Isolation and characterization of a subtype G2c variant of porcine epidemic diarrhea virus that adapts well to cell culture.

Porcine epidemic diarrhea virus (PEDV) causes the third most important disease in the pig industry, after African swine fever and porcine reproductive and respiratory syndrome, and leads to illness or death of the entire litter, causing significant economic losses. In this study, three PEDV strains (HN-1, HN-2, and SC2023) were isolated from swine farms with suspected PEDV infections in Sichuan and Henan provinces. Phylogenetic analysis based on complete S gene sequences showed that all three strains belonged to the G2c subgroup. HN-1 adapted readily to cell culture, grew to a viral titer as high as 2 × 108 TCID50/mL in Vero cells, and caused the formation of large syncytia. We analyzed the amino acid sequence of the HN-1 isolate and found that its S1 subunit contained a three-amino-acid insertion (355KRL358). A seven-amino-acid-deletion (1377FEKVHVQ1383) in the S2 subunit resulted in the partial deletion of the endocytosis signal YxxΦ and the complete deletion of the endoplasmic reticulum retrieval signal (ERRS) KVHVQ in the cytoplasmic tail of the S protein. Consequently, HN-1 is predicted to be less pathogenic than its parent strain, an attribute that facilitates rapid cell-to-cell spread by enhancing syncytium formation. In addition, strain HN-1 was found to have the mutation 884-885SG→RR, which may favor adaptation to cell culture by providing new trypsin cleavage sites. These results suggest that HN-1 is a G2c subtype variant that adapts well to cell culture and can be used to study the adaptive mechanisms of PEDV and develop attenuated vaccines.

NS2 induces an influenza A RNA polymerase hexamer and acts as a transcription to replication switch.

Genome transcription and replication of influenza A virus (FluA), catalyzed by viral RNA polymerase (FluAPol), are delicately controlled across the virus life cycle. A switch from transcription to replication occurring at later stage of an infection is critical for progeny virion production and viral non-structural protein NS2 has been implicated in regulating the switch. However, the underlying regulatory mechanisms and the structure of NS2 remained elusive for years. Here, we determine the cryo-EM structure of the FluAPol-NS2 complex at ~3.0 Å resolution. Surprisingly, three domain-swapped NS2 dimers arrange three symmetrical FluPol dimers into a highly ordered barrel-like hexamer. Further structural and functional analyses demonstrate that NS2 binding not only hampers the interaction between FluAPol and the Pol II CTD because of steric conflicts, but also impairs FluAPol transcriptase activity by stalling it in the replicase conformation. Moreover, this is the first visualization of the full-length NS2 structure. Our findings uncover key molecular mechanisms of the FluA transcription-replication switch and have implications for the development of antivirals.

Development of a novel PCV2 and PCV3 vaccine using virus-like vesicles incorporating Venezuelan equine encephalomyelitis virus-containing vesicular stomatitis virus glycoprotein.

Porcine circovirus disease (PCV) causes substantial economic losses in the pig industry, primarily from porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3). Novel vaccines are necessary to prevent and control PCV infections. PCV coat proteins are crucial for eliciting immunogenic proteins that induce the production of antibodies and immune responses. A vaccine platform utilizing Semliki Forest virus RNA replicons expressing vesicular stomatitis virus glycoprotein (VSV-G), was recently developed. This platform generates virus-like vesicles (VLVs) containing VSV-G exclusively, excluding other viral structural proteins. In our study, we developed a novel virus-like vesicle vaccine by constructing recombinant virus-like vesicles (rVLVs) that also express EGFP. These rVLVs were created using the RNA replicon of Venezuelan equine encephalomyelitis (VEEV) and New Jersey serotype VSV-G. The rVLVs underwent characterization and safety evaluation in vitro. Subsequently, rVLVs expressing PCV2d-Cap and PCV3-Cap proteins were constructed. Immunization of C57 mice with these rVLVs led to a significant increase in anti-porcine circovirus type 2 and type 3 capsid protein antibodies in mouse serum. Additionally, a cellular immune response was induced, as evidenced by high production of IFN-γ and IL-4 cytokines. Overall, this study demonstrates the feasibility of developing a novel porcine circovirus disease vaccine based on rVLVs.

Enhanced potency of an IgM-like nanobody targeting conserved epitope in SARS-CoV-2 spike N-terminal domain.

Almost all the neutralizing antibodies targeting the receptor-binding domain (RBD) of spike (S) protein show weakened or lost efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged or emerging variants, such as Omicron and its sub-variants. This suggests that highly conserved epitopes are crucial for the development of neutralizing antibodies. Here, we present one nanobody, N235, displaying broad neutralization against the SARS-CoV-2 prototype and multiple variants, including the newly emerged Omicron and its sub-variants. Cryo-electron microscopy demonstrates N235 binds a novel, conserved, cryptic epitope in the N-terminal domain (NTD) of the S protein, which interferes with the RBD in the neighboring S protein. The neutralization mechanism interpreted via flow cytometry and Western blot shows that N235 appears to induce the S1 subunit shedding from the trimeric S complex. Furthermore, a nano-IgM construct (MN235), engineered by fusing N235 with the human IgM Fc region, displays prevention via inducing S1 shedding and cross-linking virus particles. Compared to N235, MN235 exhibits varied enhancement in neutralization against pseudotyped and authentic viruses in vitro. The intranasal administration of MN235 in low doses can effectively prevent the infection of Omicron sub-variant BA.1 and XBB in vivo, suggesting that it can be developed as a promising prophylactic antibody to cope with the ongoing and future infection.

Molecular basis of hippopotamus ACE2 binding to SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a wide range of hosts, including hippopotami, which are semi-aquatic mammals and phylogenetically closely related to Cetacea. In this study, we characterized the binding properties of hippopotamus angiotensin-converting enzyme 2 (hiACE2) to the spike (S) protein receptor binding domains (RBDs) of the SARS-CoV-2 prototype (PT) and variants of concern (VOCs). Furthermore, the cryo-electron microscopy (cryo-EM) structure of the SARS-CoV-2 PT S protein complexed with hiACE2 was resolved. Structural and mutational analyses revealed that L30 and F83, which are specific to hiACE2, played a crucial role in the hiACE2/SARS-CoV-2 RBD interaction. In addition, comparative and structural analysis of ACE2 orthologs suggested that the cetaceans may have the potential to be infected by SARS-CoV-2. These results provide crucial molecular insights into the susceptibility of hippopotami to SARS-CoV-2 and suggest the potential risk of SARS-CoV-2 VOCs spillover and the necessity for surveillance. IMPORTANCE: The hippopotami are the first semi-aquatic artiodactyl mammals wherein SARS-CoV-2 infection has been reported. Exploration of the invasion mechanism of SARS-CoV-2 will provide important information for the surveillance of SARS-CoV-2 in hippopotami, as well as other semi-aquatic mammals and cetaceans. Here, we found that hippopotamus ACE2 (hiACE2) could efficiently bind to the RBDs of the SARS-CoV-2 prototype (PT) and variants of concern (VOCs) and facilitate the transduction of SARS-CoV-2 PT and VOCs pseudoviruses into hiACE2-expressing cells. The cryo-EM structure of the SARS-CoV-2 PT S protein complexed with hiACE2 elucidated a few critical residues in the RBD/hiACE2 interface, especially L30 and F83 of hiACE2 which are unique to hiACE2 and contributed to the decreased binding affinity to PT RBD compared to human ACE2. Our work provides insight into cross-species transmission and highlights the necessity for monitoring host jumps and spillover events on SARS-CoV-2 in semi-aquatic/aquatic mammals.

Isolation, identification, and proteomic analysis of outer membrane vesicles of Riemerella anatipestifer SX-1.

Riemerella anatipestifer, belonging to Weeksellaceae family Riemerella, is a bacterium that can infect ducks, geese, and turkeys, causing diseases known as duck infectious serositis, new duck disease, and duck septicemia. We collected diseased materials from ducks on a duck farm in China and then isolated and purified a strain of serotype 1 R. anatipestifer named SX-1. Animal experiments showed that SX-1 is a highly virulent strain with an LD50 value of 101 CFU/mL. The complete genome sequence was obtained. The complete genome sequence of R. anatipestifer SX-1 was 2,112,539 bp; 847 genes were involved in catalytic activity, and 445 genes were related to the cell membrane. The total length of the repetitive sequences was 8746 bp. Four CRISPR loci were predicted in R. anatipestifer strain SX-1, and 4 genomic islands were predicted. Concentration and ultra-high-speed centrifugation were used to extract the outer membrane vesicles of R. anatipestifer SX-1. The OMVs were extracted successfully. Particle size analysis revealed the size and abundance of particles: 147.4 nm, 94.9%; 293.6 nm, 1.1%; 327.2 nm, 1.1%; 397.2 nm, 0.3%; and 371.8 nm, 1.1%. The average size was 173.5 nm. Label-free proteomic technology was used to identify proteins in the outer membrane vesicles. ATCC 11845 served as the reference genome sequence, and 148 proteins were identified using proteomic analysis, which were classified into 5 categories based on their sources. Among them, 24 originated from cytoplasmic proteins, 4 from extracellular secreted proteins, 27 from outer membrane proteins, 10 from periplasmic proteins, and 83 from unknown sources. This study conducted a proteomic analysis of OMVs to provide a theoretical basis for the development of R. anatipestifer OMVs vaccines and adjuvants and lays the foundation for further research on the relationship between the pathogenicity of R. anatipestifer and OMVs.

Structural basis and analysis of hamster ACE2 binding to different SARS-CoV-2 spike RBDs.

Pet golden hamsters were first identified being infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) delta variant of concern (VOC) and transmitted the virus back to humans in Hong Kong in January 2022. Here, we studied the binding of two hamster (golden hamster and Chinese hamster) angiotensin-converting enzyme 2 (ACE2) proteins to the spike protein receptor-binding domains (RBDs) of SARS-CoV-2 prototype and eight variants, including alpha, beta, gamma, delta, and four omicron sub-variants (BA.1, BA.2, BA.3, and BA.4/BA.5). We found that the two hamster ACE2s present slightly lower affinity for the RBDs of all nine SARS-CoV-2 viruses tested than human ACE2 (hACE2). Furthermore, the similar infectivity to host cells expressing hamster ACE2s and hACE2 was confirmed with the nine pseudotyped SARS-CoV-2 viruses. Additionally, we determined two cryo-electron microscopy (EM) complex structures of golden hamster ACE2 (ghACE2)/delta RBD and ghACE2/omicron BA.3 RBD. The residues Q34 and N82, which exist in many rodent ACE2s, are responsible for the lower binding affinity of ghACE2 compared to hACE2. These findings suggest that all SARS-CoV-2 VOCs may infect hamsters, highlighting the necessity of further surveillance of SARS-CoV-2 in these animals.IMPORTANCESARS-CoV-2 can infect many domestic animals, including hamsters. There is an urgent need to understand the binding mechanism of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants to hamster receptors. Herein, we showed that two hamster angiotensin-converting enzyme 2s (ACE2s) (golden hamster ACE2 and Chinese hamster ACE2) can bind to the spike protein receptor-binding domains (RBDs) of SARS-CoV-2 prototype and eight variants and that pseudotyped SARS-CoV-2 viruses can infect hamster ACE2-expressing cells. The binding pattern of golden hamster ACE2 to SARS-CoV-2 RBDs is similar to that of Chinese hamster ACE2. The two hamster ACE2s present slightly lower affinity for the RBDs of all nine SARS-CoV-2 viruses tested than human ACE2. We solved the cryo-electron microscopy (EM) structures of golden hamster ACE2 in complex with delta RBD and omicron BA.3 RBD and found that residues Q34 and N82 are responsible for the lower binding affinity of ghACE2 compared to hACE2. Our work provides valuable information for understanding the cross-species transmission mechanism of SARS-CoV-2.

Characterization of a novel recombinant NADC30­like porcine reproductive and respiratory syndrome virus in Shanxi Province, China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens affecting the swine industry. In this report, a novel PRRSV strain SXht2012 was isolated from Shanxi province in China. To identify genetic characteristics of SXht2012, we conducted phylogenetic and homology analyses after sequencing its complete genome. The results revealed that SXht2012 belonged to NADC30-like strain and shared 91.3% nucleotide (nt) identity with strain NADC30. Notably, sequence alignment showed that a distinctive feature in the NSP2 region, where a 131-amino acid (aa) deletion was found in the hypervariable region (HVR). Additionally, variations were also detected in the GP5 protein, specifically in the decoy peptide, T cell peptide, and a potential glycosylation site (aa 32). Furthermore, we also found that SXht2012 was likely a recombination virus originating from NADC30-like and JXA1-like strains, and three recombination breakpoints were identified in the genome at nt positions 1516, 5280 and 6851, which correspond to the NSP2, NSP3, and NSP7 regions. Overall, these findings have significant implications for understanding the genetic variation and evolutionary dynamics of PRRSV strains.

Genomic Characterization and gE/gI-Deleted Strain Construction of Novel PRV Variants Isolated in Central China.

Pseudorabies virus (PRV) variants have caused substantial economic losses in the swine industry in China since 2011. To surveil the genetic variation in PRV field strains, here, two novel variant strains of PRV were isolated from Shanxi Province in central China and were designated SX1910 and SX1911. To identify the genetic characteristics of the two isolates, their complete genomes were sequenced, and phylogenetic analysis and sequence alignment revealed that field PRV variants have undergone genetic variations; notably, the protein-coding sequences UL5, UL36, US1 and IE180 exhibited extensive variation and contained one or more hypervariable regions. Furthermore, we also found that the glycoproteins gB and gD of the two isolates had some novel amino acid (aa) mutations. Importantly, most of these mutations were located on the surface of the protein molecule, according to protein structure model analysis. We constructed a mutant virus of SX1911 with deletion of the gE and gI genes via CRISPR/Cas9. When tested in mice, SX1911-ΔgE/gI-vaccinated mice were protected within a comparable range to Bartha-K61-vaccinated mice. Additionally, a higher dose of inactivated Bartha-K61 protected the mice from lethal SX1911 challenge, while a lower neutralization titer, higher viral load and more severe microscopic lesions were displayed in Bartha-K61-vaccinated mice. These findings highlight the need for continuous monitoring of PRV and novel vaccine development or vaccination program design for PRV control in China.

RAP44 phage integrase-guided 50K genomic island integration in Riemerella anatipestifer.

Bacteriophages are viruses that infect bacteria. Bacteria and bacteriophages have been fighting for survival. Over time, the evolution of both populations has been affected. Pathogenic Flavobacteriaceae species including Riemerella anatipestifer mainly infects ducklings, geese, and turkeys. However, it does not infect humans, rats, or other mammals, and is a suitable and safe research object in the laboratory. Our previous study showed that there is a 10K genomic island in R. anatipestiferIn this study, we found another integrated 50K genomic islands and focused on the relationship between R. anatipestifer genomic islands and the RAP44 phage genome. The phage RAP44 genome was integrated into R. anatipestifer chromosome, and an evolutionary relationship was evident between them in our comparative analysis. Furthermore, the integrated defective RAP44 phage sequence had the function of integration, excision, and cyclization automatically. Integrases are important integration elements. The integrative function of integrase was verified in R. anatipestifer. The integrase with the attP site can be integrated stably at the attB locus of the R. anatipestifer genome. A recombinant strain can stably inherit and express the exogenous gene. By studying the integration between host bacterium and phage, we have provided evidence for the evolution of the genomes in R. anatipestifer.

Cross-reaction of current available SARS-CoV-2 MAbs against the pangolin-origin coronavirus GX/P2V/2017.

Since the identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, multiple SARS-CoV-2-related viruses have been characterized, including pangolin-origin GD/1/2019 and GX/P2V/2017. Our previous study indicated that both viruses have the potential to infect humans. Here, we find that CB6 (commercial name etesevimab), a COVID-19 therapeutic monoclonal antibody (MAb) developed by our group, efficiently inhibits GD/1/2019 but not GX/P2V/2017. A total of 50 SARS-CoV-2 MAbs divided into seven groups based on their receptor-binding domain (RBD) epitopes, together with the COVID-19 convalescent sera, are systematically screened for their cross-binding and cross-neutralizing properties against GX/P2V/2017. We find that GX/P2V/2017 displays substantial immune difference from SARS-CoV-2. Furthermore, we solve two complex structures of the GX/P2V/2017 RBD with MAbs belonging to RBD-1 and RBD-5, providing a structural basis for their different antigenicity. These results highlight the necessity for broad anti-coronavirus countermeasures and shed light on potential therapeutic targets.

Generation of a live attenuated influenza A vaccine by proteolysis targeting.

The usefulness of live attenuated virus vaccines has been limited by suboptimal immunogenicity, safety concerns or cumbersome manufacturing processes and techniques. Here we describe the generation of a live attenuated influenza A virus vaccine using proteolysis-targeting chimeric (PROTAC) technology to degrade viral proteins via the endogenous ubiquitin-proteasome system of host cells. We engineered the genome of influenza A viruses in stable cell lines engineered for virus production to introduce a conditionally removable proteasome-targeting domain, generating fully infective PROTAC viruses that were live attenuated by the host protein degradation machinery upon infection. In mouse and ferret models, PROTAC viruses were highly attenuated and able to elicit robust and broad humoral, mucosal and cellular immunity against homologous and heterologous virus challenges. PROTAC-mediated attenuation of viruses may be broadly applicable for generating live attenuated vaccines.

Screening of Proliferation-Related Genes and Pathological Changes in Thiram-Induced Tibial Dyschondroplasia.

Materials and Methods: Three hundred sixty (n = 360) broiler chickens were equally divided into control (C) and thiram (T) groups. Furthermore, the C and T groups were dividedinto 8-, 9-, 11-, and 13-day-old chickens. Results: Clinically, it was observed that broiler chickens of group T had abnormal posture, gait, and lameness, and histopathological results revealed dead and abnormal chondrocytes of T group on day 6. Real-time qPCR results showed that HDAC1, MTA1, H4, and PCNA genes were significantly expressed (P < 0.05). HDAC1 was upregulated on days 1, 2, 4, and 6 (P < 0.01); MTA1 was upregulated on days 1 and 2 (P < 0.01); H4 was upregulated on days 2 and 4 (P < 0.01), and PCNA was downregulated on days 1, 2, and 4 (P < 0.01). Furthermore, IHC results of HDAC1 protein were significantly (P < 0.01) expressed in proliferative zone of day 1 and hypertrophic zone of day 6. MTA1 protein was significantly (P < 0.01) expressed on days 1, 2, and 6 in all zones, except prehypertrophic zone of day 2. Conclusion: In conclusion, the mRNA expressions of HDAC1, MTA1, H4, and PCNA were differentially expressed in the chondrocytes of thiram-induced TD chickens. HDAC1 and MTA1 protein expression found involved and responsible in the abnormal chondrocytes' proliferation of broiler chicken.

Transcriptome-based biomarker gene screening and evaluation of the extracellular fatty acid-binding protein (Ex-FABP) on immune and angiogenesis-related genes in chicken erythrocytes of tibial dyschondroplasia.

BACKGROUND: Tibial dyschondroplasia (TD) is a bone disorder in which dead chondrocytes accumulate as a result of apoptosis and non-vascularization in the tibial bone of broiler chickens. The pathogenicity of TD is under extensive research but is yet not fully understood. Several studies have linked it to apoptosis and non-vascularization in the tibial growth plate (GP). We conceived the idea to find the differentially expressed genes (DEGs) in chicken erythrocytes which vary in expression over time using a likelihood-ratio test (LRT). Thiram was used to induce TD in chickens, and then injected Ex-FABP protein at 0, 20, and 50 µg.kg-1 to evaluate its therapeutic effect on 30 screened immunity and angiogenesis-related genes using quantitative PCR (qPCR). The histopathology was also performed in TD chickens to explore the shape, circularity, arrangements of chondrocytes and blood vessels. RESULTS: Clinical lameness was observed in TD chickens, which decreased with the injection of Ex-FABP. Histopathological findings support Ex-FABP as a therapeutic agent for the morphology and vascularization of affected chondrocytes in TD chickens. qPCR results of 10 immunity (TLR2, TLR3, TLR4, TLR5, TLR7, TLR15, IL-7, MyD88, MHCII, and TRAF6) and 20 angiogenesis-related genes (ITGAV, ITGA2, ITGB2, ITGB3, ITGA5, IL1R1, TBXA2R, RPL17, F13A1, CLU, RAC2, RAP1B, GIT1, FYN, IQGAP2, PTCH1, NCOR2, VAV-like, PTPN11, MAML3) regulated when Ex-FABP is injected to TD chickens. CONCLUSION: Immunity and angiogenesis-related genes can be responsible for apoptosis of chondrocytes and vascularization in tibial GP. Injection of Ex-FABP protein to thiram induced TD chickens decrease the chondrocytes damage and improves vascularization.

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.

Janus kinase/signal transducer and activator of transcription signaling pathway-related genes STAT3, SOCS3 and their role in thiram induced tibial dyschondroplasia chickens.

Pathogenicity of tibial dyschondroplasia (TD) in broiler chickens is not detected yet. Janus Kinase/Signal Transducer and Activator of Transcription (JAK-STAT) signaling pathway-related genes were investigated in thiram induced TD chickens. Real-time qPCR and immunohistochemical (IHC) technique were used to observe the expression changes of STAT3 and SOSC3 gene on days 1, 2, 4, 6 after feeding 100 mg·kg-1 thiram. Morphological, pathological, and histological results of this study suggested that chondrocyte cells were observed more damaged on day 6 than day 1, 2, and 4. Therefore, Lameness and damaged chondrocytes gradually increased from day 1 to 6. The mRNA expression level of STAT3 was observed insignificant (P > 0.05) in thiram induced TD chickens' group of day 1. However, on days 2, 4, and 6, the expression was significant (P < 0.05). SOCS3 increased in thiram group on days 1, 2 and 6, decreased on day 4 (P < 0.05). The p-STAT3 and SOCS3 protein's protein localization was evaluated in the control and thiram-induced TD broiler chickens through IHC, suggesting that SOSC3 protein was observed significantly higher on days 1, 2, and 6 and down-regulated on day 4. p-STAT3 protein on thiram induced group was observed significantly upregulated on days 4 and 6. In conclusion, the differential expression of STAT3 and SOCS3 showed that the JAK-STAT signaling pathway might play an important role in regulating an abnormal proliferation, differentiation, or apoptosis of chondrocytes in TD at an early stage.