Genomic analysis of Clostridium perfringens type D isolates from goat farms.

C. perfringens type D strains are the leading cause of enterotoxaemia in ruminants such as goats, sheep, and cattle. However, there has been no prior research on the genomic characteristics of C. perfringens type D strains from various regions in China. Here, we investigated the antibiotic resistance, genomic characteristics, and phylogenetic relationship of C. perfringens type D isolates recovered from goat farms in Shaanxi, Gansu, and Ningxia provinces. The antibiotic resistance test indicated that the isolates displayed high minimum inhibitory concentration (MIC) values to sulfafurazole, whereas the other antibiotics tested, such as penicillin, enrofloxacin, and florfenicol, worked well on them. Additionally, only tetracycline resistance genes [tetA(P) and tetB(P)] were identified from the isolates. A collective of 13 toxin genes, including etx and cpe were detected among the isolates. Sequence comparison revealed that the etx and cpe genes shared high sequence identities, and they could coexist on a pCW3-like plasmid, representing a potential risk to both animal breeding and public health. Phylogenetic analysis using core genome multi-locus sequence typing (cgMLST) and core genome single nucleotide polymorphisms (SNPs) revealed the close genetic relationship and potential regional/transregional transmission of the C. perfringens type D isolates in Shaanxi and Gansu provinces. Furthermore, pan-genomic analysis suggested the functional differences at the protein-coding gene level, although isolates from the same source shared a close genetic relationship. In conclusion, this study indicated the antibiotic resistance, virulence markers, potential transregional transmission, and genomic diversity of C. perfringens type D strains from various regions in China, which could provide references for the prevention of C. perfringens foodborne diseases and further research.

The Ca2+-dependent phosphatase calcineurin dephosphorylates TBK1 to suppress antiviral innate immunity.

Tumor necrosis factor receptor-associated factor family member-associated NF-κB activator-binding kinase 1 (TBK1) plays a key role in the induction of the type 1 interferon (IFN-I) response, which is an important component of innate antiviral defense. Viruses target calcium (Ca2+) signaling networks, which participate in the regulation of the viral life cycle, as well as mediate the host antiviral response. Although many studies have focused on the role of Ca2+ signaling in the regulation of IFN-I, the relationship between Ca2+ and TBK1 in different infection models requires further elucidation. Here, we examined the effects of the Newcastle disease virus (NDV)-induced increase in intracellular Ca2+ levels on the suppression of host antiviral responses. We demonstrated that intracellular Ca2+ increased significantly during NDV infection, leading to impaired IFN-I production and antiviral immunity through the activation of calcineurin (CaN). Depletion of Ca²+ was found to lead to a significant increase in virus-induced IFN-I production resulting in the inhibition of viral replication. Mechanistically, the accumulation of Ca2+ in response to viral infection increases the phosphatase activity of CaN, which in turn dephosphorylates and inactivates TBK1 in a Ca2+-dependent manner. Furthermore, the inhibition of CaN on viral replication was counteracted in TBK1 knockout cells. Together, our data demonstrate that NDV hijacks Ca2+ signaling networks to negatively regulate innate immunity via the CaN-TBK1 signaling axis. Thus, our findings not only identify the mechanism by which viruses exploit Ca2+ signaling to evade the host antiviral response but also, more importantly, highlight the potential role of Ca2+ homeostasis in the viral innate immune response.IMPORTANCEViral infections disrupt intracellular Ca2+ homeostasis, which affects the regulation of various host processes to create conditions that are conducive for their own proliferation, including the host immune response. The mechanism by which viruses trigger TBK1 activation and IFN-I induction through viral pathogen-associated molecular patterns has been well defined. However, the effects of virus-mediated Ca2+ imbalance on the IFN-I pathway requires further elucidation, especially with respect to TBK1 activation. Herein, we report that NDV infection causes an increase in intracellular free Ca2+ that leads to activation of the serine/threonine phosphatase CaN, which subsequently dephosphorylates TBK1 and negatively regulates IFN-I production. Furthermore, depletion of Ca2+ or inhibition of CaN activity exerts antiviral effects by promoting the production of IFN-I and inhibiting viral replication. Thus, our results reveal the potential role of Ca2+ in the innate immune response to viruses and provide a theoretical reference for the treatment of viral infectious diseases.

The W195 Residue of the Newcastle Disease Virus V Protein Is Critical for Multiple Aspects of Viral Self-Regulation through Interactions between V and Nucleoproteins.

The transcription and replication of the Newcastle disease virus (NDV) strictly rely on the viral ribonucleoprotein (RNP) complex, which is composed of viral NP, P, L and RNA. However, it is not known whether other viral non-RNP proteins participate in this process for viral self-regulation. In this study, we used a minigenome (MG) system to identify the regulatory role of the viral non-RNP proteins V, M, W, F and HN. Among them, V significantly reduced MG-encoded reporter activity compared with the other proteins and inhibited the synthesis of viral mRNA and cRNA. Further, V interacted with NP. A mutation in residue W195 of V diminished V-NP interaction and inhibited inclusion body (IB) formation in NP-P-L-cotransfected cells. Furthermore, a reverse-genetics system for the highly virulent strain F48E9 was established. The mutant rF48E9-VW195R increased viral replication and apparently enhanced IB formation. In vivo experiments demonstrated that rF48E9-VW195R decreased virulence and retarded time of death. Overall, the results indicate that the V-NP interaction of the W195 mutant V decreased, which regulated viral RNA synthesis, IB formation, viral replication and pathogenicity. This study provides insight into the self-regulation of non-RNP proteins in paramyxoviruses.

Effects of a novel Bacillus subtilis GXYX crude lipopeptide against Salmonella enterica serovar Typhimurium infection in mice.

The increased rate of antibiotic resistance strongly limits the resolution of Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. Therefore, new strategies to control bacterial infections are urgently needed. Bacillus subtilis (B. subtilis) and its metabolites are desirable antibacterial agents. Here, we aimed to evaluate the antibacterial activity of the novel B. subtilis strain GXYX (No: PRJNA940956) crude lipopeptide against S. Typhimurium. In vitro, GXYX crude lipopeptides affected S. Typhimurium biofilm formation and swimming and attenuated the adhesion and invasion abilities of S. Typhimurium toward BHK-21 cells; in addition, it inhibited the mRNA expression of the filA, filC, csgA, and csgB genes, which are related to the adhesion and invasion ability of S. Typhimurium. In vivo, pretreatment with GXYX crude lipopeptide via intragastric administration improved the survival rate by 30%, which was related to reductions in organ bacterial loads and clinical signs in mice. Intragastric administration of GXYX crude lipopeptide significantly downregulated the mRNA levels of TNF-α, IL-1ß, IL-12 and IL-6 in response to S. Typhimurium-induced inflammation compared with intraperitoneal injection. Moreover, it significantly improved the intestinal barrier-related gene (ZO-1, claudin-1, occludin-1) mRNA levels in intestinal tissue damaged by S. Typhimurium infection. In conclusion, GXYX crude lipopeptides were effective at reducing S. Typhimurium colonization, laying a foundation for the further development of novel antibacterial agents.

Construction and characterization of a reverse genetics system of bovine parainfluenza virus type 3c as a tool for rapid screening of antivirals in vitro.

Bovine parainfluenza virus type 3 (BPIV3) is a key pathogen associated with bovine respiratory disease complex (BRDC). However, its specific pathogenesis mechanisms have not been fully elucidated. Reverse genetics provides a useful method for understanding the pathogenic mechanism of BPIV3. To ensure the functionality of the rescue platforms, we first constructed a minigenome (MG) system of BPIV3 utilizing a 5-plasmid system in this investigation. Then, a full-length infection clone of BPIV3 was obtained from the SX-2021 strain, and different methods were employed to identify the rescued virus. Additionally, we recovered a recombinant BPIV3 using the reverse genetics system that could express enhanced green fluorescence protein (eGFP). Through the growth curve assays, the replicate capability of rBPIV3-SX-EGFP was found to be similar to that of the parental virus. Subsequently, the rBPIV3-SX-EGFP was used to determine the antiviral activity of ribavirin. The results showed that ribavirin had an anti-BPIV3 effect in MDBK cells. In conclusion, the successful development of a reverse genetic system for the SX-2021 strain establishes a foundation for future studies on BPIV3, including investigations into its pathogenic mechanism, gene function, and antiviral screening properties.

Bovine parainfluenza virus type 3 infections induce ER stress-mediated autophagy to facilitate virus replication.

Bovine Parainfluenza Virus Type 3 (BPIV3) serves as a crucial pathogen in cattle, adept at triggering severe respiratory symptoms. This investigation explores the intricate interplay of endoplasmic reticulum stress (ER stress), unfolded protein response (UPR), and autophagy upon BPIV3 infection. In this study, we initially confirm a substantial increase in glucose regulatory protein 78 (GRP78) expression, accompanied by noticeable morphological changes and significant expansion of the ER lumen observed through transmission electron microscopy upon BPIV3 infection. Our findings indicate that ER Stress is induced during BPIV3 infection in vitro. Subsequently, we illustrate that BPIV3 triggers ER Stress to facilitate viral replication through heightened autophagy through treatment with the ER stress inhibitor 4-phenylbutyrate (4-PBA) and utilizing small interfering RNA (siRNA) technology to knock down GRP78. Additionally, we observe that the activation of ER stress initiates the UPR via PERK and ATF6 pathways, with the IRE1 pathway not contributing to the regulation of ER stress-mediated autophagy. Moreover, intervention with the PERK inhibitor GSK2606414, ATF6 inhibitor Ceapin-A7, and siRNA technology successfully reverses BPIV3-induced autophagy. In summary, these findings propose that BPIV3 induces ER stress to enhance viral replication through increased autophagy, with the PERK and ATF6 pathways playing a significant role in ER stress-mediated autophagy.

Bovine parainfluenza type 3 virus induces incomplete autophagy to promote viral replication by activated beclin1 in vitro.

Bovine Parainfluenza virus Type 3 (BPIV3) is one of the most important pathogens in cattle, capable of causing severe respiratory symptoms. Numerous studies have shown that autophagy plays a diverse role in the infection process of various pathogens. The influence of autophagy machinery on BPIV3 infection has not yet been confirmed. In the present study, we initially demonstrated that the expression of LC3 was significantly increased and exhibited a notable increase in double or single-membrane vesicles under a transmission electron microscope during BPIV3 infection. These observations unequivocally establish the induction of steady-state autophagy in vitro consequent to BPIV3 infection. Furthermore, quantification of autophagic flux substantiates the induction of an incomplete autophagic process during BPIV3 infection. Additionally, through targeted interventions, we demonstrate the regulatory impact of pharmacological agents influencing autophagy and RNA interference targeting an autophagy-associated protein on viral replication. Intriguingly, our data revealed that BPIV3 infection enhanced the phosphorylation of rapamycin kinase (mTOR). This result demonstrated that mTOR does not operate as a counteractive regulator of BPIV3-induced autophagy. Instead, we discern an augmentation in the expression of Beclin1, a key autophagy initiator, which complexes with Vps34, constituting a Class III phosphatidylinositol 3-kinase. This phenomenon serves as a hallmark in the inaugural phase of autophagy initiation during BPIV3 infection. Collectively, these discernments underscore that BPIV3 infection actively stimulates autophagy, thereby enhancing viral replication through the activation of Beclin1, independently of the mTOR signaling pathway. This nuanced comprehension significantly contributes to unraveling the intricate molecular mechanisms governing BPIV3-induced autophagy.

Porcine Epidemic Diarrhea Virus: Etiology, Epidemiology, Antigenicity, and Control Strategies in China.

Porcine epidemic diarrhea virus (PEDV) is a porcine enteric coronavirus, which is one of the main causative agents of porcine epidemic diarrhea (PED), with 100% morbidity and 80-100% mortality in neonatal piglets. Since 2010, large-scale PED caused by highly pathogenic variants of PEDV has occurred successively in China and other countries in the world, posing a great threat to the global pig industry. It has been demonstrated in many investigations that the classic attenuated vaccine strain, PEDV CV777, is insufficient to fully protect against the PEDV variants. Moreover, the maternally derived antibodies elicited by inactivated vaccines also cannot completely protect piglets from infection. In addition, feedback feeding poses a risk of periodic PEDV recurrence in pig farms, making it challenging to successfully limit the spread of PEDV in China. This review focuses on the etiology, epidemiology, antigenicity, and control strategies of PEDV in China and provides information for the formulation of effective control measures.

Erratum: MiR-375 Has Contrasting Effects on Newcastle Disease Virus Growth Depending on the Target Gene : Erratum.

[This corrects the article DOI: 10.7150/ijbs.25106.].

Characterization of Lung Microbiomes in Pneumonic Hu Sheep Using Culture Technique and 16S rRNA Gene Sequencing.

Hu sheep, a locally bred species in China known for its high productivity, is currently suffering from pneumonia. Here, we combine high-throughput 16SrRNA gene sequencing and bacterial culturing to examine the bacterial community in pneumonic Hu Sheep lungs (p < 0.05). The results showed that the abundance and diversity of lung bacteria in healthy sheep were significantly higher than those in pneumonia sheep (p = 0.139), while there was no significant difference between moderate and severe pneumonia. Furthermore, the composition of the lung microbiota community underwent significant alterations between different levels of pneumonia severity. The application of LEfSe analysis revealed a notable enrichment of Mannheimiae within the lungs of sheep afflicted with moderate pneumonia (p < 0.01), surpassing the levels observed in their healthy counterparts. Additionally, Fusobacterium emerged as the prevailing bacterial group within the lungs of sheep suffering from severe pneumonia. Integrating the results of bacterial isolation and identification, we conclusively determined that Mannheimia haemolytica was the primary pathogenic bacterium within the lungs of sheep afflicted with moderate pneumonia. Furthermore, the exacerbation of pneumonia may be attributed to the synergistic interplay between Fusobacterium spp. and other bacterial species. Our results provide new insights for guiding preventive and therapeutic measures for pneumonia of different severities in sheep.

Antigenic variation in hemagglutinin-neuraminidase of Newcastle disease virus isolated from Tibet, China.

Vaccines are widely used to prevent Newcastle disease virus (NDV). Under the pressure of immunization, NDVs with mutations among epitopes of F and HN protein were isolated, which indicates that the efficiency of vaccine may decrease in terms of preventing emerged NDV. However, the lack of evidences to support whether these mutations contribute to antigenic mutation and immune escape in NDV leading to the controversy that the matched vaccine is more effective than the mismatched vaccine. In this study, a genotype VII velogenic NDV strain (C22) was isolated from a vaccinated farm in Tibet, China. We found that this strain was close to NDV from east China, but it had a specific mutation (K138R) in one epitope (131DYIGGIGKE139) of HN protein. This mutation might change the interaction between amino acids in stalk-head link region of HN protein and then induce the specific antibody to worse recognize the C22 strain, but it did not alter viral virulence and growth ability. Then, the C22 strain was attenuated via modification of the F protein cleavage site to generate a matched vaccine. Comparing to a mismatched vaccine (LaSota), this matched vaccine showed advantages in inhibiting viral shedding and tissue damage. However, both vaccines induced chicken to generate similar level of neutralizing antibodies against C22, C22mut (R138K) and LaSota. These results suggest that the epitope mutation is insufficient to help NDV escaping neutralizing antibodies of vaccinated chicken, supporting that the merits of NDV matched vaccine are not totally related to humoral immunity.

Genetic and pathogenic characterization of an NADC-34-like isolate of porcine reproductive and respiratory syndrome virus.

In this study, an NADC34-like strain of porcine reproductive and respiratory syndrome virus (PRRSV), YC-2020, was isolated from a pig farm in Yuncheng, Shanxi Province, China. Phylogenetic and molecular evolutionary analysis showed that the genome sequence of YC-2020 was very similar to those of NADC34-like PRRSV strains in the ORF2-7 region. However, it was more closely related to NADC30-like PRRSV and highly pathogenic (HP) PRRSV in the NSP2 and NSP3-9 coding regions, respectively, suggesting that recombination had occurred between viruses belonging to lineages 1 and 8. Piglets infected with YC-2020 exhibited mild clinical signs, but they had severe histopathological lesions in their lungs. These findings reveal novel genetic and pathogenic features of this isolate.

Genome characteristics of the optrA-positive Clostridium perfringens strain QHY-2 carrying a novel plasmid type.

Clostridium perfringens is a bacterial species of importance to both public and animal health. The gene optrA is the first gene that confers resistance to the tedizolid, a last-resort antimicrobial agent in human medicine. Herein, we whole-genome sequenced and analyzed one optrA-positive C. perfringens strain QHY-2 from Tibetan sheep in Qinghai province and identified one optrA plasmid pQHY-2. The plasmid shared similar structure with the optrA-positive plasmids p2C45 and p21-D-5b previously identified in C. perfringens, demonstrating the potential horizontal transmission of the optrA plasmids among C. perfringens strains. Annotation of the optrA-positive plasmids showed optrA and erm(A) located on a segment flanked by IS element IS1216E, and fexA, optrA, and erm(A) located on a segment flanked by IS element ISVlu1, which revealed the possible dissemination mechanism. Additionally, a Tn6218-like transposon carrying aac(6')-aph(2″) and erm(B) was also detected on pQHY-2, demonstrating the transposition of Tn6218 and spread of antibiotic resistance among Clostridium bacteria. Molecular analysis indicated the optrA-positive plasmids belonged to a plasmid type distinct from the pCW3-like plasmids, pCP13-like plasmids, or pIP404-like plasmids. Further structure analysis showed they might be formed by inserting segments into plasmid pCPCPI53k-r1_1, which coexist with two pCW3-like plasmids and one pCP13-like plasmid in C. perfringens strain CPI 53k-r1 isolated from a healthy human in Finland. IMPORTANCE Antimicrobial resistance is now a global concern posing threats to food safety and public health. The pCW3-like plasmids can encode several main toxin genes and three antibiotic resistance genes (ARGs), including tetA(P), tetB(P), and erm(B), which used to be considered as the main carrier of ARGs in Clostridium perfringens. In this study, we found the optrA plasmids, which belonged to a novel plasmid type, could also harbor many other ARGs, indicating this type of plasmid might be the potential repository of ARGs in C. perfringens. Additionally, this type of plasmid could coexist with the pCW3-like plasmids and pCP13-like plasmids that encoded toxin genes associated with gastrointestinal diseases, which showed the potential threat to public health.

Newcastle disease virus forms inclusion bodies with features of liquid-liquid phase separation.

Formation of inclusion bodies (IBs) is a hallmark of infections with negative-strand RNA viruses. Although the Newcastle disease virus (NDV) IBs had been observed in the 1950s, the characteristics of NDV IBs remained largely unknown. Here, we show that NDV infection triggers the formation of IBs that contain newly synthesized viral RNA. The structures of NDV IBs, observed by electron microscopy, were not membrane-bound. Fluorescence recovery after photobleaching a region of NDV IBs occurred rapidly, and IBs were dissolved by 1,6-hexanediol treatment, demonstrating they exhibited properties consistent with liquid-liquid phase separation (LLPS). We find the nucleoprotein (NP) and phosphoprotein (P) are sufficient to generate IB-like puncta, with the N arm domain and N core region of NP and the C terminus of P playing important roles in this process. In summary, our findings suggest that NDV forms IBs containing viral RNA, and provide insights into the formation of NDV IBs.

Probiotics and vitamins modulate the cecal microbiota of laying hens submitted to induced molting.

Induced molting enables laying hens to relax, restore energy and prolong the laying hen cycle, resolving problems such as poor egg quality and minimizing economic losses caused by rising global feeding costs. However, traditional molting methods may disrupt gut microflora and promote potential pathogens infections. This study used a customized additive with a mixture of probiotics and vitamins to induce molting and examine the cecal microbiota post molting. A total of two hundred 377 day-of-ISA Brown laying hens were randomly assigned to four groups: non-molt with basal diet (C), 12-day feeding restriction (FR) in earlier-molting (B), feed again to 27.12% egg production in middle-molting (A) and reach second peak of egg production over 81.36% in post-molting (D). Sequencing 16S rRNA to analyze cecal microbial composition revealed that there is no significant change in bacterial community abundance post-molting. In contrast to group C, the number of potentially harmful bacteria such as E. coli and Enterococcus was not found to increase in groups B, A, or D. This additive keeps cecal microbiota diversity and community richness steady. In cecal contents, hens in group B had lower Lactobacillus, Lachnospiraceae and Prevotellaceae (vsC, A, and D), no significant differences were found between post-molting and the non-molting. Furthermore, cecal microbiota and other chemicals (antibodies, hormones, and enzymes, etc.) strongly affect immunological function and health. Most biochemical indicators are significantly positively correlated with Prevotellaceae, Ruminococcaceae and Subdoligranulum, while negatively with Phascolarctobacterium and Desulfovibrio. In conclusion, the additive of probiotics and vitamins improved the cecal microbiota composition, no increase in the associated pathogenic microbial community due to traditional molting methods, and enhances hepatic lipid metabolism and adaptive immunological function, supporting their application and induced molting technology in the poultry breeding industry.

IncHI1 plasmids mediated the tet(X4) gene spread in Enterobacteriaceae in porcine.

The tigecycline resistance gene tet(X4) was widespread in various bacteria. However, limited information about the plasmid harboring the tet(X4) gene spread among the different species is available. Here, we investigated the transmission mechanisms of the tet(X4) gene spread among bacteria in a pig farm. The tet(X4) positive Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Enterobacter hormaeche were identified in the same farm. The whole genome sequencing (WGS) analysis showed that the K. pneumoniae belonged to ST727 (n = 11) and ST3830 (n = 1), E. cloacae and E. hormaeche belonged to ST524 (n = 1) and ST1862 (n = 1). All tet(X4) genes were located on the IncHI1 plasmids that could be conjugatively transferred into the recipient E. coli C600 at 30°C. Moreover, a fusion plasmid was identified that the IncHI1 plasmid recombined with the IncN plasmid mediated by ISCR2 during the conjugation from strains B12L to C600 (pB12L-EC-1). The fusion plasmid also has been discovered in a K. pneumoniae (K1L) that could provide more opportunities to spread antimicrobial resistance genes. The tet(X4) plasmids in these bacteria are derived from the same plasmid with a similar structure. Moreover, all the IncHI1 plasmids harboring the tet(X4) gene in GenBank belonged to the pST17, the newly defined pMLST. The antimicrobial susceptibility testing was performed by broth microdilution method showing the transconjugants acquired the most antimicrobial resistance from the donor strains. Taken together, this report provides evidence that IncHI1/pST17 is an important carrier for the tet(X4) spread in Enterobacteriaceae species, and these transmission mechanisms may perform in the environment.

Simultaneous Expression of Chicken Granulocyte Monocyte Colony-Stimulating Factor and the Hemagglutinin-Neuraminidase Epitope of the Virulent Newcastle Disease Virus Genotype VII C22 Strain in a Functional Synthetic Recombinant Adenovirus as a Genotype-Matched Vaccine with Potential Antiviral Activity.

When it comes to the prevention of clinical signs and mortality associated with infection of the Newcastle disease virus (NDV), vaccination has been very effective. However, recent evidence has proven that more highly virulent strains are emerging that bypass existing immune protection and pose a serious threat to the global poultry industry. Here, a novel rescued adenovirus 5-coexpressed chicken granulocyte monocyte colony-stimulating factor (ChGM-CSF) bio-adjuvant and C22-hemagglutinin-neuraminidase (HN) boosted chickens' immunological genetic resistance and thus improved the immunological effectiveness of the critical new-generation vaccine in vitro and in vivo. Accordingly, the hemagglutination inhibition (HI) titers (log2) of the recombinant adenovirus (rAdv)-ChGM-CSF-HN-immunized chickens had greater, more persistent, and longer-lasting NDV-specific antibodies than the La Sota and rAdv-HN-inoculated birds. Moreover, humoral and adaptive immunological conditions were shown to be in harmony after rAdv-ChGM-CSF-HN inoculation and uniformly enhanced the expression of alpha interferon (IFN-α), IFN-ß, IFN-γ, interleukin-1ß (IL-1ß), IL-2, IL-16, IL-18, and IL-22. Postchallenge, the control challenge (CC), wild-type adenovirus (wtAdv), and rAdv-ChGM-CSF groups developed unique NDV clinical manifestations, significant viral shedding, high tissue viral loads, gross and microscopic lesions, and 100% mortality within 7 days. The La Sota, rAdv-HN, and rAdv-ChGM-CSF-HN groups were healthy and had 100% survival rates. The rAdv-ChGM-CSF-HN group swiftly regulated and stopped viral shedding and had lower tissue viral loads than all groups at 5 days postchallenge (dpc). Thus, the antiviral activity of ChGM-CSF offered robust immune protection in the face of challenge and reduced viral replication convincingly. Our advance innovation concepts, combining ChGM-CSF with a field-circulating strain epitope, could lead to the development of a safe, genotype-matched, universal transgenic vaccine that could eradicate the disease globally, reducing poverty and food insecurity. IMPORTANCE We studied the biological characterization of the developed functional synthetic recombinant adenoviruses, which showed a high degree of safety, thermostability, and genetic stability for up to 20 passages. It was demonstrated through both in vitro and in vivo testing that the immunogenicity of the proposed vaccine, which uses the T2A peptide from the Thosea asigna virus capsid protein supported by glycine and serine, helps with efficiency to generate a multicistronic vector, enables expression of two functional proteins in rAdv-ChGM-CSF-HN, and is superior to that of comparable vaccines. Additionally, adenovirus can be used to produce vaccines matching the virulent field-circulating strain epitope. Because there is no preexisting human adenoviral immunity detected in animals, the potency of adenoviral vaccines looks promising. Also, it ensures that the living vector does not carry the resistance gene that codes for the kanamycin antibiotic. Accordingly, a human recombinant adenoviral vaccine that has undergone biological improvements is beneficial and important.

9-Butyl-Harmol Exerts Antiviral Activity against Newcastle Disease Virus through Targeting GSK-3ß and HSP90ß.

The paramyxoviruses represent a large family of human and animal pathogens that cause significant health and economic burdens worldwide. However, there are no available drugs against the virus. ß-carboline alkaloids are a family of naturally occurring and synthetic products with outstanding antiviral activities. Here, we examined the antiviral effect of a series of ß-carboline derivatives against several paramyxoviruses, including Newcastle disease virus (NDV), peste des petits ruminants virus (PPRV), and canine distemper virus (CDV). Among these derivatives, 9-butyl-harmol was identified as an effective antiviral agent against these paramyxoviruses. Further, a genome-wide transcriptome analysis in combination with target validation strategies reveals a unique antiviral mechanism of 9-butyl-harmol through the targeting of GSK-3ß and HSP90ß. On one hand, NDV infection blocks the Wnt/ß-catenin pathway to suppress the host immune response. 9-butyl-harmol targeting GSK-3ß dramatically activates the Wnt/ß-catenin pathway, which results in the boosting of a robust immune response. On the other hand, NDV proliferation depends on the activity of HSP90. The L protein, but not the NP protein or the P protein, is proven to be a client protein of HSP90ß, rather than HSP90α. 9-butyl-harmol targeting HSP90ß decreases the stability of the NDV L protein. Our findings identify 9-butyl-harmol as a potential antiviral agent, provide mechanistic insights into the antiviral mechanism of 9-butyl-harmol, and illustrate the role of ß-catenin and HSP90 during NDV infection. IMPORTANCE Paramyxoviruses cause devastating impacts on health and the economy worldwide. However, there are no suitable drugs with which to counteract the viruses. We determined that 9-butyl-harmol could serve as a potential antiviral agent against paramyxoviruses. Until now, the antiviral mechanism of ß-carboline derivatives against RNA viruses has rarely been studied. Here, we found that 9-butyl-harmol exerts dual mechanisms of antiviral action, with its antiviral activities being mediated by two targets: GSK-3ß and HSP90ß. Correspondingly, the interaction between NDV infection and the Wnt/ß-catenin pathway or HSP90 is demonstrated in this study. Taken together, our findings shed light on the development of antiviral agents against paramyxoviruses, based on the ß-carboline scaffold. These results present mechanistic insights into the polypharmacology of 9-butyl-harmol. Understanding this mechanism also deepens the host-virus interaction and reveals new drug targets for anti-paramyxoviruses.

A bivalent ß-carboline derivative inhibits macropinocytosis-dependent entry of pseudorabies virus by targeting the kinase DYRK1A.

Pseudorabies virus (PRV) has become a "new life-threatening zoonosis" since the human-originated PRV strain was first isolated in 2020. To identify novel anti-PRV agents, we screened a total of 107 ß-carboline derivatives and found 20 compounds displaying antiviral activity against PRV. Among them, 14 compounds showed better antiviral activity than acyclovir. We found that compound 45 exhibited the strongest anti-PRV activity with an IC50 value of less than 40 nM. Our in vivo studies showed that treatment with 45 significantly reduced the viral loads and protected mice challenged with PRV. To clarify the mode of action of 45, we conducted a time of addition assay, an adsorption assay, and an entry assay. Our results indicated that 45 neither had a virucidal effect nor affected viral adsorption while significantly inhibiting PRV entry. Using the FITC-dextran uptake assay, we determined that 45 inhibits macropinocytosis. The actin-dependent plasma membrane protrusion, which is important for macropinocytosis, was also suppressed by 45. Furthermore, the kinase DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1A) was predicted to be a potential target for 45. The binding of 45 to DYRK1A was confirmed by drug affinity responsive target stability and cellular thermal shift assay. Further analysis revealed that knockdown of DYRK1A by siRNA suppressed PRV macropinocytosis and the tumor necrosis factor alpha-TNF-induced formation of protrusions. These results suggested that 45 could restrain PRV macropinocytosis by targeting DYRK1A. Together, these findings reveal a unique mechanism through which ß-carboline derivatives restrain PRV infection, pointing to their potential value in the development of anti-PRV agents.