Duck plague virus-encoded microRNA dev-miR-D28-3p inhibits viral replication via targeting UL27.

Herpesviruses-encoded microRNAs (miRNAs) have been discovered to be essential regulators in viral life cycle, participating in viral replication, latent or lytic infection, and immunological escape. However, the roles of miRNAs encoded by duck plague virus (DPV) are still unknown. Dev-miR-D28-3p is a miRNA uniquely encoded by DPV CHv strain. The aim of this study was to explore the effect of dev-miR-D28-3p on DPV replication and explore the potential mechanisms involved. Our findings demonstrated that transfection of dev-miR-D28-3p mimic into duck embryo fibroblasts (DEFs) effectively suppressed viral copies, viral titers and viral protein expressions during DPV infection, while the results above were reversed after transfection with dev-miR-D28-3p inhibitor. Subsequently, we further discovered that dev-miR-D28-3p specifically bound to DPV-encoded UL27 and inhibited its expression, suggesting that UL27 was the target gene of dev-miR-D28-3p. Finally, we investigated the role of UL27 in DPV replication and found the overexpression of UL27 increased viral copies, viral titers, and viral protein expressions; whereas the opposite results appear when knockdown of UL27. Our findings illustrated a novel mechanism that DPV regulated itself replication via dev-miR-D28-3p, paving the way for exploring the role of DPV-encoded miRNAs.

The precise function of alphaherpesvirus tegument proteins and their interactions during the viral life cycle.

Alphaherpesvirus is a widespread pathogen that causes diverse diseases in humans and animals and can severely damage host health. Alphaherpesvirus particles comprise a DNA core, capsid, tegument and envelope; the tegument is located between the nuclear capsid and envelope. According to biochemical and proteomic analyses of alphaherpesvirus particles, the tegument contains at least 24 viral proteins and plays an important role in the alphaherpesvirus life cycle. This article reviews the important role of tegument proteins and their interactions during the viral life cycle to provide a reference and inspiration for understanding alphaherpesvirus infection pathogenesis and identifying new antiviral strategies.

Duck CD40L as an adjuvant enhances systemic immune responses of avian flavivirus DNA vaccine.

Under the dual pressure of emerging zoonoses and the difficulty in eliminating conventional zoonoses, the strategic management of bird diseases through vaccination represents a highly efficacious approach to disrupting the transmission of zoonotic pathogens to humans. Immunization with a DNA vaccine yielded limited protection against avian pathogen infection. To improve its immunogenicity, the extracellular domain of duck-derived CD40L (designated as dusCD40L) was employed as a bio-adjuvant. Our findings unequivocally established the evolutionary conservation of dusCD40L across avian species. Notably, dusCD40L exhibited a compelling capacity to elicit robust immune responses from both B and T lymphocytes. Furthermore, when employed as an adjuvant, dusCD40L demonstrated a remarkable capacity to significantly augment the titers of neutralizing antibodies and the production of IFNγ elicited by a DNA vaccine encoding the prM-E region of an avian flavivirus, namely, the Tembusu virus (TMUV). Moreover, dusCD40L could strengthen virus clearance of the prM-E DNA vaccine in ducks post-TMUV challenge. This research study presents a highly effective adjuvant for advancing the development of DNA vaccines targeting TMUV in avian hosts. Additionally, it underscores the pivotal role of duCD40L as a potent adjuvant in the context of vaccines designed to combat zoonotic infections in avian species.

Duck STING mediates antiviral autophagy directing the interferon signaling pathway to inhibit duck plague virus infection.

Migratory birds are important vectors for virus transmission, how migratory birds recognize viruses and viruses are sustained in birds is still enigmatic. As an animal model for waterfowl among migratory birds, studying and dissecting the antiviral immunity and viral evasion in duck cells may pave a path to deciphering these puzzles. Here, we studied the mechanism of antiviral autophagy mediated by duck STING in DEF cells. The results collaborated that duck STING could significantly enhance LC3B-II/I turnover, LC3B-EGFP puncta formation, and mCherry/EGFP ratio, indicating that duck STING could induce autophagy. The autophagy induced by duck STING is not affected by shRNA knockdown of ATG5 expression, deletion of the C-terminal tail of STING, or TBK1 inhibitor BX795 treatment, indicating that duck STING activated non-classical selective autophagy is independent of interaction with TBK1, TBK1 phosphorylation, and interferon (IFN) signaling. The STING R235A mutant and Sar1A/B kinase mutant abolished duck STING induced autophagy, suggesting binding with cGAMP and COPII complex mediated transport are the critical prerequisite. Duck STING interacted with LC3B through LIR motifs to induce autophagy, the LIR 4/7 motif mutants of duck STING abolished the interaction with LC3B, and neither activated autophagy nor IFN expression, indicating that duck STING associates with LC3B directed autophagy and dictated innate immunity activation. Finally, we found that duck STING mediated autophagy significantly inhibited duck plague virus (DPV) infection via ubiquitously degraded viral proteins. Our study may shed light on one scenario about the control and evasion of diseases transmitted by migratory birds.

Integrative and conjugative elements of Pasteurella multocida: Prevalence and signatures in population evolution.

Pasteurella multocida (P. multocida) is a bacterial pathogen responsible for a range of infections in humans and various animal hosts, causing significant economic losses in farming. Integrative and conjugative elements (ICEs) are important horizontal gene transfer elements, potentially enabling host bacteria to enhance adaptability by acquiring multiple functional genes. However, the understanding of ICEs in P. multocida and their impact on the transmission of this pathogen remains limited. In this study, 42 poultry-sourced P. multocida genomes obtained by high-throughput sequencing together with 393 publicly available P. multocida genomes were used to analyse the horizontal transfer of ICEs. Eighty-two ICEs were identified in P. multocida, including SXT/R391 and Tn916 subtypes, as well as three subtypes of ICEHin1056 family, with the latter being widely prevalent in P. multocida and carrying multiple resistance genes. The correlations between insertion sequences and resistant genes in ICEs were also identified, and some ICEs introduced the carbapenem gene blaOXA-2 and the bleomycin gene bleO to P. multocida. Phylogenetic and collinearity analyses of these bioinformatics found that ICEs in P. multocida were transmitted vertically and horizontally and have evolved with host specialization. These findings provide insight into the transmission and evolution mode of ICEs in P. multocida and highlight the importance of understanding these elements for controlling the spread of antibiotic resistance.

HSP70 positively regulates translation by interacting with the IRES and stabilizes the viral structural proteins VP1 and VP3 to facilitate duck hepatitis A virus type 1 replication.

The maintenance of viral protein homeostasis depends on the interaction between host cell proteins and viral proteins. As a molecular chaperone, heat shock protein 70 (HSP70) has been shown to play an important role in viral infection. Our results showed that HSP70 can affect translation, replication, assembly, and release during the life cycle of duck hepatitis A virus type 1 (DHAV-1). We demonstrated that HSP70 can regulate viral translation by interacting with the DHAV-1 internal ribosome entry site (IRES). In addition, HSP70 interacts with the viral capsid proteins VP1 and VP3 and promotes their stability by inhibiting proteasomal degradation, thereby facilitating the assembly of DHAV-1 virions. This study demonstrates the specific role of HSP70 in regulating DHAV-1 replication, which are helpful for understanding the pathogenesis of DHAV-1 infection and provide additional information about the role of HSP70 in infection by different kinds of picornaviruses, as well as the interaction between picornaviruses and host cells.

E-M349E and NS2A/2B-P1(T) are compensatory mutations of rDTMUV-NS2AB-P1P1'(AA), which regain virus proliferation by enhancing the virus package and restoring NS2A/2B cleavage.

Duck Tembusu virus (DTMUV) belongs to the Flaviviridae family and mainly infects ducks. The genome of DTMUV is translated into a polyprotein, which is further cleaved into several protein by viral NS2B3 protease and host proteases. Crucially, the cleavage of the NS2A/2B precursor during this process is essential for the formation of replication complexes and viral packaging. Previous research has demonstrated that alanine mutations in NS2A/2B (P1P1' (AA)) result in an attenuated strain (rDTMUV-NS2A/2B-P1P1' (AA)) by disrupting NS2A/2B cleavage. In this study, we investigate the effects of the P1P1' (AA) mutation on the viral life cycle and explore compensatory mutations in rDTMUV-NS2A/2B-P1P1' (AA). Infected ducklings exhibit similar body weight gain and viral tissue loads to DTMUV-WT. Compensatory mutations E-M349E and P1(T) emerge, restoring proliferation levels to those of rDTMUV-WT. Specifically, E-M349E enhances viral packaging, while P1(T) reinstates NS2A/2B proteolysis in vitro. Thus, our findings reveal novel compensatory sites capable of restoring the attenuated DTMUV during polyprotein cleavage and packaging.

The topological model of NS4B and its TMD3 in duck TMUV proliferation.

Duck Tembusu virus (DTMUV) belongs to the Flaviviridae family and mainly infects ducks. Duck Tembusu virus genome encodes one polyprotein that undergoes cleavage to produce 10 proteins. Among these, NS4B, the largest transmembrane protein, plays a crucial role in the viral life cycle. In this study, we investigated the localization of NS4B and found that it is located in the endoplasmic reticulum, where it co-localizes with DTMUV dsRNA. Subsequently, we confirmed 5 different transmembrane domains of NS4B and discovered that only its transmembrane domain 3 (TMD3) can traverse ER membrane. Then mutations were introduced in the conserved amino acids of NS4B TMD3 of DTMUV replicon and infectious clone. The results showed that V111G, V117G, and I118G mutations enhanced viral RNA replication, while Q104A, T106A, A113L, M116A, H120A, Y121A, and A122G mutations reduced viral replication. Recombinant viruses with these mutations were rescued and studied in BHK21 cells. The findings demonstrated that A113L and H120A mutations led to higher viral titers than the wild-type strain, while Q104A, T106A, V111G, V117G, and Y121A mutations attenuated viral proliferation. Additionally, H120A, M116A, and A122G mutations enhanced viral proliferation. Furthermore, Q104A, T106A, V111G, M116A, V117G, Y121A, and A122G mutants showed reduced viral virulence to 10-d duck embryos. Animal experiments further indicated that all mutation viruses resulted in lower genome copy numbers in the spleen compared to the WT group 5 days postinfection. Our data provide insights into the topological model of DTMUV NS4B, highlighting the essential role of NS4B TMD3 in viral replication and proliferation.

Iron efflux by IetA enhances ß-lactam aztreonam resistance and is linked to oxidative stress through cellular respiration in Riemerella anatipestifer.

BACKGROUND: Riemerella anatipestifer encodes an iron acquisition system, but whether it encodes the iron efflux pump and its role in antibiotic resistance are largely unknown. OBJECTIVES: To screen and identify an iron efflux gene in R. anatipestifer and determine whether and how the iron efflux gene is involved in antibiotic resistance. METHODS: In this study, gene knockout, streptonigrin susceptibility assay and inductively coupled plasma mass spectrometry were used to screen for the iron efflux gene ietA. The MIC measurements, scanning electron microscopy and reactive oxygen species (ROS) detection were used to verify the role of IetA in aztreonam resistance and its mechanism. Mortality and colonization assay were used to investigate the role of IetA in virulence. RESULTS: The deletion mutant ΔietA showed heightened susceptibility to streptonigrin, and prominent intracellular iron accumulation was observed in ΔfurΔietA under excess iron conditions. Additionally, ΔietA exhibited increased sensitivity to H2O2-produced oxidative stress. Under aerobic conditions with abundant iron, ΔietA displayed increased susceptibility to the ß-lactam antibiotic aztreonam due to heightened ROS production. However, the killing efficacy of aztreonam was diminished in both WT and ΔietA under anaerobic or iron restriction conditions. Further experiments demonstrated that the efficiency of aztreonam against ΔietA was dependent on respiratory complexes Ⅰ and Ⅱ. Finally, in a duckling model, ΔietA had reduced virulence compared with the WT. CONCLUSION: Iron efflux is critical to alleviate oxidative stress damage and ß-lactam aztreonam killing in R. anatipestifer, which is linked by cellular respiration.

Ultrasonic-assisted extraction of polysaccharide from Paeoniae Radix alba: Extraction optimization, structural characterization and antioxidant mechanism in vitro.

Paeoniae Radix alba is used in Traditional Chinese Medicine for the treatment of gastrointestinal disorders, immunomodulatory, cancer, and other diseases. In the current study, the yield of Paeoniae Radix alba polysaccharide (PRP) was significantly increased with optimal ultrasound-assisted extraction compared to hot water extraction. Further, an acidic polysaccharide (PRP-AP) was isolated from PRP after chromatographic separation and was characterized as a typical pectic polysaccharide with side chains of arabinogalactans types I and II. Moreover, it showed antioxidant effects on LPS-induced damage on IPEC-J2 cells determined by qRT-PCR and ELISA, including decreasing the pro-inflammatory factors' expressions and increasing the antioxidant enzymes activities, which was shown to be related to the Nrf2/Keap1 pathway modulated by PRP-AP. The metabolites change (such as itaconate, cholesterol sulfate, etc.) detected by untargeted metabolomic analysis in cells was also shown to be modulated by PRP-AP, and these metabolites were further utilized and protected cells damaged by LPS. These results revealed the cellular active mechanism of the macromolecular PRP-AP on protecting cells, and supported the hypothesis that PRP-AP has strong benefits as an alternative dietary supplement for the prevention of intestinal oxidative stress by modulating cellular metabolism.

Multiple functions of the nonstructural protein 3D in picornavirus infection.

3D polymerase, also known as RNA-dependent RNA polymerase, is encoded by all known picornaviruses, and their structures are highly conserved. In the process of picornavirus replication, 3D polymerase facilitates the assembly of replication complexes and directly catalyzes the synthesis of viral RNA. The nuclear localization signal carried by picornavirus 3D polymerase, combined with its ability to interact with other viral proteins, viral RNA and cellular proteins, indicate that its noncatalytic role is equally important in viral infections. Recent studies have shown that 3D polymerase has multiple effects on host cell biological functions, including inducing cell cycle arrest, regulating host cell translation, inducing autophagy, evading immune responses, and triggering inflammasome formation. Thus, 3D polymerase would be a very valuable target for the development of antiviral therapies. This review summarizes current studies on the structure of 3D polymerase and its regulation of host cell responses, thereby improving the understanding of picornavirus-mediated pathogenesis caused by 3D polymerase.

Molecular characterization of a virulent goose astrovirus genotype-2 with high mortality in vitro and in vivo.

Goose astrovirus (GAstV) is a newly identified viral pathogen threatening waterfowl, exhibiting a high prevalence across various regions in China. Notably, the Guanghan District of Deyang City, situated in Sichuan Province, has faced a outbreak of GAstV, resulting in significant mortality among goslings due to the induction of gout-like symptoms. In our research, we successfully isolated a GAstV strain known as GAstV SCG3. This strain exhibits efficient replication capabilities, proving virulent in goslings and goose embryos. Our study delved into the characteristics of GAstV SCG3 both in vitro and in vivo. Additionally, we examined tissue phagocytosis and the distribution of GAstV SCG3 in deceased goslings using H&E staining and IHC techniques. According to the classification established by the ICTV, GAstV SCG3 falls under the category of GAstV genotype-2. Notably, it demonstrates the highest homology with the published AHAU5 sequences, reaching an impressive 98%. Furthermore, our findings revealed that GAstV SCG3 exhibits efficient proliferation exclusively in goose embryos and in LMH cells, while not manifesting in seven other types of avian and mammalian cells. Significantly, the mortality of GAstV on goslings and goose embryos are 93.1 and 80%, respectively. Moreover, the viral load in the livers of infected goslings surpasses that in the kidneys when compared with the attenuated strain GAstV SCG2. The mortality of GAstV is usually between 20% and 50%, our study marks the first report of a virulent GAstV strain with such a high mortality.

Klebicin E, a pore-forming bacteriocin of Klebsiella pneumoniae, exploits the porin OmpC and the Ton system for translocation.

Bacteriocins, which have narrow-spectrum activity and limited adverse effects, are promising alternatives to antibiotics. In this study, we identified klebicin E (KlebE), a small bacteriocin derived from Klebsiella pneumoniae. KlebE exhibited strong efficacy against multidrug-resistant K. pneumoniae isolates and conferred a significant growth advantage to the producing strain during intraspecies competition. A giant unilamellar vesicle leakage assay demonstrated the unique membrane permeabilization effect of KlebE, suggesting that it is a pore-forming toxin. In addition to a C-terminal toxic domain, KlebE also has a disordered N-terminal domain and a globular central domain. Pulldown assays and soft agar overlay experiments revealed the essential role of the outer membrane porin OmpC and the Ton system in KlebE recognition and cytotoxicity. Strong binding between KlebE and both OmpC and TonB was observed. The TonB-box, a crucial component of the toxin-TonB interaction, was identified as the 7-amino acid sequence (E3ETLTVV9) located in the N-terminal region. Further studies showed that a region near the bottom of the central domain of KlebE plays a primary role in recognizing OmpC, with eight residues surrounding this region identified as essential for KlebE toxicity. Finally, based on the discrepancies in OmpC sequences between the KlebE-resistant and sensitive strains, it was found that the 91st residue of OmpC, an aspartic acid residue, is a key determinant of KlebE toxicity. The identification and characterization of this toxin will facilitate the development of bacteriocin-based therapies targeting multidrug-resistant K. pneumoniae infections.

Functional characterization of RhuB as a second TonB2-dependent hemin receptor in Riemerella anatipestifer CH-1.

In the previous study, it was shown that Riemerella anatipestifer (R. anatipestifer, RA), a pathogen in ducks and some other birds, encodes a hemin uptake system. The R. anatipestifer hemin uptake receptor RhuR is a TonB2-dependent hemin transporter. However, it remains unclear whether R. anatipestifer encodes additional TonB-dependent hemin transporters. Herein, we demonstrated that R. anatipestifer hemin uptake receptor B (RhuB) of R. anatipestifer CH-1 (RA CH-1) was negatively regulated by iron and mediated by the Fur protein, and knocking out rhuB damaged the ability of RA CH-1 to utilize iron from duck hemoglobin (Hb) but not that from duck serum. Moreover, the ability to use iron from Hb was restored by the expression rhuB in trans. Furthermore, the RhuB of RA CH-1 is a membrane protein, and recombinant RhuB could bind hemin at a 1:1 molar ratio in vitro. Compared to that of ΔtonB1ΔrhuR, the ability of ΔtonB1ΔrhuRΔrhuB to utilize hemin was impaired; meanwhile, compared to that of ΔtonB2ΔrhuR, the hemin utilization ability of ΔtonB2ΔrhuRΔrhuB was not affected, indicating that RhuB is a TonB2-dependent receptor. Compared to ΔrhuB, ΔrhuBΔrhuA did not affect hemin utilization. However, compared to ΔrhuA, ΔrhuBΔrhuA had reduced ability to utilize hemin, suggesting that RhuA relies on RhuB for its activity. Finally, the deletion of rhuB did not affect the virulence of RA CH-1. These results suggested that RhuB encodes a TonB2-dependent hemin receptor. The characterization of the second TonB-dependent receptor in R. anatipestifer enriches our understanding of the hemin uptake system of this bacterium.IMPORTANCEIron is essential for the survival of most bacteria, and hemin of hemoglobin can serve as an important iron source. In our previous studies, we showed that R. anatipestifer CH-1 encodes a TonB2-dependent hemin receptor RhuR, which is involved in hemin uptake. The deletion of rhuR did not abolish hemin utilization by RA CH-1. We hypothesized that additional hemin uptake systems exist in this bacterium. In this study, we identified the second TonB2-dependent hemin receptor RhuB in RA CH-1 through hemin utilization, protein localization, and hemin-binding experiments. The duck infection model showed that the deletion of rhuB did not affect the virulence of RA CH-1. This study is not only important for further understanding the hemin utilization mechanism of R. anatipestifer, but also for enriching the hemin uptake transporters of gram-negative bacteria.

Characterization of antibiotic resistance genes and mobile genetic elements in Escherichia coli isolated from captive black bears.

The objective of this study was to analyze the antimicrobial resistance (AMR) characteristics produced by antibiotic resistance genes (ARGs), mobile genetic elements (MGEs) and gene cassettes in Escherichia coli isolated from the feces of captive black bears. Antimicrobial susceptibility testing was performed by using the disk diffusion method, and both MGEs and integron gene cassettes were detected by polymerase chain reaction. Our results showed that 43.7% (62/142) of the isolates were multidrug resistant strains and 97.9% (139/142) of the isolates were resistant to at least one antibiotic. The highest AMR phenotype was observed for tetracycline (79.6%, 113/142), followed by ampicillin (50.0%, 71/142), trimethoprim-sulfamethoxazole (43.7%, 62/142) and cefotaxime (35.9%, 51/142). However, all isolates were susceptible to tobramycin. tetA had the highest occurrence in 6 ARGs in 142 E. coli isolates (76.8%, 109/142). Ten mobile genetic elements were observed and IS26 was dominant (88.0%, 125/142). ISECP1 was positively associated with five ß-lactam antibiotics. ISCR3/14, IS1133 and intI3 were not detected. Seventy-five E. coli isolates (65 intI1-positive isolates, 2 intI2-positive isolates and 8 intI1 + intI2-positive isolates) carried integrons. Five gene cassettes (dfrA1, aadA2, dfrA17-aadA5, aadA2-dfrA12 and dfrA1-aadA1) were identified in the intI1-positive isolates and 2 gene cassettes (dfrA1-catB2-sat2-aadA1 and dfrA1-catB2-sat1-aadA1) were observed in the intI2-positive isolates. Monitoring of ARGs, MGEs and gene cassettes is important to understand the prevalence of AMR, which may help to introduce measures to prevent and control of AMR in E. coli for captive black bears.

NS1: a promising novel target antigen with strong immunogenicity and protective efficacy for avian flavivirus vaccine development.

Tembusu virus (TMUV), an avian pathogenic flavivirus, has emerged as a significant threat to the duck industry in Southeast Asia, causing substantial economic losses. Due to the antibody-dependent enhancement (ADE) effect of TMUV subneutralizing antibodies, there is a pressing need to further develop new TMUV vaccine target antigens that ensure both safety and efficacy. Here, the TMUV non-structural protein 1 (NS1) as a target for development of effective anti-TMUV vaccines was unveiled. The amino acid sequences of TMUV NS1 exhibit a high degree of conservation across different strains (92.63-100%). To investigate the potential of TMUV NS1 as a vaccine target, the TMUV NS1-based plasmids were constructed and identified the C-terminal 30 amino acids residues of TMUV E (EC30) as an effective signal peptide for promoting NS1 expression and secretion. Subsequently, the plasmid pVAX1-EC30-NS1 was employed to immunize ducks, resulting in specific anti-NS1 IgG responses being stimulated, while without inducing anti-TMUV neutralizing antibodies. Furthermore, the cellular immune responses triggered by the TMUV NS1 were evaluated, observing a notable increase in lymphocyte proliferation at 4 wk and 6 wk postinjection with the pVAX1-EC30-NS1. Additionally, there was a significant up-regulation of NS1-specific Il-4 and Ifnγ levels at these time points. Following this, ducks from different groups were challenged with TMUV, and remarkably, those immunized with the NS1 vaccine displayed significantly lower viral copies both at 3 d postinfection (dpi) and 7 dpi (P < 0.05) compared to ducks immunized with the control vector. Notably, the NS1 demonstrated remarkable protection against TMUV challenge without causing severe gross lesions. Collectively, these findings highlighted the impressive immunogenicity and protectivity of the TMUV NS1. Consequently, NS1 holds great promise as a novel antigen target for the development of efficient and safe TMUV vaccines.

An alpha-herpesvirus employs host HEXIM1 to promote viral transcription.

Although it is widely accepted that herpesviruses utilize host RNA polymerase II (RNAPII) to transcribe viral genes, the mechanism of utilization varies significantly among herpesviruses. With the exception of herpes simplex virus 1 (HSV-1) in alpha-herpesviruses, the mechanism by which RNAPII transcribes viral genes in the remaining alpha-herpesviruses has not been reported. In this study, we investigated the transcriptional mechanism of an avian alpha-herpesvirus, Anatid herpesvirus 1 (AnHV-1). We discovered for the first time that hexamethylene-bis-acetamide-inducing protein 1 (HEXIM1), a major inhibitor of positive elongation factor B (P-TEFb), was significantly upregulated during AnHV-1 infection, and its expression was dynamically regulated throughout the progression of the disease. However, the expression level of HEXIM1 remained stable before and after HSV-1 infection. Excessive HEXIM1 assists AnHV-1 in progeny virus production, gene expression, and RNA polymerase II recruitment by promoting the formation of more inactive P-TEFb and the loss of RNAPII S2 phosphorylation. Conversely, the expression of some host survival-related genes, such as SOX8, CDK1, MYC, and ID2, was suppressed by HEXIM1 overexpression. Further investigation revealed that the C-terminus of the AnHV-1 US1 gene is responsible for the upregulation of HEXIM1 by activating its promoter but not by interacting with P-TEFb, which is the mechanism adopted by its homologs, HSV-1 ICP22. Additionally, the virus proliferation deficiency caused by US1 deletion during the early infection stage could be partially rescued by HEXIM1 overexpression, suggesting that HEXIM1 is responsible for AnHV-1 gaining transcription advantages when competing with cells. Taken together, this study revealed a novel HEXIM1-dependent AnHV-1 transcription mechanism, which has not been previously reported in herpesvirus or even DNA virus studies.IMPORTANCEHexamethylene-bis-acetamide-inducing protein 1 (HEXIM1) has been identified as an inhibitor of positive transcriptional elongation factor b associated with cancer, AIDS, myocardial hypertrophy, and inflammation. Surprisingly, no previous reports have explored the role of HEXIM1 in herpesvirus transcription. This study reveals a mechanism distinct from the currently known herpesvirus utilization of RNA polymerase II, highlighting the dependence on high HEXIM1 expression, which may be a previously unrecognized facet of the host shutoff manifested by many DNA viruses. Moreover, this discovery expands the significance of HEXIM1 in pathogen infection. It raises intriguing questions about whether other herpesviruses employ similar mechanisms to manipulate HEXIM1 and if this molecular target can be exploited to limit productive replication. Thus, this discovery not only contributes to our understanding of herpesvirus infection regulation but also holds implications for broader research on other herpesviruses, even DNA viruses.

Duck plague virus Us3 regulates the expression of pUL48.

Duck plague (DP) is one of the contagious diseases caused by Duck plague virus (DPV), which is a serious threat to the development of duck farming. Us3 is a PKA-like protein kinase in alphaherpesvirus, which can regulate the biological functions of many viral proteins, but whether Us3 regulates pUL48 protein has not been reported. In this paper, Western Blot, qRT-PCR, dual luciferase reporter system and Co-IP were used to investigate the relationship between pUL48 and Us3. The results showed that: 1) pUL48 interacted with Us3 at 138-256aa through its DBD region. 2) Us3 enhanced the protein expression of pUL48 in a dose-dependent manner. 3) Us3 promoted the mRNA level of pUL48 by activating its promoter activity. 4) Us3 inhibited the transcriptional activation function of pUL48. The results can provide scientific data for perfecting and supplementing the function of alpha herpesvirus Us3 and pUL48.

CCCP inhibits DPV infection in DEF cells by attenuating DPV manipulated ROS, apoptosis, and mitochondrial stability.

Duck plague virus (DPV) is extremely infectious and lethal, so antiviral drugs are urgently needed. Our previous study shows that DPV infection with duck embryo fibroblast (DEF) induces reactive oxygen species (ROS) changes and promotes apoptosis. In this study, we tested the antiviral effect of the carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a common mitochondrial autophagy inducer. Our results demonstrated a dose-dependent anti-DPV effect of CCCP, CCCP-treatment blocked the intercellular transmission of DPV after infection, and we also proved that CCCP could have an antiviral effect up to 48 hpi. The addition of CCCP reversed the DPV-induced ROS changes, CCCP can inhibit virus-induced apoptosis; meanwhile, CCCP can affect mitochondrial fusion and activate mitophagy to inhibit DPV. In conclusion, CCCP can be an effective antiviral candidate against DPV.

Characteristics of MDR E. coli strains isolated from Pet Dogs with clinic diarrhea: A pool of antibiotic resistance genes and virulence-associated genes.

The increasing number of multi-drug resistant (MDR) bacteria in companion animals poses a threat to both pet treatment and public health. To investigate the characteristics of MDR Escherichia coli (E. coli) from dogs, we detected the antimicrobial resistance (AMR) of 135 E. coli isolates from diarrheal pet dogs by disc diffusion method (K-B method), and screened antibiotic resistance genes (ARGs), virulence-associated genes (VAGs), and population structure (phylogenetic groups and MLST) by polymerase chain reaction (PCR) for 74 MDR strains, then further analyzed the association between AMRs and ARGs or VAGs. Our results showed that 135 isolates exhibited high resistance to AMP (71.11%, 96/135), TET (62.22%, 84/135), and SXT (59.26%, 80/135). Additionally, 54.81% (74/135) of the isolates were identified as MDR E. coli. In 74 MDR strains, a total of 12 ARGs in 6 categories and 14 VAGs in 4 categories were observed, of which tetA (95.95%, 71/74) and fimC (100%, 74/74) were the most prevalent. Further analysis of associations between ARGs and AMRs or VAGs in MDR strains revealed 23 significant positive associated pairs were observed between ARGs and AMRs, while only 5 associated pairs were observed between ARGs and VAGs (3 positive associated pairs and 2 negative associated pairs). Results of population structure analysis showed that B2 and D groups were the prevalent phylogroups (90.54%, 67/74), and 74 MDR strains belonged to 42 STs (6 clonal complexes and 23 singletons), of which ST10 was the dominant lineage. Our findings indicated that MDR E. coli from pet dogs carry a high diversity of ARGs and VAGs, and were mostly belong to B2/D groups and ST10. Measures should be taken to prevent the transmission of MDR E. coli between companion animals and humans, as the fecal shedding of MDR E. coli from pet dogs may pose a threat to humans.