatory responses and less cell death, yet showed interferon antagonist activity and the ability to partially escape adaptive immune barriers to SARS-CoV-2. Collectively, these animal viruses did not fully adapt to spread or cause severe diseases, thus causing successful zoonoses in humans. We believe that this experimental framework provides a path to identifying animal coronaviruses with the potential to cause future zoonoses. IMPORTANCE Evaluation of the zoonotic risk of animal SARSr-CoVs is important for future disease preparedness. However, there are misconceptions regarding the risk of animal viruses. For example, an animal SARSr-CoV could readily infect humans. Alternately, human receptor usage may result in spillover risk. Here, we established an analytical framework to assess the zoonotic risk of SARSr-CoV by testing a series of virus-host interaction profiles. Our data showed that the pre-emergent bat BtCoV-WIV1 and pangolin PCoV-GX were less adapted to humans than SARS-CoV-2 was, suggesting that it may be extremely rare for animal SARSr-CoVs to break all bottlenecks and cause successful zoonoses.

A SARS-CoV-2-Related Virus from Malayan Pangolin Causes Lung Infection without Severe Disease in Human ACE2-Transgenic Mice.

Coronavirus disease 2019 (COVID-19), which is caused by the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the most severe emerging infectious disease in the current century. The discovery of SARS-CoV-2-related coronaviruses (SARSr-CoV-2) in bats and pangolins in South Asian countries indicates that SARS-CoV-2 likely originated from wildlife. To date, two SARSr-CoV-2 strains have been isolated from pangolins seized in Guangxi and Guangdong by the customs agency of China, respectively. However, it remains unclear whether these viruses cause disease in animal models and whether they pose a transmission risk to humans. In this study, we investigated the biological features of a SARSr-CoV-2 strain isolated from a smuggled Malayan pangolin (Manis javanica) captured by the Guangxi customs agency, termed MpCoV-GX, in terms of receptor usage, cell tropism, and pathogenicity in wild-type BALB/c mice, human angiotensin-converting enzyme 2 (ACE2)-transgenic mice, and human ACE2 knock-in mice. We found that MpCoV-GX can utilize ACE2 from humans, pangolins, civets, bats, pigs, and mice for cell entry and infect cell lines derived from humans, monkeys, bats, minks, and pigs. The virus could infect three mouse models but showed limited pathogenicity, with mild peribronchial and perivascular inflammatory cell infiltration observed in lungs. Our results suggest that this SARSr-CoV-2 virus from pangolins has the potential for interspecies infection, but its pathogenicity is mild in mice. Future surveillance among these wildlife hosts of SARSr-CoV-2 is needed to monitor variants that may have higher pathogenicity and higher spillover risk. IMPORTANCE SARS-CoV-2, which likely spilled over from wildlife, is the third highly pathogenic human coronavirus. Being highly transmissible, it is perpetuating a pandemic and continuously posing a severe threat to global public health. Several SARS-CoV-2-related coronaviruses (SARSr-CoV-2) in bats and pangolins have been identified since the SARS-CoV-2 outbreak. It is therefore important to assess their potential of crossing species barriers for better understanding of their risk of future emergence. In this work, we investigated the biological features and pathogenicity of a SARSr-CoV-2 strain isolated from a smuggled Malayan pangolin, named MpCoV-GX. We found that MpCoV-GX can utilize ACE2 from 7 species for cell entry and infect cell lines derived from a variety of mammalian species. MpCoV-GX can infect mice expressing human ACE2 without causing severe disease. These findings suggest the potential of cross-species transmission of MpCoV-GX, and highlight the need of further surveillance of SARSr-CoV-2 in pangolins and other potential animal hosts.

Altered host protease determinants for SARS-CoV-2 Omicron.

Successful severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection requires proteolytic cleavage of the viral spike protein. While the role of the host transmembrane protease serine 2 in SARS-CoV-2 infection is widely recognized, the involvement of other proteases capable of facilitating SARS-CoV-2 entry remains incompletely explored. Here, we show that multiple members from the membrane-type matrix metalloproteinase (MT-MMP) and a disintegrin and metalloproteinase families can mediate SARS-CoV-2 entry. Inhibition of MT-MMPs significantly reduces SARS-CoV-2 replication in vitro and in vivo. Mechanistically, we show that MT-MMPs can cleave SARS-CoV-2 spike and angiotensin-converting enzyme 2 and facilitate spike-mediated fusion. We further demonstrate that Omicron BA.1 has an increased efficiency on MT-MMP usage, while an altered efficiency on transmembrane serine protease usage for virus entry compared with that of ancestral SARS-CoV-2. These results reveal additional protease determinants for SARS-CoV-2 infection and enhance our understanding on the biology of coronavirus entry.

Multiplication of defective Ebola virus in a complementary permissive cell line.

In an effort to develop safe and innovative in vitro models for Ebola virus (EBOV) research, we generated a recombinant Ebola virus where the glycoprotein (GP) gene was substituted with the Cre recombinase (Cre) gene by reverse genetics. This defective virus could multiply itself in a complementary permissive cell line, which could express GP and reporter protein upon exogenous Cre existence. The main features of this novel model for Ebola virus are intact viral life cycle, robust virus multiplication and normal virions morphology. The design of this model ensures its safety, excellent stability and maneuverability as a tool for virology research as well as for antiviral agent screening and drug discovery, and such a design could be further adapted to other viruses.

Functional Peroral Infectivity Complex of White Spot Syndrome Virus of Shrimp.

White spot syndrome virus (WSSV) is a major cause of disease in shrimp cultures worldwide. The infection process of this large circular double-stranded DNA virus has been well studied, but its entry mechanism remains controversial. The major virion envelope protein VP28 has been implicated in oral and systemic viral infection in shrimp. However, genetic analysis of viral DNA has shown the presence of a few genes related to proteins of per os infectivity factor (PIF) complex in baculoviruses. This complex is essential for the entry of baculoviruses, large terrestrial circular DNA viruses, into the midgut epithelial cells of insect larvae. In this study, we aimed to determine whether a PIF complex exists in WSSV, the components of this complex, whether it functions as an oral infectivity complex in shrimp, and the biochemical properties that contribute to its function in a marine environment. The results revealed a WSSV PIF complex (~720 kDa) comprising at least eight proteins, four of which were not identified as PIF homologs: WSV134, VP124 (WSV216), WSSV021, and WSV136. WSV134 is suggested to be a PIF4 homolog due to predicted structural similarity and amino acid sequence identity. The WSSV PIF complex is resistant to alkali, proteolysis, and high salt, properties that are important for maintaining infectivity in aquatic environments. Oral infection can be neutralized by PIF-specific antibodies but not by VP28-specific antibodies. These results indicate that the WSSV PIF complex is critical for WSSV entry into shrimp; the complex's evolutionary significance is also discussed. IMPORTANCE White spot disease, caused by the white spot syndrome virus (WSSV), is a major scourge in cultured shrimp production facilities worldwide. This disease is only effectively controlled by sanitation. Intervention strategies are urgently needed but are limited by a lack of appropriate targets. Our identification of a per os infectivity factor (PIF) complex, which is pivotal for the entry of WSSV into shrimp, could provide new targets for antibody- or dsRNA-based intervention strategies. In addition, the presence of a PIF complex with at least eight components in WSSV, which is ancestrally related to the PIF complex of invertebrate baculoviruses, suggests that this complex is structurally and functionally conserved in disparate virus taxa.

ACE2-Independent Bat Sarbecovirus Entry and Replication in Human and Bat Cells.

Hundreds of sarbecoviruses have been found in bats, but only a fraction of them have the ability to infect cells using angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV and -2. To date, only ACE2-dependent sarbecoviruses have been isolated from field samples or grown in the laboratory. ACE2-independent sarbecoviruses, comprising the majority of the subgenus, have not been propagated in any type of cell culture, as the factors and conditions needed for their replication are completely unknown. Given the significant zoonotic threat posed by sarbecoviruses, cell culture models and in vitro tools are urgently needed to study the rest of this subgenus. We previously showed that the exogenous protease trypsin could facilitate cell entry of viral-like particles pseudotyped with spike protein from some of the ACE2-independent sarbecoviruses. Here, we tested if these conditions were sufficient to support bona fide viral replication using recombinant bat sarbecoviruses. In the presence of trypsin, some of the spike proteins from clade 2 viruses were capable of supporting bat sarbecovirus infection and replication in human and bat cells. Protease experiments showed a specific viral dependence on high levels of trypsin, as TMPRSS2 and furin had no effect on clade 2 virus entry. These results shed light on how sarbecoviruses transmit and coexist in their natural hosts, provide key insights for future efforts to isolate and grow these viruses from field samples, and further underscore the need for broadly protective, universal coronavirus vaccines. IMPORTANCE Our studies demonstrate that some unexplored sarbecoviruses are capable of replicating in human and bat cells in an ACE2-independent way but need a high trypsin environment. We found that trypsin is not compensated by other known proteases involved in some coronavirus entry. This work provides important information that the trypsin-dependent entry may be a widely employed mechanism for coronaviruses and will help for further understanding the biological features of the less-studied viruses.

A triple-RBD-based mucosal vaccine provides broad protection against SARS-CoV-2 variants of concern.

The rapid mutation and spread of SARS-CoV-2 variants urge the development of effective mucosal vaccines to provide broad-spectrum protection against the initial infection and thereby curb the transmission potential. Here, we designed a chimeric triple-RBD immunogen, 3Ro-NC, harboring one Delta RBD and two Omicron RBDs within a novel protein scaffold. 3Ro-NC elicits potent and broad RBD-specific neutralizing immunity against SARS-CoV-2 variants of concern. Notably, intranasal immunization with 3Ro-NC plus the mucosal adjuvant KFD (3Ro-NC + KFDi.n) elicits coordinated mucosal IgA and higher neutralizing antibody specificity (closer antigenic distance) against the Omicron variant. In Omicron-challenged human ACE2 transgenic mice, 3Ro-NC + KFDi.n immunization significantly reduces the tissue pathology in the lung and lowers the viral RNA copy numbers in both the lung (85.7-fold) and the nasal turbinate (13.6-fold). Nasal virologic control is highly correlated with RBD-specific secretory IgA antibodies. Our data show that 3Ro-NC plus KFD is a promising mucosal vaccine candidate for protection against SARS-CoV-2 Omicron infection, pathology and transmission potential.

A strategy to assess spillover risk of bat SARS-related coronaviruses in Southeast Asia.

Emerging diseases caused by coronaviruses of likely bat origin (e.g., SARS, MERS, SADS, COVID-19) have disrupted global health and economies for two decades. Evidence suggests that some bat SARS-related coronaviruses (SARSr-CoVs) could infect people directly, and that their spillover is more frequent than previously recognized. Each zoonotic spillover of a novel virus represents an opportunity for evolutionary adaptation and further spread; therefore, quantifying the extent of this spillover may help target prevention programs. We derive current range distributions for known bat SARSr-CoV hosts and quantify their overlap with human populations. We then use probabilistic risk assessment and data on human-bat contact, human viral seroprevalence, and antibody duration to estimate that a median of 66,280 people (95% CI: 65,351-67,131) are infected with SARSr-CoVs annually in Southeast Asia. These data on the geography and scale of spillover can be used to target surveillance and prevention programs for potential future bat-CoV emergence.

Lethal Swine Acute Diarrhea Syndrome Coronavirus Infection in Suckling Mice.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a recently emerging bat-borne coronavirus responsible for high mortality rates in piglets. In vitro studies have indicated that SADS-CoV has a wide tissue tropism in different hosts, including humans. However, whether this virus potentially threatens other animals remains unclear. Here, we report the experimental infection of wild-type BALB/c and C57BL/6J suckling mice with SADS-CoV. We found that mice less than 7 days old are susceptible to the virus, which caused notable multitissue infections and damage. The mortality rate was the highest in 2-day-old mice and decreased in older mice. Moreover, a preliminary neuroinflammatory response was observed in 7-day-old SADS-CoV-infected mice. Thus, our results indicate that SADS-CoV has potential pathogenicity in young hosts. IMPORTANCE SADS-CoV, which likely has originated from bat coronaviruses, is highly pathogenic to piglets and poses a threat to the swine industry. Little is known about its potential to disseminate to other animals. No efficient treatment is available, and the quarantine strategy is the only preventive measure. In this study, we demonstrated that SADS-CoV can efficiently replicate in suckling mice younger than 7 days. In contrast to infected piglets, in which intestinal tropism is shown, SADS-CoV caused infection and damage in all murine tissues evaluated in this study. In addition, neuroinflammatory responses were detected in some of the infected mice. Our work provides a preliminary cost-effective model for the screening of antiviral drugs against SADS-CoV infection.

The Glycan-Binding Trait of the Sarbecovirus Spike N-Terminal Domain Reveals an Evolutionary Footprint.

The spike protein on sarbecovirus virions contains two external, protruding domains: an N-terminal domain (NTD) with unclear function and a C-terminal domain (CTD) that binds the host receptor, allowing for viral entry and infection. While the CTD is well studied for therapeutic interventions, the role of the NTD is far less well understood for many coronaviruses. Here, we demonstrate that the spike NTD from SARS-CoV-2 and other sarbecoviruses binds to unidentified glycans in vitro similarly to other members of the Coronaviridae family. We also show that these spike NTD (S-NTD) proteins adhere to Calu3 cells, a human lung cell line, although the biological relevance of this is unclear. In contrast to what has been shown for Middle East respiratory syndrome coronavirus (MERS-CoV), which attaches sialic acids during cell entry, sialic acids present on Calu3 cells inhibited sarbecovirus infection. Therefore, while sarbecoviruses can interact with cell surface glycans similarly to other coronaviruses, their reliance on glycans for entry is different from that of other respiratory coronaviruses, suggesting sarbecoviruses and MERS-CoV have adapted to different cell types, tissues, or hosts during their divergent evolution. Our findings provide important clues for further exploring the biological functions of sarbecovirus glycan binding and adds to our growing understanding of the complex forces that shape coronavirus spike evolution. IMPORTANCE Spike N-terminal domains (S-NTD) of sarbecoviruses are highly diverse; however, their function remains largely understudied compared with the receptor-binding domains (RBD). Here, we show that sarbecovirus S-NTD can be phylogenetically clustered into five clades and exhibit various levels of glycan binding in vitro. We also show that, unlike some coronaviruses, including MERS-CoV, sialic acids present on the surface of Calu3, a human lung cell culture, inhibit SARS-CoV-2 and other sarbecoviruses. These results suggest that while glycan binding might be an ancestral trait conserved across different coronavirus families, the functional outcome during infection can vary, reflecting divergent viral evolution. Our results expand our knowledge on the biological functions of the S-NTD across diverse sarbecoviruses and provide insight on the evolutionary history of coronavirus spike.