DYRK1A is a multifunctional host factor that regulates coronavirus replication in a kinase-independent manner.

Coronaviruses (CoVs) pose a major threat to human and animal health worldwide, which complete viral replication by hijacking host factors. Identifying host factors essential for the viral life cycle can deepen our understanding of the mechanisms of virus-host interactions. Based on our previous genome-wide CRISPR screen of α-CoV transmissible gastroenteritis virus (TGEV), we identified the host factor dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), but not DYRK1B, as a critical factor in TGEV replication. Rescue assays and kinase inhibitor experiments revealed that the effect of DYRK1A on viral replication is independent of its kinase activity. Nuclear localization signal modification experiments showed that nuclear DYRK1A facilitated virus replication. Furthermore, DYRK1A knockout significantly downregulated the expression of the TGEV receptor aminopeptidase N (ANPEP) and inhibited viral entry. Notably, we also demonstrated that DYRK1A is essential for the early stage of TGEV replication. Transmission electron microscopy results indicated that DYRK1A contributes to the formation of double-membrane vesicles in a kinase-independent manner. Finally, we validated that DYRK1A is also a proviral factor for mouse hepatitis virus, porcine deltacoronavirus, and porcine sapelovirus. In conclusion, our work demonstrated that DYRK1A is an essential host factor for the replication of multiple viruses, providing new insights into the mechanism of virus-host interactions and facilitating the development of new broad-spectrum antiviral drugs.IMPORTANCECoronaviruses, like other positive-sense RNA viruses, can remodel the host membrane to form double-membrane vesicles (DMVs) as their replication organelles. Currently, host factors involved in DMV formation are not well defined. In this study, we used transmissible gastroenteritis virus (TGEV) as a virus model to investigate the regulatory mechanism of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) on coronavirus. Results showed that DYRK1A significantly inhibited TGEV replication in a kinase-independent manner. DYRK1A knockout (KO) can regulate the expression of receptor aminopeptidase N (ANPEP) and endocytic-related genes to inhibit virus entry. More importantly, our results revealed that DYRK1A KO notably inhibited the formation of DMV to regulate the virus replication. Further data proved that DYRK1A is also essential in the replication of mouse hepatitis virus, porcine deltacoronavirus, and porcine sapelovirus. Taken together, our findings demonstrated that DYRK1A is a conserved factor for positive-sense RNA viruses and provided new insights into its transcriptional regulation activity, revealing its potential as a candidate target for therapeutic design.

Crystal structure of the dimerized of porcine circovirus type II replication-related protein Rep'.

Porcine circovirus type 2 (PCV2) can cause porcine circovirus-associated disease (PCVAD), which causes significant economic losses to the global pig industry annually. There are no effective antiviral drugs used to control and treat PCV2, and prevention is mainly obtained through vaccination. PCV2 genome replicates through the rolling circle replication (RCR) mechanism involving Rep and Rep', so analyzing the holistic structure of Rep and Rep' will help us better understand the replication process of PCV2. However, there are no reports on the integral structure of Rep' and Rep, which seriously hinders the research of the viral replication. By using the x-ray diffraction method, the structure of the Rep' dimer was resolved by us in this study. Structural analysis revealed that Rep' is a dimer formed by the interaction of the C-terminal domain. The two Rep' form a positively charged groove, which may play an essential role in the viral binding of dsDNA. Together, this study help to understand the replication process of the virus and may also provide new insights into the development of antiviral drugs.

Identification of a Novel Neutralizing Epitope on the N-Terminal Domain of the Human Coronavirus 229E Spike Protein.

The receptor binding domain (RBD) of the coronavirus spike protein (S) has been verified to be the main target for potent neutralizing antibodies (nAbs) in most coronaviruses, and the N-terminal domain (NTD) of some betacoronaviruses has also been indicated to induce nAbs. For alphacoronavirus HCoV-229E, its RBD has been shown to have neutralizing epitopes, and these epitopes could change over time. However, whether neutralizing epitopes exist on the NTD and whether these epitopes change like those of the RBD are still unknown. Here, we verified that neutralizing epitopes exist on the NTD of HCoV-229E. Furthermore, we characterized an NTD targeting nAb 5H10, which could neutralize both pseudotyped and authentic HCoV-229E VR740 in vitro. Epitope mapping indicated that 5H10 targeted motif E1 (147-167 aa) and identified F159 as critical for 5H10 binding. More importantly, our results revealed that motif E1 was highly conserved among clinical isolates except for F159. Further data proved that mutations at position 159 gradually appeared over time and could completely abolish the neutralizing ability of 5H10, supporting the notion that position 159 may be under selective pressure during the human epidemic. In addition, we also found that contemporary clinical serum has a stronger binding capacity for the NTD of contemporary strains than historic strains, proving that the epitope on the NTD could change over time. In summary, these findings define a novel neutralizing epitope on the NTD of HCoV-229E S and provide a theoretical basis for the design of vaccines against HCoV-229E or related coronaviruses. IMPORTANCE Characterization of the neutralizing epitope of the spike (S) protein, the major invasion protein of coronaviruses, can help us better understand the evolutionary characteristics of these viruses and promote vaccine development. To date, the neutralizing epitope distribution of alphacoronaviruses is not well known. Here, we identified a neutralizing antibody that targeted the N-terminal domain (NTD) of the alphacoronavirus HCoV-229E S protein. Epitope mapping revealed a novel epitope that was not previously discovered in HCoV-229E. Further studies identified an important residue, F159. Mutations that gradually appeared over time at this site abolished the neutralizing ability of 5H10, indicating that selective pressure occurred at this position in the spread of HCoV-229E. Furthermore, we found that the epitopes within the NTD also changed over time. Taken together, our findings defined a novel neutralizing epitope and highlighted the role of the NTD in the future prevention and control of HCoV-229E or related coronaviruses.

Adaptive Mutation in the Main Protease Cleavage Site of Feline Coronavirus Renders the Virus More Resistant to Main Protease Inhibitors.

The rapid global emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused serious health problems, highlighting the urgent need for antiviral drugs. The viral main protease (Mpro) plays an important role in viral replication and thus remains the target of choice for the prevention or treatment of several viral diseases due to high sequence and structural conservation. Prolonged use of viral protease inhibitors can lead to the development of mutants resistant to those inhibitors and to many of the available antiviral drugs. Here, we used feline infectious peritonitis virus (FIPV) as a model to investigate its development of resistance under pressure from the Mpro inhibitor GC376. Passage of wild-type (WT) FIPV in the presence of GC376 selected for a mutation in the nsp12 region where Mpro cleaves the substrate between nsp12 and nsp13. This mutation confers up to 3-fold resistance to GC376 and nirmatrelvir, as determined by EC50 assay. In vitro biochemical and cellular experiments confirmed that FIPV adapts to the stress of GC376 by mutating the nsp12 and nsp13 hydrolysis site to facilitate cleavage by Mpro and release to mediate replication and transcription. Finally, we demonstrate that GC376 cannot treat FIP-resistant mutants that cause FIP in animals. Taken together, these results suggest that Mpro affects the replication of coronaviruses (CoVs) and the drug resistance to GC376 by regulating the amount of RdRp from a distant site. These findings provide further support for the use of an antiviral drug combination as a broad-spectrum therapy to protect against contemporary and emerging CoVs. IMPORTANCE CoVs cause serious human infections, and antiviral drugs are currently approved to treat these infections. The development of protease-targeting therapeutics for CoV infection is hindered by resistance mutations. Therefore, we should pay attention to its resistance to antiviral drugs. Here, we identified possible mutations that lead to relapse after clinical treatment of FIP. One amino acid substitution in the nsp12 polymerase at the Mpro cleavage site provided low-level resistance to GC376 after selection exposure to the GC376 parental nucleoside. Resistance mutations enhanced FIPV viral fitness in vitro and attenuated the therapeutic effect of GC376 in an animal model of FIPV infection. Our research explains the evolutionary characteristics of coronaviruses under antiviral drugs, which is helpful for a more comprehensive understanding of the molecular basis of virus resistance and provides important basic data for the effective prevention and control of CoVs.

Structural comparisons of host and African swine fever virus dUTPases reveal new clues for inhibitor development.

African swine fever, caused by the African swine fever virus (ASFV), is among the most significant swine diseases. There are currently no effective treatments against ASFV. ASFV contains a gene encoding a dUTPase (E165R), which is required for viral replication in swine macrophages, making it an attractive target for inhibitor development. However, the full structural details of the ASFV dUTPase and those of the comparable swine enzyme are not available, limiting further insights. Herein, we determine the crystal structures of ASFV dUTPase and swine dUTPase in both their ligand-free and ligand-bound forms. We observe that the swine enzyme employs a classical dUTPase architecture made up of three-subunit active sites, whereas the ASFV enzyme employs a novel two-subunit active site. We then performed a comparative analysis of all dUTPase structures uploaded in the Protein Data Bank (PDB), which showed classical and non-classical types were mainly determined by the C-terminal ß-strand orientation, and the difference was mainly related to the four amino acids behind motif IV. Thus, our study not only explains the reason for the structural diversity of dUTPase but also reveals how to predict dUTPase type, which may have implications for the dUTPase family. Finally, we tested two dUTPase inhibitors developed for the Plasmodium falciparum dUTPase against the swine and ASFV enzymes. One of these compounds inhibited the ASFV dUTPase at low micromolar concentrations (Kd = 15.6 µM) and with some selectivity (∼2x) over swine dUTPase. In conclusion, our study expands our understanding of the dUTPase family and may aid in the development of specific ASFV inhibitors.

Insight into vaccine development for Alpha-coronaviruses based on structural and immunological analyses of spike proteins.

Coronaviruses that infect humans belong to the Alpha-coronavirus (including HCoV-229E) and Beta-coronavirus (including SARS-CoV and SARS-CoV-2) genera. In particular, SARS-CoV-2 is currently a major threat to public health worldwide. The spike (S) homotrimers bind to their receptors via the receptor-binding domain (RBD), which is a major target to block viral entry. In this study, we selected Alpha-coronavirus (HCoV-229E) and Beta-coronavirus (SARS-CoV and SARS-CoV-2) as models. Their RBDs exist two different conformational states (lying or standing) in the prefusion S-trimer structure. Then, the differences in the immune responses to RBDs from these coronaviruses were analyzed structurally and immunologically. Our results showed that more RBD-specific antibodies (antibody titers: 1.28×105; 2.75×105) were induced by the S-trimer with the RBD in the "standing" state (SARS-CoV and SARS-CoV-2) than the S-trimer with the RBD in the "lying" state (HCoV-229E, antibody titers: <500), and more S-trimer-specific antibodies were induced by the RBD in the SARS-CoV and SARS-CoV-2 (antibody titers: 6.72×105; 5×105) than HCoV-229E (antibody titers:1.125×103). Besides, we found that the ability of the HCoV-229E RBD to induce neutralizing antibodies was lower than S-trimer, and the intact and stable S1 subunit was essential for producing efficient neutralizing antibodies against HCoV-229E. Importantly, our results reveal different vaccine strategies for coronaviruses, and S-trimer is better than RBD as a target for vaccine development in Alpha-coronavirus Our findings will provide important implications for future development of coronavirus vaccines.Importance Outbreak of coronaviruses, especially SARS-CoV-2, poses a serious threat to global public health. Development of vaccines to prevent the coronaviruses that can infect humans has always been a top priority. Coronavirus spike (S) protein is considered as a major target for vaccine development. Currently, structural studies have shown that Alpha-coronavirus (HCoV-229E) and Beta-coronavirus (SARS-CoV and SARS-CoV-2) RBDs are in "lying" and "standing" states in the prefusion S-trimer structure. Here, we evaluated the ability of S-trimer and RBD to induce neutralizing antibodies among these coronaviruses. Our results showed that the S-trimer and RBD are both candidates for subunit vaccines in Beta-coronavirus (SARS-CoV and SARS-CoV-2) with a RBD "standing" state. However, for Alpha-coronavirus (HCoV-229E) with a RBD "lying" state, the S-trimer may be more suitable for subunit vaccines than the RBD. Our results will provide novel ideas for the development of vaccines targeting S protein in the future.

The trypsin-enhanced infection of porcine epidemic diarrhea virus is determined by the S2 subunit of the spike glycoprotein.

Porcine epidemic diarrhea virus (PEDV) is an enteric pathogen in the swine industry, causing high mortality in neonatal piglets. Efficient PEDV infection usually relies on the presence of trypsin, yet the mechanism of trypsin dependency is ambiguous. Here, we identified two PEDV strains, trypsin-enhanced YN200 and trypsin-independent DR13, in which the spike (S) protein of YN200 exhibits a stronger ability to induce syncytium formation and cleaved by trypsin than that of DR13. Using a full-length infectious YN200 cDNA clone, we confirmed that the S protein is a trypsin dependency determinant by comparison of rYN200 and rYN200-SDR13 To explore the trypsin-associated sites of the YN200 S protein, we then constructed a series of mutations adjacent to the fusion peptide. The results show that the putative S2' cleavage site (R892G) is not the determinant for virus trypsin dependency. Hence, we generated viruses carrying chimeric S proteins: the S1 subunit, S2 subunit, and S2720∼892 aa domain (NS2') were individually replaced by the corresponding DR13 sequences. Intriguingly, only the S2 substitution, not the S1 or NS2' substitutions, provides trypsin-independent growth of YN200. Additionally, the NS2' recombinant virus significantly abrogated effective infection, indicating a vital role for NS2' in viral entry. These findings suggest that the trypsin dependency of PEDV is mainly controlled by mutations in the S2 subunit rather than directly trypsin cleavage site.ImportanceWith the emergence of new variants, PEDV remains a major problem in the global swine industry. Efficient PEDV infection usually requires trypsin, while the mechanism of trypsin dependency is complex. Here, we used two PEDV strains, trypsin-enhanced YN200 and trypsin-independent DR13, and results showed that the S protein determined PEDV trypsin dependency by using a reverse genetic system of YN200. The S2 subunit was verified as the main portion of PEDV trypsin dependency, though the putative S2' site mutation cannot render trypsin-independent growth of YN200. Finally, these results provide some different insight to the PEDV trypsin dependency and might inspire vaccine development.

Establishment of Full-Length cDNA Clones and an Efficient Oral Infection Model for Feline Coronavirus in Cats.

Feline infectious peritonitis virus (FIPV) is the etiologic agent of feline infectious peritonitis (FIP) and causes fatal disease in cats of almost all ages. Currently, there are no clinically approved drugs or effective vaccines for FIP. Furthermore, the pathogenesis of FIP is still not fully understood. There is an urgent need for an effective infection model of feline infectious peritonitis induced by FIPV. Here, we constructed a field type I FIPV full-length cDNA clone, pBAC-QS, corresponding to the isolated FIPV QS. By replacing the FIPV QS spike gene with the commercially available type II FIPV 79-1146 (79-1146_CA) spike gene, we established and rescued a recombinant virus, designated rQS-79. Moreover, we constructed 79-1146_CA infectious full-length cDNA pBAC-79-1146_CA, corresponding to recombinant feline coronavirus (FCoV) 79-1146_CA (r79-1146_CA). In animal experiments with 1- to 2-year-old adult cats orally infected with the recombinant virus, rQS-79 induced typical FIP signs and 100% mortality. In contrast to cats infected with rQS-79, cats infected with 79-1146_CA did not show obvious signs. Furthermore, by rechallenging rQS-79 in surviving cats previously infected with 79-1146_CA, we found that there was no protection against rQS-79 with different titers of neutralizing antibodies. However, high titers of neutralizing antibodies may help prolong the cat survival time. Overall, we report the first reverse genetics of virulent recombinant FCoV (causing 100% mortality in adult cats) and attenuated FCoV (causing no mortality in adult cats), which will be powerful tools to study pathogenesis, antiviral drugs, and vaccines for FCoV. IMPORTANCE Tissue- or cell culture-adapted feline infectious peritonitis virus (FIPV) usually loses pathogenicity. To develop a highly virulent FIPV, we constructed a field isolate type I FIPV full-length clone with the spike gene replaced by the 79-1146 spike gene, corresponding to a virus named rQS-79, which induces high mortality in adult cats. rQS-79 represents the first described reverse genetics system for highly pathogenic FCoV. By further constructing the cell culture-adapted FCoV 79-1146_CA, we obtained infectious clones of virulent and attenuated FCoV. By in vitro and in vivo experiments, we established a model that can serve to study the pathogenic mechanisms of FIPV. Importantly, the wild-type FIPV replicase skeleton of serotype I will greatly facilitate the screening of antiviral drugs, both in vivo and in vitro.

The structural basis of African swine fever virus core shell protein p15 binding to DNA.

African swine fever (ASF) is an acute, hemorrhagic, and highly contagious disease caused by African swine fever virus (ASFV). The mortality rate of acute infection up to 100% have posed an unprecedented challenge of the swine industry. Currently no commercial antiviral drug is available for the control and treatment of ASFV. The structural resolution of ASFV virions reveals the details of ASFV morphogenesis, providing a new perspective for the research and promotion of the development of ASFV vaccines. Although the architecture of ASFV have been solved via cryo-EM, the structural details of four of the five viral layers remain unclear (except the outer capsid). In this study, we resolved the crystal structure of the ASFV core shell protein p15. The secondary structural elements of a protomer include four α-helix structures and six antiparallel ß-strands. Further analysis revealed that ASFV p15 forms disulfide-linked trimers between the Cys9 from one protomer and Cys30 from other protomer. Additionally, the nucleic acid-binding property was characterized by electrophoretic mobility shift assay. Two critical amino acid Lys10 and Lys39 have been identified which is essential to the nucleic acid-binding affinity of ASFV p15. Together, these findings may provide new insight into antiviral drug development.

Structural Characterization of the Helicase nsp10 Encoded by Porcine Reproductive and Respiratory Syndrome Virus.

Currently, an effective therapeutic treatment for porcine reproductive and respiratory syndrome virus (PRRSV) remains elusive. PRRSV helicase nsp10 is an important component of the replication transcription complex that plays a crucial role in viral replication, making nsp10 an important target for drug development. Here, we report the first crystal structure of full-length nsp10 from the arterivirus PRRSV, which has multiple domains: an N-terminal zinc-binding domain (ZBD), a 1B domain, and helicase core domains 1A and 2A. Importantly, our structural analyses indicate that the conformation of the 1B domain from arterivirus nsp10 undergoes a dynamic transition. The polynucleotide substrate channel formed by domains 1A and 1B adopts an open state, which may create enough space to accommodate and bind double-stranded RNA (dsRNA) during unwinding. Moreover, we report a unique C-terminal domain structure that participates in stabilizing the overall helicase structure. Our biochemical experiments also showed that deletion of the 1B domain and C-terminal domain significantly reduced the helicase activity of nsp10, indicating that the four domains must cooperate to contribute to helicase function. In addition, our results indicate that nidoviruses contain a conserved helicase core domain and key amino acid sites affecting helicase function, which share a common mechanism of helicase translocation and unwinding activity. These findings will help to further our understanding of the mechanism of helicase function and provide new targets for the development of antiviral drugs.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is a major respiratory disease agent in pigs that causes enormous economic losses to the global swine industry. PRRSV helicase nsp10 is a multifunctional protein with translocation and unwinding activities and plays a vital role in viral RNA synthesis. Here, we report the first structure of full-length nsp10 from the arterivirus PRRSV at 3.0-Å resolution. Our results show that the 1B domain of PRRSV nsp10 adopts a novel open state and has a unique C-terminal domain structure, which plays a crucial role in nsp10 helicase activity. Furthermore, mutagenesis and structural analysis revealed conservation of the helicase catalytic domain across the order Nidovirales (families Arteriviridae and Coronaviridae). Importantly, our results will provide a structural basis for further understanding the function of helicases in the order Nidovirales.

Insight into the evolution of nidovirus endoribonuclease based on the finding that nsp15 from porcine Deltacoronavirus functions as a dimer.

Nidovirus endoribonucleases (NendoUs) include nonstructural protein 15 (nsp15) from coronaviruses and nsp11 from arteriviruses, both of which have been reported to participate in the viral replication process and in the evasion of the host immune system. Results from a previous study of coronaviruses SARS-CoV, HCoV-229E, and MHV nsp15 indicate that it mainly forms a functional hexamer, whereas nsp11 from the arterivirus PRRSV is a dimer. Here, we found that porcine Deltacoronavirus (PDCoV) nsp15 primarily exists as dimers and monomers in vitro Biological experiments reveal that a PDCoV nsp15 mutant lacking the first 27 amino acids of the N-terminal domain (Asn-1-Asn-27) forms more monomers and displays decreased enzymatic activity, indicating that this region is important for its dimerization. Moreover, multiple sequence alignments and three-dimensional structural analysis indicated that the C-terminal region (His-251-Val-261) of PDCoV nsp15 is 10 amino acids shorter and forms a shorter loop than that formed by the equivalent sequence (Gln-259-Phe-279) of SARS-CoV nsp15. This result may explain why PDCoV nsp15 failed to form hexamers. We speculate that NendoUs may have originated from XendoU endoribonucleases (XendoUs) forming monomers in eukaryotic cells, that NendoU from arterivirus gained the ability to form dimers, and that the coronavirus variants then evolved the capacity to assemble into hexamers. We further propose that PDCoV nsp15 may be an intermediate in this evolutionary process. Our findings provide a theoretical basis for improving our understanding of NendoU evolution and offer useful clues for designing drugs and vaccines against nidoviruses.

A Dimerization-Dependent Mechanism Drives the Endoribonuclease Function of Porcine Reproductive and Respiratory Syndrome Virus nsp11.

UNLABELLED: Porcine reproductive and respiratory syndrome virus (PRRSV) RNA endoribonuclease nsp11 belongs to the XendoU superfamily and plays a crucial role in arterivirus replication. Here, we report the first crystal structure of the arterivirus nsp11 protein from PRRSV, which exhibits a unique structure and assembles into an asymmetric dimer whose structure is completely different from the hexameric structure of coronavirus nsp15. However, the structures of the PRRSV nsp11 and coronavirus nsp15 catalytic domains were perfectly superimposed, especially in the "active site loop" (His129 to His144) and "supporting loop" (Val162 to Thr179) regions. Importantly, our biochemical data demonstrated that PRRSV nsp11 exists mainly as a dimer in solution. Mutations of the major dimerization site determinants (Ser74 and Phe76) in the dimerization interface destabilized the dimer in solution and severely diminished endoribonuclease activity, indicating that the dimer is the biologically functional unit. In the dimeric structure, the active site loop and supporting loop are packed against one another and stabilized by monomer-monomer interactions. These findings may help elucidate the mechanism underlying arterivirus replication and may represent great potential for the development of antiviral drugs. IMPORTANCE: Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the family Arteriviridae, order Nidovirales PRRSV is a major agent of respiratory diseases in pigs, causing tremendous economic losses to the swine industry worldwide. The PRRSV nsp11 endoribonuclease plays a vital role in arterivirus replication, but its precise roles and mechanisms of action are poorly understood. Here, we report the first dimeric structure of the arterivirus nsp11 from PRRSV at 2.75-Å resolution. Structural and biochemical experiments demonstrated that nsp11 exists mainly as a dimer in solution and that nsp11 may be fully active as a dimer. Mutagenesis and structural analysis revealed NendoU active site residues, which are conserved throughout the order Nidovirales(families Arteriviridae and Coronaviridae) and the major determinants of dimerization (Ser74 and Phe76) in Arteriviridae Importantly, these findings may provide a new structural basis for antiviral drug development.

No association between Y chromosomal haplogroups and severe acne in the Han Chinese population.

Severe acne presents sexual dimorphism in its incidence in Chinese population. It is more prevalent in males. To assess the possible Y chromosomal contribution to severe acne risk in Han Chinese males, we analyzed 2041 Y chromosomal SNPs (Y-SNPs) in 725 severe acne cases and 651 controls retrieved from our recent genome-wide association study data. After data filtering, we assigned 585 cases and 494 controls into 12 Y chromosomal haplogroups based on 307 high-confidence Y-SNPs. No statistically significant difference in the distribution of Y chromosomal haplogroup frequencies was observed between the case and control groups. Our results showed a lack of association between the incidence of severe acne and the different Y chromosomal haplogroup in the Han Chinese population.

A potential dual protection vaccine: Recombinant feline herpesvirus-1 expressing feline parvovirus VP2 antigen.

Recently, herpesvirus viral vectors that stimulate strong humoral and cellular immunity have been demonstrated to be the most promising platforms for the development of multivalent vaccines, because they contain various nonessential genes and exhibit long-life latency characteristics. Previously, we showed that the feline herpesvirus-1 (FHV-1) mutant WH2020-ΔTK/gI/gE, which was safe for felines and provided efficacious protection against FHV-1 challenge, can be used as a vaccine vector. Moreover, previous studies have shown that the major neutralizing epitope VP2 protein of feline parvovirus (FPV) can elicit high levels of neutralizing antibodies. Therefore, to develop a bivalent vaccine against FPV and FHV-1, we first generated a novel recombinant virus by CRISPR/Cas9-mediated homologous recombination, WH2020-ΔTK/gI/gE-VP2, which expresses the VP2 protein of FPV. The growth characteristics of WH2020-ΔTK/gI/gE-VP2 were similar to those of WH2020-ΔTK/gI/gE, and WH2020-ΔTK/gI/gE-VP2 was stable for at least 30 generations in CRFK cells. As expected, we found that the felines immunized with WH2020-ΔTK/gI/gE-VP2 produced FPV-neutralizing antibody titers (27.5) above the positive cutoff (26) on day 14 after single inoculation. More importantly, recombinant WH2020-ΔTK/gI/gE-VP2 exhibited severely impaired pathogenicity in inoculated and cohabiting cats. The kittens immunized with WH2020-ΔTK/gI/gE and WH2020-ΔTK/gI/gE-VP2 produced similar levels of FHV-specific antibodies and IFN-ß. Furthermore, felines immunized with WH2020-ΔTK/gI/gE-VP2 were protected against challenge with FPV and FHV-1. These data showed that WH2020-ΔTK/gI/gE-VP2 appears to be a potentially safe, effective, and economical bivalent vaccine against FPV and FHV-1 and that WH2020-ΔTK/gI/gE can be used as a viral vector to develop feline multivalent vaccines.

Feline infectious peritonitis virus ORF7a is a virulence factor involved in inflammatory pathology in cats.

A hyperinflammatory response is a prominent feature of feline infectious peritonitis (FIP), but the mechanisms behind the feline infectious peritonitis virus (FIPV)-induced cytokine storm in the host have not been clarified. Studies have shown that coronaviruses encode accessory proteins that are involved in viral replication and associated with viral virulence, the inflammatory response and immune regulation. Here, we found that FIPV ORF7a gene plays a key role in viral infection and host proinflammatory responses. The recombinant FIPV strains lacking ORF7a (rQS-79Δ7a) exhibit low replication rates in macrophages and do not induce dramatic upregulation of inflammatory factors. Furthermore, through animal experiments, we found that the rQS-79Δ7a strain is nonpathogenic and do not cause symptoms of FIP in cats. Unexpectedly, after three vaccinations with rQS-79Δ7a strain, humoral and cellular immunity was increased and provided protection against virulent strains in cats, and the protection rate reaches 40%. Importantly, our results demonstrated that ORF7a is a key virulence factor that exacerbates FIPV infection and inflammatory responses. Besides, our findings will provide novel implications for future development of live attenuated FIPV vaccines.

Analysis of molecular variation of porcine reproductive and respiratory syndrome virus in Central China from 2006 to 2012.

To analyze the epidemiology of PRRSV in Hubei Province of China, 668 serum samples collected from 14 pig-breeding farms were tested. We found that the PRRSV-positive rate was 5.24 % and that HP-PRRSV had become the dominant strain. To further investigate the genetic variation of PRRSV strains in this region, the complete gene sequences of nsp2, orf5, and orf7 from nine PRRSV strains collected during 2011-2012 were determined and compared with 33 known sequences. The results revealed that diverse HP-PRRSV strains are present in this region. An analysis of orf5 gene sequences showed that the strains collected during 2009-2010 formed a tightly clustered branch. When compared with the JXA1 strain, they had one mutation (V29 → A29) in a decoy epitope. Furthermore, we found that the number of potential N-glycosylation sites had apparently increased since 2006. These findings increase our knowledge of PRRSV epidemiology in Central China.

mLST8 is essential for coronavirus replication and regulates its replication through the mTORC1 pathway.

Coronaviruses (CoVs), which pose a serious threat to human and animal health worldwide, need to hijack host factors to complete their replicative cycles. However, the current study of host factors involved in CoV replication remains unknown. Here, we identified a novel host factor, mammalian lethal with sec-13 protein 8 (mLST8), which is a common subunit of mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), and is critical for CoV replication. Inhibitor and knockout (KO) experiments revealed that mTORC1, but not mTORC2, is essential for transmissible gastroenteritis virus replication. Furthermore, mLST8 KO reduced the phosphorylation of unc-51-like kinase 1 (ULK1), a factor downstream of the mTORC1 signaling pathway, and mechanistic studies revealed that decreased phosphorylation of the mTORC1 downstream factor ULK1 promoted the activation of autophagy, which is responsible for antiviral replication in mLST8 KO cells. Then, transmission electron microscopy indicated that both mLST8 KO and autophagy activator inhibited the formation of double-membrane vesicles in early viral replication. Finally, mLST8 KO and autophagy activator treatment could also inhibit the replication of other CoVs, indicating a conserved relationship between autophagy activation and CoV replication. In summary, our work reveals that mLST8 is a novel host regulator of CoV replication, which provides new insights into the mechanism of CoV replication and can facilitate the development of broad-spectrum antiviral drugs. IMPORTANCE CoVs are highly variable, and existing CoV vaccines are still limited in their ability to address mutations in CoVs. Therefore, the need to improve our understanding of the interaction of CoVs with the host during viral replication and to find targets for drugs against CoVs is urgent. Here, we found that a novel host factor, mLST8, is critical for CoV infection. Further studies showed that mLST8 KO inhibited the mTORC1 signaling pathway, and we found that autophagy activation downstream of mTORC1 was the main cause of antiviral replication in mLST8 KO cells. Autophagy activation impaired the formation of DMVs and inhibited early viral replication. These findings deepen our understanding of the CoV replication process and provide insights into potential therapeutic applications.

Better therapeutic effect of oral administration of GS441524 compared with GC376.

FIP is a fatal feline disease caused by FIPV. Two drugs (GS441524 and GC376) target FIPV and have good therapeutic effect when administered by subcutaneous injection. However, subcutaneous injection has limitations compared with oral administration. Additionally, the oral efficacy of the two drugs has not been determined. Here, GS441524 and GC376 were shown to efficiently inhibit FIPV-rQS79 (recombination virus with a full-length field type I FIPV and the spike gene replaced with type II FIPV) and FIPV II (commercially available type II FIPV 79-1146) at a noncytotoxic concentration in CRFK cells. Moreover, the effective oral dose was determined via the in vivo pharmacokinetics of GS441524 and GC376. We conducted animal trials in three dosing groups and found that while GS441524 can effectively reducing the mortality of FIP subjects at a range of doses, GC376 only reducing the mortality rate at high doses. Additionally, compared with GC376, oral GS441524 has better absorption, slower clearance and a slower rate of metabolism. Furthermore, there was no significant difference between the oral and subcutaneous pharmacokinetic parameters. Collectively, our study is the first to evaluate the efficacy of oral GS441524 and GC376 using a relevant animal model. We also verified the reliability of oral GS441524 and the potential of oral GC376 as a reference for rational clinical drug use. Furthermore, the pharmacokinetic data provide insights into and potential directions for the optimization of these drugs.

Safety and immunogenicity of a TK/ gI/gE gene-deleted feline herpesvirus-1 mutant constructed via CRISPR/Cas9 in feline.

Feline herpesvirus-1 (FHV-1) is the aetiological agent of feline viral rhinotracheitis, which accounts for approximately 50 % of all viral upper respiratory diseases in cats. Commercially available modified live vaccines containing FHV-1 are generally safe and effective, but these FHV-1 vaccines retain full virulence genes and can establish latency and reactivate to cause infectious rhinotracheitis in vaccine recipients, raising safety concerns. To address this shortcoming, we constructed a novel TK/gI/gE -gene-deleted recombinant FHV-1 (WH2020-ΔTK/gI/gE) through CRISPR/Cas9-mediated homologous recombination. The growth kinetics of WH2020-ΔTK/gI/gE were slightly delayed compared to those of the parent strain WH2020. Recombinant FHV-1 had severely impaired pathogenicity in cats. Felines immunized with WH2020-ΔTK/gI/gE produced high levels of gB-specific antibodies, neutralizing antibodies and IFN-ß. Additionally, WH2020-ΔTK/gI/gE provided greater protection against challenge with FHV-1 field strain WH2020 than did the commercial modified live vaccine. After challenge, the cats vaccinated with WH2020-ΔTK/gI/gE showed significantly fewer clinical signs, pathological changes, viral shedding, and viral loads in the lung and trigeminal ganglia than those vaccinated with the commercial vaccine or unvaccinated. Our results suggest that WH2020-ΔTK/gI/gE is a promising candidate as a safer and more efficacious live FHV-1 vaccine, with a decreased risk of vaccine-related complications, and could inform the design of other herpesvirus vaccines.