Binding and molecular basis of the bat coronavirus RaTG13 virus to ACE2 in humans and other species.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading worldwide, causing a global pandemic. Bat-origin RaTG13 is currently the most phylogenetically related virus. Here we obtained the complex structure of the RaTG13 receptor binding domain (RBD) with human ACE2 (hACE2) and evaluated binding of RaTG13 RBD to 24 additional ACE2 orthologs. By substituting residues in the RaTG13 RBD with their counterparts in the SARS-CoV-2 RBD, we found that residue 501, the major position found in variants of concern (VOCs) 501Y.V1/V2/V3, plays a key role in determining the potential host range of RaTG13. We also found that SARS-CoV-2 could induce strong cross-reactive antibodies to RaTG13 and identified a SARS-CoV-2 monoclonal antibody (mAb), CB6, that could cross-neutralize RaTG13 pseudovirus. These results elucidate the receptor binding and host adaption mechanisms of RaTG13 and emphasize the importance of continuous surveillance of coronaviruses (CoVs) carried by animal reservoirs to prevent another spillover of CoVs.

Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2.

The recent emergence of a novel coronavirus (SARS-CoV-2) in China has caused significant public health concerns. Recently, ACE2 was reported as an entry receptor for SARS-CoV-2. In this study, we present the crystal structure of the C-terminal domain of SARS-CoV-2 (SARS-CoV-2-CTD) spike (S) protein in complex with human ACE2 (hACE2), which reveals a hACE2-binding mode similar overall to that observed for SARS-CoV. However, atomic details at the binding interface demonstrate that key residue substitutions in SARS-CoV-2-CTD slightly strengthen the interaction and lead to higher affinity for receptor binding than SARS-RBD. Additionally, a panel of murine monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) against SARS-CoV-S1/receptor-binding domain (RBD) were unable to interact with the SARS-CoV-2 S protein, indicating notable differences in antigenicity between SARS-CoV and SARS-CoV-2. These findings shed light on the viral pathogenesis and provide important structural information regarding development of therapeutic countermeasures against the emerging virus.

Molecular basis of cross-species ACE2 interactions with SARS-CoV-2-like viruses of pangolin origin.

Pangolins have been suggested as potential reservoir of zoonotic viruses, including SARS-CoV-2 causing the global COVID-19 outbreak. Here, we study the binding of two SARS-CoV-2-like viruses isolated from pangolins, GX/P2V/2017 and GD/1/2019, to human angiotensin-converting enzyme 2 (hACE2), the receptor of SARS-CoV-2. We find that the spike protein receptor-binding domain (RBD) of pangolin CoVs binds to hACE2 as efficiently as the SARS-CoV-2 RBD in vitro. Furthermore, incorporation of pangolin CoV RBDs allows entry of pseudotyped VSV particles into hACE2-expressing cells. A screen for binding of pangolin CoV RBDs to ACE2 orthologs from various species suggests a broader host range than that of SARS-CoV-2. Additionally, cryo-EM structures of GX/P2V/2017 and GD/1/2019 RBDs in complex with hACE2 show their molecular binding in modes similar to SARS-CoV-2 RBD. Introducing the Q498H substitution found in pangolin CoVs into the SARS-CoV-2 RBD expands its binding capacity to ACE2 homologs of mouse, rat, and European hedgehog. These findings suggest that these two pangolin CoVs may infect humans, highlighting the necessity of further surveillance of pangolin CoVs.

Molecular Epidemiology of Bovine Enteroviruses and Genome Characterization of Two Novel Bovine Enterovirus Strains in Guangxi, China.

Bovine enterovirus (BEV) is a highly infectious pathogen that may cause respiratory and gastrointestinal disease outbreaks in cattle. This study aimed to investigate the prevalence and genetic characteristics of BEVs in Guangxi Province, China. A total of 1,168 fecal samples from 97 different bovine farms were collected between October 2021 and July 2022 in Guangxi Province, China. BEV was confirmed using a reverse transcription-PCR (RT-PCR) method targeting the 5' untranslated region (UTR), and isolates were genotyped by sequencing their genomes. The nearly complete genome sequences of eight BEV strains showing cytopathic effects in MDBK cells were determined and analyzed. In total, 125 (10.7%) of 1,168 fecal samples were positive for BEV. BEV infection was significantly associated with farming patterns and clinical symptoms (P < 0.05; odds ratio [OR] > 1). Molecular characterization indicated that five BEV strains from this study belonged to EV-E2 and one strain to EV-E4. Two BEV strains (GXNN2204 and GXGL2215) could not be assigned to a known type. Strain GXGL2215 showed the closest genetic relationship with GX1901 (GenBank accession number MN607030; China) in its VP1 (67.5%) and P1 (74.7%) and with NGR2017 (MH719217; Nigeria) in its polyprotein (72.0%). It was also close to the EV-E4 strain GXYL2213 from this study when the complete genome (81.7%) was compared. Strain GXNN2204 showed the closest genetic relationship with Ho12 (LC150008; Japan) in the VP1 (66.5%), P1 (71.6%), and polyprotein (73.2%). Genome sequence analysis suggested that strains GXNN2204 and GXGL2215 originated from the genomic recombination of EV-E4 and EV-F3 and EV-E2 and EV-E4, respectively. This study reports the cocirculation of multiple BEV types and the identification of two novel BEV strains in Guangxi, China, and it will provide further insights into the epidemiology and evolution of BEV in China. IMPORTANCE Bovine enterovirus (BEV) is a pathogen that causes intestinal, respiratory, and reproductive disease infections in cattle. This study reports on the widespread prevalence and biological characteristics of the different BEV types which currently exist in Guangxi Province, China. It also provides a reference for the study of the prevalence of BEV in China.

Insertion of exogenous genes within the ORF1b coding region of porcine astrovirus.

Porcine astrovirus (PAstV) is a common cause of diarrhea in swine farms. The current understanding of the molecular virology and pathogenesis of PAstV is incomplete, especially due to the limited functional tools available. Here, ten sites in the open reading frame 1b (ORF1b) of the PAstV genome were determined to tolerate random 15 nt insertions based on the infectious full-length cDNA clones of PAstV using transposon-based insertion-mediated mutagenesis of three selected regions of the PAstV genome. Insertion of the commonly used Flag tag into seven of the ten insertion sites allowed the production of infectious viruses and allowed their recognition by specifically labeled monoclonal antibodies. Indirect immunofluorescence showed that the Flag-tagged ORF1b protein partially overlapped with the coat protein within the cytoplasm. An improved light-oxygen-voltage (iLOV) gene was also introduced into these seven sites, and only one viable recombinant virus that expressed the iLOV reporter gene at the B2 site was recovered. Biological analysis of the reporter viruses showed that these exhibited similar growth characteristics to the parental virus, but they produced fewer infectious virus particles and replicated at a slower rate. The recombinant viruses containing iLOV fused to ORF1b protein, which maintained their stability and displayed green fluorescence for up to three generations after passaging in cell culture. The porcine astroviruses (PAstVs) expressing iLOV were then used to assess the in vitro antiviral activities of mefloquine hydrochloride and ribavirin. Altogether, the recombinant PAstVs expressing iLOV can be used as a reporter virus tool for the screening of anti-PAstV drugs as well as the investigation of PAstV replication and the functional activities of proteins in living cells.

Isolation and Phylogenetic Analysis of a Hunnivirus Strain in Water Buffaloes From China.

In recent years, hunniviruses have been reported in a variety of animal species from many countries. Here, hunnivirus was detected in fecal samples from water buffaloes and named as BufHuV-GX-2106. The samples were inoculated into cultures of MDBK cells supplemented with TPCK trypsin and the BufHuV-GX-2106 strain was stably passaged and replicated. Electron microscopic analysis showed the BufHuV-GX-2106 virus particles were spherical and 20~30 nm in diameter. The complete genome of a plaque purified sample of BufHuV-GX-2106 was determined and analyzed. Genomic analysis revealed that the whole sequence of BufHuV-GX-2106 was ~7,601 nucleotides (nt) in length and consisted of a large open reading frame of 6,759nt, a 5'UTR, a 3'UTR and a poly(A) tail. The complete genome sequence of BufHuV-GX-2106 shares 68-85% nucleotide identities with other known hunnivirus strains, indicating high genetic heterogeneity among these viruses. Phylogenetic analysis showed that BufHuV-GX-2106 belonged to the Hunnivirus A species and was more closely related to ovine hunnivirus than other known viruses of this type. This study describes the first isolation and complete genome sequence of a hunnivirus strain from water buffaloes. In addition, this study will help to understand the mechanisms involved in the pathogenesis of Hunnivirus A among different animal species.

Molecular insights into receptor binding of recent emerging SARS-CoV-2 variants.

Multiple SARS-CoV-2 variants of concern (VOCs) have been emerging and some have been linked to an increase in case numbers globally. However, there is yet a lack of understanding of the molecular basis for the interactions between the human ACE2 (hACE2) receptor and these VOCs. Here we examined several VOCs including Alpha, Beta, and Gamma, and demonstrate that five variants receptor-binding domain (RBD) increased binding affinity for hACE2, and four variants pseudoviruses increased entry into susceptible cells. Crystal structures of hACE2-RBD complexes help identify the key residues facilitating changes in hACE2 binding affinity. Additionally, soluble hACE2 protein efficiently prevent most of the variants pseudoviruses. Our findings provide important molecular information and may help the development of novel therapeutic and prophylactic agents targeting these emerging mutants.

Broad host range of SARS-CoV-2 and the molecular basis for SARS-CoV-2 binding to cat ACE2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the recent pandemic COVID-19, is reported to have originated from bats, with its intermediate host unknown to date. Here, we screened 26 animal counterparts of the human ACE2 (hACE2), the receptor for SARS-CoV-2 and SARS-CoV, and found that the ACE2s from various species, including pets, domestic animals and multiple wild animals, could bind to SARS-CoV-2 receptor binding domain (RBD) and facilitate the transduction of SARS-CoV-2 pseudovirus. Comparing to SARS-CoV-2, SARS-CoV seems to have a slightly wider range in choosing its receptor. We further resolved the cryo-electron microscopy (cryo-EM) structure of the cat ACE2 (cACE2) in complex with the SARS-CoV-2 RBD at a resolution of 3 Å, revealing similar binding mode as hACE2 to the SARS-CoV-2 RBD. These results shed light on pursuing the intermediate host of SARS-CoV-2 and highlight the necessity of monitoring susceptible hosts to prevent further outbreaks.